«2 United States Patent Furusako et al 'US007465547B2 (10) Patent No.: US 7,465,547 B2 (45) Date of Patent: Dec. 16, 2008 (54) METHODS FOR DETECTING HUMAN LOW MOLECULAR WEIGHT CD14 (75) Inventors: Shoji Furusako, Shizuoka (JP); Kamon. Shirakawa, Shizuoka (JP) (93) Assignee: Mochida Pharmaceutical Co, Ltd. Tokyo (1P) (4). Notice: Subject to any diselaimer, the tem of this patent is extended or adjusted under 35 USC. 154(b) by 384 days. an @) (66) Appl.Nos — 100834,257 PCT Filed: Now. 12, 2003 PCT Nos PCTISPO3/14369 $371 (oA). (2),(4) Date: May 10, 2008 (87) PCT Pub. Nos Wo2004/044005 PCT Pubs, Date: May 27, 2004 3) Prior P US 200710106067 A ication Duta May 10, 2007 G0) Now: 12, 2002 Sep. 22,2008 orsign Application Priority Data (e) 2002-328866 cP) 2005-33077 (1) Ince OTK 1628 C12? 2108 GOIN 33/50 GOIN 33/543, GIN 33/577 (2006.01) us.cl, 435/741; 435)7.24; 435/78: 530/388,221 530/388.23; S30°388.7;$301389.2 Field of Classification Seareh None ‘Se pplication ile for complete search history. References Cited (2006.01) (2006.01) (2006.01) (2006.01) (2) (8) 66) USS. PATENT DOCUMENTS 5766599 A 6.1998 Lichemseine a 6016628 BI 772005 Ferwsako ea 20080141917 AL 72004 Achen eta FOREIGN PATENT DOCUMENTS: ro19ss ‘62002 12003 2003 711996 280876 AS 1aas86 AL US71 AL 1336620 AL W09620956 AL WO-0172093 AL 10-2001 wo.0242333 AL 5/2002 OTHER PUBLICATIONS Levenson Clinical Laboratory News 2008, vol. 34, No.l fstreved ‘on Feb. 2, 2008], Retrieved fom the Internet: tse wo wo wo Weinisch etal. Clin Exp Immunol 1996, 10874-78° ion ea. Blood 1988, 92208-2002." ‘Manocha, Susy etal: Expat Opa. avestg, Drugs sol. 1,No. 12 pp. 1795-1812 (2002, Terman ct al, Molecular Immunology, vol 28, pp. 117-118 con, Teta Siochemisey: Pre. Natl Acad Sei, USA, vl 77,No. 6,9. “dun, 198), Journal of Protein Chemistry, vol, 11, No.5, pp 433 444.1992), Coleman eta, Research in Immusoogy, vol 148,No.L pp. 38°36, (on, Endo ot al, The Japanese Association for Infectious Disease, S2nd ast apan Mecing held Oct. 31,2003 English translation). Endo ctal. Th Joumal of Japanese Associaton fr Acute Main, ol 14, No. 10.p 602 (Ost 2005), English rans. The Oil Journal of the Shock Sit, Abstract othe 6th Wold Congres om Tra, Shock, inflammation and Sepsis (Mar. 200). Endo et al Jourmal of the Japanese Society of Intensive Care Mod cine, ol. 1 Supplement, p44, Jan 2004, English vasa The Oficial oural ofthe Criteal Care Forum v9, Soppeteat pp. S994 (Mar 7004), ‘Asc ofthe 104th General Assembly of the Japan Surgical So fy, Ap 2004 English alton (Ottcial Joumal ofthe European Society of ltensive Care Medicine ‘nd the European Society of Pediatrie & Neonatal Intensive Car, ol 30, Supplement Lp S192, Sep. 2004 Edo eal Program and Absracts ofthe 10th Conference ofthe Japan Endotoxin Society, No. 15, 2004, Fglsh easton Soural of Enotosin Research, v.10, No, p. 373, Now. 2008 Endo ea, Journal ofthe Japanese Society of Intensive Care Me cine, ol 12, Supplement, p21, Jan 2008, English uanslaion Endo ot al, The Joural ofthe lpanese Assocation To Infectious Diseases, vol 80, Supplement, p- 271, Mar 2006, English rans Endo otal, Japan Journal of Critical Care for Endotoxemi, vol. 9, No.1 pp. 6-80, 2005 English Abstract on p50. ‘Yacht al, Japanese Seityof Chemotherapy pp 11008) ‘Landmann, Repel: The Journal of afetious Disease va. 71 pp. 630-544 1995). Ste, Fl eta; Furopean Journal of Biochemistry (German): vol. 236 pp. 437-464 (1098). ‘Bazil, Vladimir et al; European Jounal of Immunology (German); ‘ol 16, pp 1883-1589 (1086). (Continved) Primary Examiner Philip Gambet “Assistant ExaminerSharon Wen (74) Attorney, Agent, or Firm—Bireh, Stewart, Kolasch & Birch, LLP PUD No. on ABSTRACT ‘The present invention provides an antibody prepared using a peptide as an antigen, the peptide having & to 30 amino acid residues selected rom an amino acid sequence at positions | ‘o 68 of humaa high-molecula-weight CD14, oran antibody that binds to peptide having a specific amino aeid sequence ata position among the positions 1 (0 68. An assay kit for ‘human low-moleeularweight CD14 using the antibody and ‘an assay method ofthe present invention, proferably a sand- ‘wich method, are able t quantitatively o qualitatively deter ‘ine human lowe-molectlae-weight CD14 with high sensitv~ ty and specificity ina simple manne, so that they are useful {or the diagnosis of patient suffering from sepsis £8 Claims, 7 Drawing SheetsUS 7,465,547 B2 Page 2 OTHER PUBLICATIONS chit, Chsine ot ls Allesgc und Immunologie (Germs), vo 2p 17-26 18). Van Voorhis, Wesley C. et als Journal of Experimental Medicine (USA), vol 158 pp. 126-145 1983). Bazil, Vadimir eal; Molecular Immunology (UK), vol 26, 1557.62 (1989. ‘Grand, Ut al; Joural of Immunological Methods (Holland vol 188, pp. 228-232 (1992}. ‘Brginan, in ct a Clinical Immunology an Immanopathol ‘ogy (USA). v0 $0. pp. 307-310 (1996) Shigeatsu Endo eal Journal of Japanese Associaton for ewe Meticine, Oct 15,2003, vl I, No 10, pp. 692, Op28S ‘Yaegishctal Japanese Scity of Chemotherapy an Te Japanese Asociation forthe nfxtions Diseases, ol. 11. No. S, 2005) Bp. mae Bra, uropcan Journal of Immunology, Fb, 1995, vl 25, No.2 XP-OO24SS84, pp 6410, Majete eta Europea Journal of Physiology 2000, vl 439, No.3, ‘XP-£02453846 pp, RIOO-RIIO, Stele tal European Journal of Biochemistry, vo. 243, No. | (1997), XP-002458847, pp. 100-109. Majel etal, Protein Expression and Purifstion, vol 17, No. 1999, pp. 96-104, Iwaki tal: Biochemical and Biophysical Research Communia- tions, vo 328, No.1, 2008), pp. 178176. ‘Kim tal; The Jounal of Bilogial Chemis: vl. 280, No.1, 11347-11381 2008), * cited by examinerUS 7,465,547 B2 Sheet 1 of 7 Dec. 16, 2008 U.S, Patent Auvd 3NI1 TOULNOD aa ALVONPNOO aNvuaNaN a azn vd ONiddoud ONIGYOSEY ta qyyyy <_e>/NOLLOILIA ~TIdWVS 7 f NoLugay @) AGOSLNYWNOTOAIOG* A N ee ASNON-LINY (i)- ACOBILLNY _ g9s())- 4S G3TIGVI-GIOTION a109 Luvd aN a ee aNvuaWan Qvd azn vd ONIddoua DNIGHOSBY iggy /NOLLAL8A “Hd NVS

f AQOBLNVIWNOTOMIOU AX @ a ASNOW-LLNY (i)- AGOSLLNY e9g(l)- £-€1-90114 G3738V1-GlOTIO G09 <}> T’OIadUS 7,465,547 B2 Sheet 2 of 7 Dec. 16, 2008 U.S, Patent (usj6u) L OL O00'} NOlLwavdaud GYVONVLS += + ++ Hed luonoe}0q oul loquog e°ordUS 7,465,547 B2 Sheet 3 of 7 Dec. 16, 2008 U.S, Patent M3171) Adu. dad ju W a 14/871) 30Ldad 0 oO s 0 0 soo B 8 soo ro z ro ro} sro mM oN sv0 SIS? s¢0 — sz0 SISdES WOUs ONESaaNS ANALLYd WOUS WNYAS TWNGIAIGNI TVWHON WOYs WNYaS “TOULNOO BALLVDEN 40 ACILdad (v9-6S SNOLUSOd LY) 30LLdad #109 —o— (29-LS SNOLLISOd LY) 30ILd3d bdo (8S-€5 SNOIISOd 1) 30LLd3d #100 —o— (@9-€5 SNOILISOd L¥) 301Ld3d PL -— (wugsp) SONVaYOSEY €°OIdU.S. Patent Dec. 16, 2008 Sheet 4 of 7 US 7,465,547 B2 + a ABSORBANCE (450nm) 2° oa 0 50 100 150 CD14(1-307)S286C (ng/mL)U.S, Patent CD14(1-307)S286C(ng/mL) 50 40 30 20 10 0 Dee. 16, 2008 Sheet 5 of 7 US 7,465,547 B2 FIG.5 [oN et est Pn 0 1 2 3 4 SOLUBLE CD14 FROM SERUM OF NORMAL INDIVIDUAL (2 g/mL)US 7,465,547 B2 Sheet 6 of 7 Dec. 16, 2008 U.S, Patent “ON YORE oy Ge oF gz Oe sh OL ¥ ; Basses r oor = 002 > | — oog & =f — = o0v | 00s LUM VIA-p109 LHOISM-YV INDFIOW-MO1-C-] LD VIB FIGVUVAY ATIVIONaWNOO -0-| - oo t ft eoioy BAAS 9° DIAUS 7,465,547 B2 Sheet 7 of 7 Dec. 16, 2008 U.S, Patent uonoely st O& 8 4 SL [LM VI3-b19 LHDISM-YVINDATON-MOT -o-] AM YIS SIEVIVAY ATIVIOUSWNO9 —0-] t.2 2O4 SED 9 tttt ease BG4Lo BaNZ'eL L°o1a eqnep og oor ost 002 osz 00 ose oor Osh 00S (1m /3u)US 7,465,547 B2 1 METHODS FOR DETECTING HUMAN LOW ‘MOLECULAR WEIGHT CD14 ‘TECHNICAL FIFLD ‘The presentiavention relates (oan antibody prepared using peptides an antigen, the peptide having 8 to 30 amino acid residues selected Irom specific amino acid sequences for human high molecular weight CD14, Furthemore, the present invention elates tan antibody that binds toa peptide having a specific amino acid sequence in amino acid sequences for human high molecular weight CD14, TInaddition, the present invention relates to an assay’ for human low-moleeularveight CD14 and a method of mea- suring the same. Furthermore, the present invention relates to ‘4 novel diagnostic method for sepsis in which human low= ‘molecular-weight CDI‘ isdetennined directly. Furthermore, the present invention relates toa peptide useful for the prepae ration ofthe above antibody anda method of preparing the ‘antibody, BACKGROUND ART A.CDI4 molecule was named as a protein identified by 3 amily of antibodies that recognize glycoproteins expressed ‘on the membrane surface of monocytes in Thied Leukocyte “Typing Conference, 1986, Ia 1990, Wright et a. elucidated that the CDI4 molecule iso receptor for LPS, endotoxin Science”, vol. 249, p, 1431-1433, 1990), The CD14 mol- ‘cule is a glyeaprocein having a molecular weight of 3-55 Da, and analyses on cDNA revealed that 1.4 kb mRNA has coding sequence of 356 amino (“Nucleic Acids Research” (CK), vol. 16, p. 4173, 1988), It was reported that human CD14 molecules include soluble CDI4 molecules in addition to: membrane-bound ‘CD14 molecules and blood contains solubleCD4moleciles having different molecular weights (“European Journal of Immunology” (Germany), vol. 23, p. 2144-2151, 1998). In addition, Landmann etal. conducted Wester blot analyseson soluble CD14 in serum of patients suffering from sepsis and reported that soluble CDI of about $5 kDa isat high levels Jn non-suevval sepsis patients and patients with paroxysmal actual hemoglobinria (PNT) and that i normal sera, this molecule was aot detected but soluble CD14 of 49-4Da, 9 slightly lower molecular weight than the former, was detected (The Joumal of Infectious Disease”, vol. 171, 9. 639-644, 1995), Stelter reported that the difference in sugar chains is Jnvolved in those subtypes having different molecular weights and two soluble CD14 subtypes having diferent ‘molecular weights are found in blood even aller removal of N= and O-linked sugar chains ("Puropean Joumal of Bio- chemistry” (Germans), vol, 236, p. 457-464, 1996). In ad tion, Buller etal. conducted the C-terminal analysis on soluble CD14 and reported that a GPI group binds to serine residue at postion 327 of soluble CD14 and that a soluble ‘CD1d molecule having a molecular weight ofabout S6KDa is ‘oneof the molecular species from which GPl isnot anchored (European foural of Immunology” (Germany), vol. 25, p. 604-610, 1995), Antibodies against CD14 molecules include many ant ‘CD14 atiboties, which have been prepared and used in ‘identification of CD14 proteins, such as MEM-I8 prepared by Bazil etal. (“European Journal of Immunology” (Ger- many), vol. 16, p- 1588-1589, 1986), RoMo-1 prepared by butt etal. Allergic und Immunologie" (Germany), vol. 34, 0 o 2 ».17-26, 1988),and 3C10 prepared by Steinman etal “Jour ‘al of Experimental Medicine” (US.A.), vol. 158, p. 126- 145, 1983), Furtermore, soluble-CD14 assay systems using those antibodies have been reported by Shut etal. (DE-286876-A), Bazil et al. (*Molecolar Immunology” (U'K.), vol. 26, 9 657-662, 1989), and Grunwald et al. (“Jounal of Immuno- logical Methods” (Holland), vol. 155, p. 225-232, 1992), allowing the assay of soluble CD14 in human body uid, Furbermore, soluble CDIMELISA. kits have been released on the market from IBL-Haahurg, Medgenix, and & D Systems, and the assay of soluble CD14 hns been per Tormed for many diseases sch as sepsis (“Clinical Immunol- ogy And Immunopathology” (US.A.}, vol. 80, p. 307-310, 1996; and “Rinshokensa", vo. 38, p. 341-344, 1994), However, it was found that soluble CD14 is nota sep specific marker hecause of increases in levels of soluble CD14 molecules of about $5 kDa aud 49 kDa (lm report to report, the molecular weights are differnt and not limite to bout §5 kDa and 49 kDe, andthe same will be appli in the {ollowing description) depending on thedegreeof proceeding ‘of diseases even in diseases except sopsis (“Infection and Immunity” (US.A.), vol. 67, p. 417-420, 1999; “Clinical and Experimental Immunology” (U.K.), vol. 120, p. 483-487, 2000; and “Clinical Experimental Immunology” (U.K.), vl. 96, p. 15-19, 1994). Funhermore, the soluble CD14 was expected to be a marker for the severity of sepsis, However, the soluble CD14 has not heen provided as a diagnostic pode vet for sepsis because of no correlation With septic shock (Pediatrie allergy and immunology) (Denmark), vel 8p 194-199, 1997) and also no correlation with systemic inflam: matory response syndrome (SIRS) (“European Joumal of Clinical Investigation” (U.X.), vol. 28, p. 672-678, 1998), ‘The inventors of the present iaventon have found out the presence of @ soluble CD14 molecule with a ow molecular ‘Weight of about 36 Da i blood in addition to others such as to kinds of soluble CD14 molecules described above of about $5 kDa and 49 kDa reported by Landmann etal. (high ‘molecular weight CD14 (from report to report, the molecular ‘weights are different and not limited to about 35 kDa and 49 kDa, and the same willbe applied in the Following desrip- tion). The inventors ofthe present invention have also found ‘out the presence of a small amount ofthe low-molecular- ‘weight CD14 in noemal individuals and of an increased ‘amount of the Iow-molecular-weight CD14 in patients suf- {ering from sepsis, Consequently, the inventors of the present invention have validated the clinical efficacy ofthe assay on a soluble low-moleculareweight CD14, However, the ant CD14 antibodies known inthe art are those that recpize & high-molecular-weight sohible CD14 protein or those resog- sizeboth high-and low-molecula- weight soluble CD14 pro- teins. Thus, no antibody that recognizes only a low-molecu- Jr-weight CD14 bas been known in the art. Besides, the aminoacid sequence ofthe low-molecular-weight CD14 pro- tein las been considered to be identical with a part of the amino acid sequence of the highsmolecularweight soluble CD14 protein, so that the preparation of an antibody as described above and a direct immunological assay on the Jow-molecular-weight CD14 using the antibody’ have beea considered to be difficult, Therefore, as an assay for the soluble low-molecular-weight CD14, there isa proposal in ‘which the level of low-molecular-weight CD14 in blood is indirectly obtained by subtracting the level of high-molecu- lar-weight CD14 i blood from the total level of the soluble CD14 in blood (International publication WO 01/22085).US 7,465,547 B2 3 DISCLOSURE OF THE INVENTION ‘Under such circumstances, an assy for qualitatively oF ‘quantitatively determining human low-moleculr-weight, ‘CD14 with high seositivity and specificity in a convenient ‘manner, the assay allowing direct determination of the human Jow-molecular-weight CD14 and being useful forthe diag- nosis of a patient suffering from sepsis, and an assay it for the assay have Been desired. Furthermore, an antibody against the human low-molecular-weight CD14 useful forthe assay has heen desired. ‘The inventors ofthe preset invention have invented, as 3 result of the extensive study, an atibody prepared using & peptide as an antigen, the peptide having 8 to 30 amino acid residues selected from amino acid sequences at positions 110 68 of human high-moleculae-weight CD14 as an antibody which can be used for qualitatively or quantitatively deter rmning human los-moleculae-weight CD14 with high sensi tivity and specificity ina convenient manner. In addition, the Jnventors ofthe present invention have invented an antibody that binds toa peptide having a specific amino acid sequence jin the amino seid sequence for the human high-molecula- weight CD14. Furthermore the inventors ofthe present invention have invented at assay for specifically determining human low- moleculae-weight CD14 and an assay kit for human l= molecular-weight CD14, Sill furthermore the inventors of the present invention have invented a novel diagnostic method for sepsis in which human low-molecular-weight ‘CD14 is determined directly. Besides, the inventors of the present invention have iavented peptide usefl forte prepa- Fation ofthe above antibody’ and a method of preparing the above antibody. In other words, the present invention provides the fllow= ing novel antibodies anda assay kit for man low-molec- weDIA, fies as described inthe following (1-1) t0 (1-5: (1-1) An antibody prepared using a peptide as an antigen, the peptide having consecutive & to 30 amino acid residues selected roman amino acid sequence described in SEQID Nord (1-2) An antibody prepared using a peptide as an antigen, the peplide having consecutive & 10 20 amino acid residues selected! roman amino acid sequence described in SEQID NO: I 3) An antibody prepared using a peptide as an antigen, the peplide having consecutive & to 16 amino acid residues Selecta rom amino aed sequences a positions 530 68 in the amino acid Sequence described in SEQ ID: 1 -4) An antibody prepared using a peptide as an antigen, the peptidehaving amino acid resides described in aay oneof SEQ ID NOS: 2 to 4 (1-5) An antibody prepared using a peptide asan antigen, the peplidehaving ls amino cid residues described in SEQ ID. NO: 2. (2) Antibodies as described in the following (2-1) and (2-2) (2-1) An antibody that binds toa peptide having amino acid residues described in ay one of SEQ ID NOS: 210 4. (2-2) An antibody that binds to a peptide having 16 amino ‘acid residues deserbed in SEQ ID NO: 2 @) Assay kits for human low-moleculaeweight CD14, rep- resented inthe following (3-1) to (3-22) (G-1) Anassay kit for human low-molecular-weight CD14 for tiretly assaying human lows-molecelar-weight CD14 ina specimen without detecting human high-molecula-weight 4 CD14, comprising an antibody that binds tat least one of the human low-molectlacweight CD14 or « fragment thereot (6-2) Theassaykit for haman fow-molecular-weight CD14 of 5 Gel), wherein the antibody that binds o the human low- ‘molecular-weight C14 or the fragment thereof isthe an body described in any oneoftheabove (1-1)t0(1+5)}, (2-1), oF 2-2),0r a fragment thereol (6-3) Theassay'kit for human fow-moleculae-weight CD14 of {G-1), wherein the antibody that binds to the human low- ‘molecular-weight CD14 or the Feagment there's the an body described in any ane ofthe above (1-4), (1-5), 2-, or (2-2), ora fragment thereof (6-4) Theassay'kit for niman low-moleculae-weight CD14 oF {G-1), wherein the antibody that binds tothe human low- ecular-weight CD14 or the fragment thereof isthe ant ‘body described in any one of the above (I-5) oF (2-2), oF ‘rapment thereof (G-5) Theassay kit for human fow-molecular-weight CD14 of any one of 3-1) 10 G4), wherein the human low-molecu- Tarweight CD14 is assayed by a sandwich immunoassay method (6-6) Theassay kit for sman low-molecular-weight CD14 of {G-5), further compesing a second binding substance that ‘binds to the human low-molecular-weight CD (G-7) Theassay'kitfor himan low-moleculag-weight CD40 {G.6), wherein the secon binding substance isan utibody ‘tha binds to the hunan low-moleculae-weight CD14 oF & fragment thersof, 20-8) Theassay kit for human low-molecular-weight CDI4of (G6), wherein the second binding substance is mono- clonal antibody that binds to the human low-molecular- wweight CD14. G-9) The assay kit of (3-6), wherein the second binding substance is an antibody that binds to any one region of: Amino acid residues a positions 1 to 52 of human high- ‘molecular-weight CD14 a fragment thereof an antibody tat competes with or shows eross-reaetvity with nan ‘ody that binds to any one region of amino acid resis at positions 1 to 52 of the human high-molecular-weight CD14: or a fragment thereof. G-10) The assay kit of (3-6), wherein the second biding Substance is: an antibody that binds to any one of amino acid residues at positions 17 to 26 of humaa high-molect- larweight CD14: a figment thereof, an antibody that competes with or shows emss-reactivity with an antibody ‘ha hinds to any one of amin acid resides at positions 17 0 26 ofthe human high-molecular-weight CD14; othe ‘ragmeat thereof, 50 G-11) The assay kit for human low-molecular-weight CD14 fof any one of (2-6) to (3-10), wherein the antibody phase direct method or solid-phase binding method, compe- tition method, or the like. Those methods will be deseribed Jat. “The high-molecular-weight CD14 may be prepared wsing an antibody specifi to the high-molecularweight CD14, ‘hich is described in Example 5 of WO 01/22085. Tris also conceivable that the antibody may be prepared by preparing and selecting an antibody that dacs not bind to the high-molecular-weight CD14 by the same way as described above using a peptide as an antigen, the peptide having a part ‘of the amino aeid sequence of human CD14. The “peptide having « part of the amino aeid sequence of human CD14” means, for example, each peptide that contains consecutive 8 ‘oF more amino acide in the sequence of 16 amino acids ‘deseribed in SEQ ID NO: 2. ‘According toa third aspect ofthe presen invention theres provided a assay kit for aman low-molecul-weight CD14, ‘hich contains an antibody that binds toateastone of human Jow-molecular-weight CDI4 or a fragment ofthe antibody ‘and which direlly assays the human low-molecular-weight ‘CD1d ina specimen without detecting human high-moleca- Jar-weight CDI. ‘The kt of the present invention contains an antibody that binds toat least one ofhuman lo-moleculae-weight CD14 or a frogment of the antibody and diretly assays the human low-molecular-weight CDI in a specimen. In addition, the kitdeteets the human fow-molecular-weight CD14 3s an ans Jytebut doesnot detect human high-molecular-weight CDI, 0 that the human low-molectlaeweight CDI4 ean. be rely assayed. The “fragment of the antibody” means Fab, Fab’ or F(ab), ofthe antibody. ‘The assay Kit for human low-molecular-weight CDI4 of the present invention is not specifically limited a far as it ‘contains an antibody that binds to at least one of human Jow-molecular-weight CDM or fragment ofthe antibody ‘and dieetly assays the human low-molecular-weight CDI jn specimen, Preferably, itis an assay kt for human lw= molecular-weight CD14 containing the antibody of the present invention ofa fragment ofthe antibody asthe anti body that binds to the human low-molecularsveight CD14 oF the fragment ofthe antibody. More preferably. iti an assay Kit for hua low-molecula-weight CD14 including an an o 14 body prepared using a peptide as an antigen, the peptide bhaving amino acid resides described i any one of SEQ ID NOS: 2 to 4, oF a fragment of the antibody. In addition, preferably, it san assay kit for human low-molocular-weight CD14 including an antibody that binds toa peptide having amino acid residues described in any one of SEQ ID NOS: 2 {to ora fragment of the antibody as an antibody that binds to the human low-molecular-weight CD14 ora tragment of the antibody, Particularly preferably, itis an assay kt for human Jow-molectlar-weight CD14 incising an antibody prepared using a peptide as an antigen, the peptide having amino acid residues described in SEQ ID NO: 2 or a fragment of the antibody, or an antibody that binds to peptide having. 16 ‘aming acid residues described in SEQ IDNO: 2 or a fragment ‘of the antibody, as an antibody that binds to human low volecularaveight molecule ofa fragment ofthe antibody FFurtermore the principe ofthe assay is not specifically limited as far asthe assay i a method of immunologically assaying human low-molecular-weight CD14 using the ant body or the fragment theo, "Asan example the principle ofthe assay, an assay kit for ‘human low-moleculae-weight CD14 (hereinafter, it may be described as a sandwich immunoassay kit) which determines the human low-molecular-weight CD14 by a sandwich ‘immunoassay using the “antibody that binds to a peptide ‘having [6aino acid residues described ia SEQ ID NO: 2” as preferred example of the antibody aecording tothe second faspeet of the present invention, will be deseribed concretely. ‘A well-known method may be sedas the sandwich im ‘oassay. The prneipl, application, and modification of the assay are described in, forexample,"Hypersensiive Fazyme Immunoassay”, Fiji Ishikawa Fa, Cente for Academie Pub- lications Japan (1993), "New Utilization Examples and Applications to Diagnostic Reagent/Drug Development of Immunoassay”, Immunoassay Development Research Soci- ey, Keiei-Kyoiku Shuppan, and “Enzyme Immunoassay (3rd Fd, Pili Ishikawa Fa, Igak-Shoin Ld (1987). Furtermore the sandwich immunoassay kit of the present invention contains an antibody that binds o a peptide having 16 amino acid residues described in SEQ ID NO: 2. The chareterstic features ofthe antibody that binds to the pep- tide having 16 amino acids deseribed in SEQ ID NO: 2, a method of preparing such an antibody, andso one just asthe same as those aecording to the first aspeet of the present invention. Tbe antibody may be, but not spevifically limited a polyclonal antibody or a monoclonal antibody. ‘The sandwich immunoassay isa method using Wo or more kinds of antibodies that recognize different sites on a protein tobe usually assayed, where the assay is performed by form- ‘ng an antibody-antigen-antibody complex, ‘First, an insoluble easier coupled with a fist antibody is prepared and is then provided as a solid phase or a reaction lave. specimen is added wo the insoluble carrier provided Asthe solid phase and then they are allowed o react with each other. After they have heea reacted fora prodtemnined time Period, the solid phase is washed to remove an unbound Substance therefrom, Subsequently, a labeled second ant body is added. After the mixture has heen reacted for 2 pre- determined time period, the labeled antibody that didnot orm a complex is removed by washing and then the amount ‘of the complex bound w the solid pase is qualitatively or ‘quantitatively determined onthe basis of the labeled product ina specific manner. The sandwich method may use any one ‘of a method inching two steps as deseribed above (double- step method) and method including the step of simulta- ronusly aking both an antigen and a labeled antibody (Gingle-step method),US 7,465,547 B2 15, Inthe sandwich immunoassay kt ofthe present invention the assay is performed by forming. complex of the “antibody that binds to peptide having 16 amino seid residues described in SEQ ID NO: 2° human low-molecular-weight CD14—the “second binding substance that binds to the human low-molecular-weight CD14" The format ofthe sandwich immunoassay kt ofthe present Jnvention includes: an insoluble carer bound with an anti body that binds o a peptide having 16 amino acid residues described in SEQ ID: 2 and a second binding substance that binds toa labeled low-moleculae-weight CD14 (hereinafter it may be simply described as a sevond binding substance): oF ‘an insoluble carrier bound with a second binding substance ‘and an antibody that binds to a labeled peptide having 16, amino acid residues described in SEQ ID NO: 2. Examples of the second binding substance include an a body that binds tthe low-molecular-weight CDI4. The ani body that binds tothe low-molecula-weight CDI4 may be @ polyclonal antibody or 4 monoclonal autibody and is not specifically limited, The monoclonal antibody is preferable th respect to affinity to the sandwich immunoassay wing the antibody that binds to the peptide having 16 amino acid residues described in SEQIDNO- 2. Furthermore, thesecond binding substance may be a fragment of the monoclonal antibody. The fragment ofthe antibody is Fab, Fab’ or F(ab’): ‘ofthe monoclonal antibody ‘he antibody thatbindsto the low- molecular-weight CD14 (hereinafter, itmay be deseribedasa second antibody) may be an antibody that specifically binds to the low-molecular ‘weight CD14 or an antibody that binds to high-molecular ‘weight CD14 and isnot specifically limited. Preferably, ts ‘an antibody that binds 10a site different from that of the antibody ofthe present invention. The scnd antibody ie an tibody that binds toa region excep a region comresponding to 16 amino acids deseribed in SEQ ID NO: 2 of the low molecular-weight CD14 when an antibody that binds to 8 Peptide having 16 amino acid residues described in SEQ ID NO: 2s used as the antibody ofthe present invention. More preferably. the socond binding substance described above is ‘an antibody that binds to any one region of amino aeid resi ‘duesat postions Ito $2 of the human high-molecular-weight ‘CDldora fragment ofthe antibody; or an antibody compet ing with or showing cross-reactivity with an antibody that binds to any one region of amino acid residues at positions 1 1o 52 of the human high-molecular-weight CD14 or a Irag- ‘ment ofthe antibody. Particularly preferably, the second bind- ing substance deseribed above is: an antibody that binds to any oneamino acid residue at positions 17 26 of the human low-molectlar-weight CD14 a fragment ofthe antibody: or an antibody competing with (showing cross-reactivity with) sn antibody that binds to any one amino acid residue at Positions 17 to 26of the human low-molecular-weight CD14 ‘ora fragment ofthe antibody. ‘A polyclonal antibody or monoclonal antibody may be prepared, for example, using high-molecular-weight CD14, fovemolecularveight CD14, a mixture of high-molecular ‘weight CD14 with low-molecular-weight CD14, o combi nant CD14 as an antigen, as in the case with the method ‘acconting (0 the first aspect of the present invention. An ‘exemplified method of preparing second antibody wsing 3 mixture of high-molecularweight CDI4 with low-molecu- Jar-weight CD14, and recombinant CD14 as antigens will be shown in Example 3 described below TInaditon, itis preferable to select socondantibody such that, before actually conducting the assay, just as inthe ease ‘with Example 3 deseribed later, «system forthe sandwich ‘method including an antibody that bind t a peptide having 0 o 16 16 amino acid resides described in SEQ ID NO: 2 and an antibody which is a candidate for the Second antibody is preliminary constructed 10 confinn the sensitivity of the Funbermore the fragments ofthe antibody: Fab, Fab), and (ab) can be prepared by well-known method (“iypersen- sitive Enzyme Immunoassay”, writen by Eiji Ishikawa, p. 25.40, Center for Academic Publications Japan (1993) Inthe sandwich immunoassay, the assay may be performed bya competition method as an alternate of the above method. samethod ofallowing an antigen ina specimento compete witha labeled antigen ora labeled antigen analogue during the formation of an antibody antigen-antibody compe. Inthe sandwich immunoassay kit of the present invention, the assay is performed by forminga complex ofthe “antibody that binds to a peptide having 16 amino acid residues described in SEQID NO: 2°-labeled human low-molecular- ‘weight CDI4 (or an analogue thereo!)—the “second binding substance that binds to human low-molecular-weight CD14" “The format of the competition method forthe sandwich ‘immunoassay kit of the present invention inludes: an insoluble easier hounded with an antibody that binds to @ peptide having 16 amino acid residves described in SEQ ID ‘NO: 2; a second binding substance: and labeled human lov ‘molecular-weight CD14 of labeled human low-molecula- ‘weight CD14 analogue, or includes: an antibody that binds to ‘peptide having 16 amino acid resides deseribed in SEQ ID NO? 2; an insoluble carrier bound with « second binding substance; and labeled human fow-molecular-weight CD14 fr Taboled human low-molecular-ejght CD14 analogue. Examples of the human low-molecular-weight CD14 ana- logue include a soluble polypeptide having amino acids at positions 1 t9 285 on the Nerminal of the human CD14 (hervinafter, described as SCD14 (1-285) and a recombine polypeptide having amino acid at positions 1to 307 on the ‘N-terminal of the humsan CD14 where seein at position 286 js substituted with eysteine (hereinafter, deserbed as sCDI4 (1-307) $2860). In the assay system, however, the human Jowemolectlar-weight CD14 analogues not specifically Him ited as faras it sa substance capable of competing with the human low-molectlaraveight CTDI4 in a specimen. The methods of preparing sCDI4 (1-285) and sCDI4 (1-307) S2S6C are described in WO 01/72998. -Furtermore inthe sandwich immunoassay, the assay may be performed by taking advantage of the second specific bindings an alternative method. tis method of conducting fn assay by forming a complex of anibody-antigen-ant body—second specific binding substance—spevtie binding partner ofthe second specific binding substance (hereinafter, Stmay be desribed asa second specific binding partner) Inthe sandwich immunoassay kt of the presen inveation, the ssay’is performed by forming complex ofthe “antibody that binds to @ peptide having 16 amino acid residues {described in SEQ IDNO: 2” human low-molocular-weight CD14—the “Second binding substance that binds to human Jow-molectlar-weight CD14" second specific binding sub- stance—second specific binding partner, or by forming a complex of the “second binding substance that binds to ‘human low-molecular-weight CD14”——human low. Jar-weight CD14 the “antibody that binds to a pepi jing 16 amino acid residues described in SEQ ID NO: 2° second specific binding. substance second specific binding partner. "The format taking advantageof the second specifi binding of the sandwich immunoassay kit of the present invention Jncldes: an antibody labeled witha second spesifie binding substance that binds to peptide having 16 amino acid reUS 7,465,547 B2 17 ‘dues described in SEQ ID NO: 2; a second specific binding substance that binds o labeled low-molecular weight CDI: ‘and an soluble career bound with asecond specific binding partner, oF includes: labeled antibody that binds w peptide having 16 amino acid resides described in SEQ TD NO: 2:8 second binding substance that binds to -molocular-wight ‘CD14 labeled with a second specific binding substance; and tn insoluble carrer bound with a second specific binding partner. "Examples of the combination ofthe second specifi biding substance and the second specifi binding partner include: an ‘antigen and an antibody thereof, a igand and a receptor thereof substance containing some sugars and lectin; and biotin and avidin or sireptavidin, urthermore, examples of the sandwich immunoassay include: an assay with the formation of a complex of a body-antigen-antibody—anti-immunoglobulin antibody by taking advantage of an antibody against an antibody, ie. an nj-immonoglobulin antibody; and an assay with the Forms tion of anti-immunoglobulin antibody-antibody-antigen-an- tibody ~second specific binding substance second specific binding partner by aking advantage of ant-immunoglobulin antibody and scond specific binding The sandwich immunoassay kit of the present invention ‘conducts an assay by: forming a complex of the “antibod that binds 10 a peptide having 16 amino seid residues «described in SEQ ID NO: 2° human ow-moleculr-weight, CD14 the “second binding substance tht binds t human Jow-molectlarweight CD14" anti-immunoglobulin anti body; forming a complex of the “antibody that binds 10.3 Peptide having 16 amino acid residues described in SEQ ID NO: 2°—human low-molecular-weight CDL4— the “second binding substance that hinds to human low-molocular-weight CD14" ant-immunoglobulin antibody; forming an ‘munoglobulin antibody the “antibody tht binds to a pep- tide having 16 amino acid residues describe in SEQ ID NO: 2° human low-molecwlarsweight CD14 the “second binding substance that binds tohnman jow-moleculae-weight CD14" second specific binding substance—second spe cific binding partner; forming anti-immunoglobulin ant body—the “second hinding substance that binds to human Jow-molecular-weight —CD14"™—human fow-molectlar~ ‘weight CD14—the “antibody that binds to a peptide having aminoacid residues described in SEQ ID NO: 2" second specific binding substance -sovond specific binding partner, or the lke, Any sandwich immunoassay is within the seope of the assay of the present vention eventhough 2 soli phase, a labeled substance,or the ikeis formed by taking advantage of the second specific binding as far a it performs an assay by orming a complex ofthe “antibody tht binds to a peptide having 16 amino seid residues deseribod in SEQ ID NO: 2" human low-moleculur-weight CD14—the “Second ‘binding substance that binds to human low-molecular-weight cpl, In other wonds, any sandsvich immunoassay kit of the present invention is within the scope ofthe kit ofthe present fnvention as far as it includes an antibody that binds 10 2 peptide having 16 amino acid residues described in SEQ ID NO: 2. Anvinsoluble eerie usedin the sandwich immunoassay kit ‘of the present invention may be beads, latex particles, mag- netic particles, a plate, a tbe, membrane, or the ike. Mate- rials ofthe beads, plate, or tube inchude polystyrene, nylon, plas, silicone rubber, stainless steel, and plastic. The mem brane may be cellulose, acelose derivative, nitocelislose ‘a porous synthetic polymer, a glass flber, loth, a nonwoven, o 18 fabric, filter paper, or the like. The beads, latex particles, magnetic particles. or the like may be used in a spherical shape A spherical shape is advantageous in saving a space ia storage. The plate or tube may’ be wed inthe form ofa wel ‘A well form is advantageous ia that it will be accepted 0 8 ‘commercial automatic measuring instrument, plate reader, oF thelike. The membrane canbe used for an immtunochromato- araphie method or flow through method described later. The antibody that binds o a peptide having 16 amino acid residues described in SEQ ID NO: 2, the second binding substance, the second specific binding substance or the par fer thereof, oF the aoli-immunoglobulia antibody can be bound to the insoluble carrier by a thermal adsomption ‘method, chemical binding method, or the ike. In addition, iis preferable to subject the non-adsorption surface of the insoluble carrier being fre of the above sub- stance to a blocking treatment with any substance that does pot affect the assay system because the treatment will impart ‘increased specificity or sensitivity to the assay system. The substances that do not affect the assay system includ: pro- teins such as BSA and casein: and surfactants suchas Toon 20 and NP-40, Tahelsto he use in the sandwich immunoassay kt ofthe present inwention include: enaymes sch as peroxidase, alkali Phosphatase, Pp-galactosidase, oxidase, and urokinase; heniluminesceat substances such a aridinium ora deriva. tivethoreof and aoquorin oF a modified product thereof fuo- escent substances such as FITC; dyestall, gold colloid; col ‘fed latex; and isotopes. For instance, in the cave of using peroxidase asan enzyme, 33,5.Stictrabensidine or 1.2-phenylene diamine may be ‘exemplified asa chromogenie substrate. In the case of using alkali phospatese, 4-nitropheny phosphate may be exempl Tied a8 a chromogenic substrate la the ease of using Bere salactosidase, 2-nitrophenylf-,-galactoside may be exem- plified asa chromogenie substrate nzyme-labeling t the antibody that binds to a peptide having 16 amino aeid residues described in SEQ ID NO: 2, the sccond binding substance, the second specific binding substance or the partner thereof, or the ant-immunoglobulin Antibody can be performed by a tworstep glutaraldehyde ‘method, periodic Seid method, maleimide method, pyridy] disulfide method, or the like. ‘Apart from tbe enzyme, a well-known technology such as «thermal adsorption method or chemical binding method ‘may be available in the labeling. Enzyme-labeling is preferable because it can be assayed using conventional chromometry system ifany chromogenic substrate exemplified above is sel and because the sensitiv ity thereof is comparatively high. Furthermore, the labeling toed in a simple kit such as a kit utilizing an immunochro- ‘matographie mothod or flow through method described later is prefenble because dye stu, gold colloid, or colored latex can be visually observed ‘The sandwich immunoassay Kt ofthe present invention is charaeterizd in that an assay is performed by a sandwich mnunoassay andl includes an antibody that binds to peptide hhaving 16 amino acid residues described in SEQ ID NO: 2 The sandvich immminoassay can use the well-known echnal- ‘oy as described above. In addition to the above concrete ‘description, aay kit based on the sandwich immunoassay is ‘within the scope of the sandivich immunoassay kit of the present invention and is not specifically imited as far a the kit includes an antibody that binds to a peptide having. 16 ‘amino acid residues deseribed in SEQ 1D NO: 2. Io other ‘words, itis enough forthe kit contain an antibody that hinds toa peptide having 16 amino acid residues desribed in SEQUS 7,465,547 B2 19 IDNO:2 aad a reagent required forthe sandwich inmunoss- say. In addition, no conten is restricted as far as it does not ‘inhibit the assay’ results based on the principle ofthe assay. Fr instance, a bulfer or diluent of a spocimen, labeled antibody, oF the Tike, a chromogenic substrate (see the above ‘descrption) stable fer an enzyme when the enzyme is sed ora labeled antibody, blocking agent, astopping reagent, or washing solution may be exemplified as an optional eons tutional element. In addition, a standard substance may’ be also exemplified as an optional constitutional element. The Standard substances include human los-moleculae-weight ‘CD14 and human low-molecularsweight CD14 analogues Funhermore, a kit that utilizes an immanochromato- znaphic method oF a flow through method on the basis of a Sandwich immunoassay as a principle of the assay is also Within the scope of the sandiich immunoassay kit of the present invention The immunochromatographic method is a method where san antigen provided as test substance in a specimen moves ‘long atest strip to an insoluble easier on which an aatibody js immobilized while the antigen reacts with a labeled ani body being arranged in the tes trp so as io be able to move, tnd then a complex of the antibody-nntigen-antibody is ormed on the insoluble carrier. In general, the antigen can be assayed by a single step of dropping the specimen on the test step. For instance, apparatuses forthe immunochromatographic method ave disclose in JP 01-063865 A, WO 88/08534, and Wo 90109502. In ation, apparatases forthe immisnocho- matographie method having flow channels with diferent developing speeds are disclosed in WO 89/03993 and WO 99/27364, and for example, the upparatuses can be applied sch that a abeled antibody can bereactedatfer the formation ‘ofa complex by allowing the ration between the immobi lized antibody and the antigen. ‘An example that utilizes the immunochromatographic method of the sandwich immunoassay kit of the present vention will be described below. Tor instance, adevice (ic, 2 kit) isa test strip on which 3 samplecading par, a reagent part a detection part, and an absorbing part are provided stich that liquid spoimen added ‘on the sample~sding pat is allowed 0 move along those pars in that order. Iti ullcient that labeled second binding ubstance he impregnated in the reagent prt and an insoluble ‘arti bound with an antibody tha bind wo a peptide having 16 amino acid residues described in SEQ ID NO: 2 bo arranged on the detection par. ‘The specimen added on the sample-adding part absorbs the labeled second binding substance at the reagent part. The human low-molecular-weight CD14 reacts with the labeled second binding substance to form a complex while they move to thedetoetion part. On lie detection part, thecomplex reacts ith the antigen that binds to the peptide having 16 amino ji residues described in SEQ ID NO* 2, resulting inthe Tormation of a complex of the “aatibody that binds to the peptide having 16 amino acid residues described in SEQ ID NO: 2"-—hhuman low-molecular weight CDL4—the “Second binding substance that binds to nman los-molecular-weght CD14" on an insoluble carter. Any substance and reagent in the specimen, whieh are not involved in the reaction, move to the absorbing part, The lbel of the complex formed on the detection part may be determined, particulanly may be visi ally determines. ‘A porous carier or the like may be used asa test strip. The porous carrier may be, for example, nitrocellulose cellulose cellulose denvatve, sylon, a nylon fiber, a glass fiber of @ porous syntbetie polymer 0 o 20 Part of the test strip may be directly used asthe sample- adding part or reagent part. Alternatively, for example, cellt- lose iter paper, las fiber, cloth, non-woven fabric, porous synthetic polymer, or the like may be used depending on the mount ofthe sample or the dose of the reagent Celulose, a cellulose derivative, nitrocelilose, a porous symtlietic polymer, a glass fiber, cloth, non-woven fabric, fier paper, othe like may be used forthe detection part as described above. ‘A water-absorbable material may be used for the absorbing part, Examples of the waterabsorbable material include: an ‘ahsorbent polymer such a sponge; cellulose filter paper: and titer paper Theabove is one ofthe examples ofthe immuncehromato- _taphie method. A reference part forconfiming the progress ‘fa reaction may be added, or the test strip may be provided ‘with a support or covered with an extemal cover. However, the kit ofthe present invention is aot limited to them, ‘Funtbermote, as described in the explanation about the sandwich immunoassay, a kit for an_immuncchromate- saraphic method, by which a complex ofthe “antibody that binds toa peptide having 16amino aeid residues described in SEQID NO! 2" human low-molecular-weight CDI4—the “second binding substance that binds to human low-molecw- Jar-weight CD14" is formed on an insoluble cartier and an assay is performed by fomning a complex that utilizes an ‘ant-immunoglobutin antibody and second specific binding. js also with the scope of the sandwich immunoassay kit of the present invention. “The fas through method is a method by which an antigen provided as test substance forms an anibody-antigen-ant body complex together with a solution in a specimen on a semibrane provided as an insoluble carrer. At this time, a substance Failed to be fixed on the membrane is generally removed by perpendicularly passing through the membrane from the front tothe back, ‘WO 8801605 discloses an apparatus based on.a multi-step method by which a specimen, a regent, and a cleaner are dropped ont a membrane JP 06-273419 A dlscloses a method being improved as 2 single-step method in which « multilayered! membrane is Formed and 2 regent partis provided thereon so as to conduct the assay only by dropping a specimen, “Hereinafter an exaniple ofthe sandwich immunoassay kit of the preseat invention using w flow through method will be eseribe Fr instance, a device (Le, a Kit) is @ kit on which a samplecadding part, a reagent par, a detection part and an hsorbing part sie layers one on top of another such that 9 Tiquid specimen aided on the sample-acding pats allowed to move along those parts in that order. It is sulicent that a labeled second binding substance be impregnated in the eigen part und an inoluble eartier bound with an antibody that binds to a peptide having 16 amino acid residues scribed in SEQID NO: 2he atranged on the devetion par. ‘The specimen added on the sample-adding par passes ‘through the sample-adding part perpendicularly from the top to hack o the membrane (hereinafter, the same holds ve for the sample movement) and then absorbs the second binding substance at the reagent part. The human low-molecular- ‘weight CD14 reacts with the labeled second binding sub- stage to farm a complex while they move to the detection part, On the detection part, the complex reacts with the ant ‘gen that binds to the peptide having 16 amino acid residues ‘eseribed in SEQ TD NO: 2, resulting inthe formation of @ ‘complex ofthe “antibody tht binds to the peptide having 16 no acid residues described in SEQ ID NO: 2”—human,US 7,465,547 B2 2 low-molecular weight CD14—the “second binding sub- stance that binds to human low-molecula-weight CD14" on an insoluble carrier. Any substance and reagent in the speci men, which are not involved in the reaction, move 10 the “absorbing part Thelabelof the complex formed an the detee= tion part may be determined, particularly may be visually ‘determined. The label can be visually observed in a simple ‘manner ita device is designed such thatthe detection partis ‘detachable from the sample-adling part and reagent part oF fom the absorbing part. ln addition, the label ca be visually ‘observed from the sample-adding part i'each ofthe sample ‘adding part and reagent partis made of translucent material, ‘or from the lower side ithe absorbing par is arranged above the detection part (he sample-ading par side) in the case ‘of JP 06-0273419 A. The same members as those of the immunochromato- araphic method can be applied and each member may be {omed like w membrane toallow the solution ina specimen to “The above is one of the examples of the flow through method, reference part for eonfirming the progress of @ reaction may be added, or each member may be provided with f support or covered with an extemal cover However, the Sandwich immunowssay kit of the present invention is aot Timited to them. Furthermore, as described in the explanation about the sandwielt immunoassay, in addition to the formation of & ‘complex of the “antibody that binds to a peptide having 16 amino acid rescues described in SEQ ID NO: 2° human Tow-molecular-weight CD14—the “second binding. sub- stance that binds to human low-molecular-weight CDI4" on ‘an insoluble camer, a kt for the flow through method tat ‘conducts the assay by forming a complex that wiles the ‘immunoglobulin antibody and the second specifi bind- ing is also within the scope ofthe sandwich immunoasssy kit ‘ofthe present invention, Funhermore, the sandwich immunoassay it ofthe present Jnvention can be available 10 an assay based on a MEDIA method (JP 0.264552 A) of electrochemically measuring sjgnals from a label and an assay based on an immuncassay method ("Bioscience and Industry", wo. 61, p. 440-484 2003) singamicrochip. The assay kts using those principles fare within he scope ofthe sandivich immunoassay’ kit ofthe present invention af far as they are characterized by theie ‘assays based on the sandwich immuaoastay and include aa ‘antibody that binds toa peptide having l6amino ail resides ‘described in SEQ ID NO: 2. ‘The sandwich immunoassay kt ofthe present invention is ‘characterized by including an antibody that binds to a peptide having 16 amino acid residues deseribed in SEQ ID NO: 2 and is capable of specifically determining low-molecuar- ‘weight CDI4. A specimen o be used in the sandwich immo- roassay kit ofthe present iavention is preferably an aqueous specimen. Particularly preferable examples ofthe specimen include blood, blood component such a5 serum or plasm, urine or other body Fads, cel culture supernatant, and eol- lumi eluent, They are useful foe the determination on lxs= ‘molecular-weight CDI in them. However, from the speci ‘mens except the human blood component, such as human urine or other body fads, blood components, urine, or ather body fluids form species except a human being, eel culture supernatant, and column eluent, proteins, polypeptides, orthe Tike which are analogous tothe low-molecularweight CD14 may be also assayed as well as the low-molecdlar-weght ‘CD14, Any assay’ kit forthe above polypeptides, or the ike ‘which are analogows to the low-molecolar-weight CD14 is also withia the scope ofthe sandwich immunoassay kitof the 0 o 2 present invention as far as they each inelude an antibody that binds toa peptide having 1G nino acid residues describes in SEQ ID NO: 2, Furtbermore in the above explanation, the fragment Fab, Fabor (Fab)),of the “antibody that binds toa peptide having 6 amino acid residues deseribed in SEQ ID NO: 2" may be cused instead ofthe “antibody that binds a peptide having 16 amino acid residues described in SEQ ID NO: 2” In the above description, the concrete examples using the “antibody that binds to a peptide having 16 amino acid re dues described in SEQ ID NO: 2” have been described as preferred examples ofthe antibody according to the second aspect of the present invention. However, the antibody according to the first aspect of the present invention, the ‘antibody sovording to the second aspect of the presen iaven- ‘ion except the “antibody that binds 1 a peptide having 16 faming aeid residues deseribed in SEQ ID NO: 2", oF the {ragment Fab, Fab’, or (Fab) of hose antibodies may be also sed, Preferable isa sandwich imnmnoassay’ kit using the an body of the sceond aspect of the present invention. More preferable is sandwich immunoassay kit using the “ant body that binds toa peptide having 16 amino acid residues described in SEQ ID NO: 2” Furtermore prineipls forthe assay include an agalutina- ‘ion method, solid-phase binding method, and solution reae- jan method in addition to the sandwich immunoassay pethod. Depending on those methods, therespectivekitsmay ‘be constructed such that each of them contains the antibody tat binds to at least one of human low-moleculae-weight (CDi 4orthe fragmento the antibody, preferably theantibody ‘of the present invention or the fragment ofthe antibod, In the agglutination method, the antibody is bound on the surlace of particles and the presence of the antigen cause the particles 10 agglutinate, so that the antigen ean be quali Lively or quantitatively determined in a specific manner in reference tothe degree of agglutination ofthe particles. An agglutination immunoassay kit ofthe present invention jgondts the assay by fomaing the “antibody of the present Jvention”—human low-imolecularsweight CD14 and eaus- ing the agglutination thereot “The format of the aggltination immunoessay kit ofthe present invention includes particles to the surface of whieh the antibody of the present invention binds "The panicles used may be those generally used, including latex, red blood cells (@..,shoep red blood eels), gelatin, snicto beads, carbon particles, othe like ‘The solid-phase binding method isa method of conducting theassay by the formation ofa complex between an antibody and an antigen ona solid phase, Anantigen-containing speci- ‘men is adsorbed in an insoluble carer (4c, solid phase, the same shall apply hereinafter). Next, a labeled antibody is ‘added and the mixture is reacted to qualitatively or quanti tively determine the amount of the complex bound on the solid phase in specific manner on the basis ofthe labelod product, Furtermore, a6 @ competition method, an antigen anae Jogue is adsorbed on an insoluble carier to allow te labeled antigen to compete with the rexction with the antigen in the specimen to determine the amount of the labeled antibody bound to the antigen analogue. Furthermore, san altemative ‘method ofthe competition metho, the antibody i adsorbed ‘nthe insoluble carrier andthe reation withthe antigen inthe specimen is competed with the labeled antigen analogue 10 ‘Tobind a peptide having the sequence described in SEQID NO: 2 (corresponding to a sequence at postions $3 10 68 ‘described in SEQ ID NO: 5) hereinafter, described as S68 Peptide) to a carier protein a the N-terminal thereof trough fan SH group, the peptide was synthesized by inserting eys- teine into the N-terminal, Thats, using a peptide synthesizer ABH33A (Applied), amino acid columas were aligned ‘ecording tthe amino acid sequence and an amino ackd column for eysteine was placed on the N-terminal, followed by eandocting automatic synthesis. The synthesized peptide ‘Was cutout froma resin by a conventional procedure ana was thea precipitated with ether, recovered, and dissolved in tilled water again, followed by feeze drying. fer the result ing eaude peptide had been dissolved, the peptide was eluted ‘with linear gradient of 5-70% acetonitrile concentration using a C18 revere phase HPLC (CAPCELL-PAK, Shiseido Corp), followed by collecting a lraction containing. a target peptide, The collected fmction was freeze-dried and 2103 mg Df purified peptide was obtained. 1-2) Preparation of Peptide Carier Antigen Using Synthetic Peptide Fach of two kinds of peptides prepared in I-(1) was dis- solved in distilled water to 10 mg/ml. and the solution was ‘mixed with IO mg/ml. of maleimide-ativated Keyhole limpet Fbemocyanin (Inject Maleimide Activated Mariculture Key- hole Limpet Hemocyanin (KLE) (PIERCE)) in equivalent amounts. After the mixture had heen reacted for 2 houes at ‘oom lemperature, the reaction mixture was desalted by an [NAP-10 column (Amersham Bioscience) being equilibrated ‘with physiological saline to obtain mp of S68-peptide car ier antigen (hereinafter, describe as S68 pepide-KLH). The ‘concentration of proteins deseribedin the following examples was oblained by dividing the amount of used KLH by the Amount of figuid. 13) Preparation of Peptide as Antigen <2> ‘Two Kinds of peptide sequences represented ia Table 1 were synthesized using a pepiide syuthesizer (PSSH-8, Shi- ‘mada! Corporation) By the same way as tha of 1-1) and Purified, respectively. Each of the obtained peptides was bout Simg in amount By the way, the “number” in the table represents the name of a peptide explained below and the “position” represents the postion thereof fond in the amino atid sequence deseribed in SEQ ID NO: 5. TABLE 1 30 po nu tw tw nega oee Gia teu Aap Asp Gla Phe Arg Cys Val CysUS 7,465,547 B2 27 TABLE 1-continued ‘seo Poor 24-32 Arg cye Val cye Ron Phe 4 Ser Glu bro Gln Pro Aap {ep Ser Glu Ala Phe Gn oF 1.4) Preparation of Peptide Carier Antigen Using Synthetic Peptide <2> Fach of the peptides prepared in 1-(3) was dissolved in BS (pH 7.2) containing 0.1 M EDTA. and as inthe case of 1(2) 3 mg of each of peptide carrer antigens where KLH. bound tothe respective peptides was obtained. 1.(5) Preparation of Polyclonal Antibody Using Synthetic Peptide <1> For preparing « polyclonal antibody against S68 peptide KLH prepared in (2), @ mbit was immunized using S68 Peptide-KLH. That is, 100 yg of each of S68 peptide-KLH ‘a diluted with 500 of physiological saline and the so tion was mixed with $00 of Freund's complete adjuvant (DIFCO) inequivalent amounts, followesdby subcutaneously ‘administering the mixture tothe back of New Zealand white emale rabbit (Ritayama Labes) weighing 2.1 to 2.2 ky. Alter 2weeks, 100 gof each of S68 peptide-KLH was diluted with 500 pl of physiological saline and the solution was mixed ‘with 500 jL of Freund's incomplete adjuvant (DIFCO) ia ‘equivalent amounts, fllowed by subcutaneously sdminister- ing the mixture tothe back. ARer additional 2 weeks from that, 100 ug of S68 peptide-KLH was diluted with 1 ml of physiological saline and the solution was administered in an After 1 week fom the completion of administration, blood ‘was collected from te ear vein and antiserum was separated from the blood by routine procedures and an antibody was purified. First, ammonium sulfate was ade to the antiseram Up (0 final saturation concentration of 33%. After the mix- ture lad hee stirred for 1 hour at 4° C., the separate pre- iptate was centrifuged. Then, the precipitate was dissolved ina 76-mM phosphate buffer (hereinafter, described as PBS (pH 6.4) andthe solution was dialyzed overnight. ARer the dialysate had been tered, the filtrate was applied to aprote AA column (Prosep-A, Millipore). Then, «binding 1gG frae- tion Was eluted witha0.1 Mglyeine hydrochloride butler (pH 3.0)toobiaina purifid anibody. After dialysis with PBS (pl 644), the protein concentration was calculated fom the absor- bance at's wavelength of 280 nm (absorption coefficient: (0.533 mg/m). Hereinater, the obtained antibody will be described ss an S68 peptide polyclonal antibody 1.46) Preparation of Polyclonal Antibody Using Synthetic Peptide as Antigen <2 Using each of the peptide carrier antivens prepared is 1.44), i the ease of 1-3), he immunization and the puri- ‘cation of antiserum were performed to prepare each of pep- fide polyclonal antibodies (POO| and POO2 polyelonal anti bodies). Furthermore, the immunization was performed sich thatthe peptide carrier antigen (0.5 mg/rabbit) was adminis- tered $ times in two monthsAfler the whole blood had beea collected, each ofthe antisera antiserum POOL anc POO2) was ‘obiained, 0 o 28 1-7) Preparation of Specific Purified Polyelonal Antibody For purifying only an antibody against S68 peptide from the S68-peptide polyclonal antibodies, specife purification was performed by the following method, Firs, for biding the ‘S68 peptide inserted with eysteine(hereinsier, described as C-S68 peptide) toa cartier through an SH group, 200 ug of (C-868 peptide was mixed per | ml ofSuloLink Coupling Gel (PIERCE) and reacted according tothe manvalthereot Aer the completion of the reaction, the remaining active eroup was blocked and then an S68 peptide-biding aiity column was prepared. Next, 792 mg of the purified IgG fraction described in 1-{3) was applied and thon the column was ‘washed with a phosphate buffer (pH 7.4) (Dulbecco, herein- er, described as D-PBS (pH 7.4), fllowed by eluting an ant-S68-peptide antibody with 0.1 M glycine hydrochloride buffer (pH13.0)-After the ction, pH was readjusted to neu- tral and then dialysis was performed with PBS, followed by calculating the protein concentration ffom an absorbance at 280 nm (absorption coetficieat: 0.533 mg/m). Asa result (052mg of an anti-S68-peptide antibody. (hereinator eseribed as $68 aniboxy) was obtained Example 2 repuration of Monoclonal Antibody Using ‘Synthetic Peptide as Antigen 201g of S68 peptide-KLH prepared in Fxample 1-(2) was dissolved in 100 u-of physiological saline and mixed with an equivalent amount of Freund's complete adjuvant (DIFCO), followed by administering 100 af the mixture to each of the rearfoot pads ofa female Wister rat aged 8 Weeks, Aer 2 weds, the Hise Iymph node was surgically excised and cell Tusion was performed. The ell fusion was conducted aecord- ing to Tamie Ando and Takeshi Chiba: “Initoduction t0 ‘Monoclonal Antibody Experimental Manipulation”, page 83, 1991 (Kedlansha). In other words, lymphocytes were sepa- rated from the lymph node using & cell strainer (Faleon) and sixes with myeloma cells (Sp2/0-Agi4) al a nitio of 5:1 followed by cel fusion using polyethylene glyco. Fused cells were suspended in an HAT medium and hybridomas were selected, followed by screening hybridomas producing the target antibody. “The sereening was performed by an ELISA method ia CD14 (1-307) S286C was dieectly immobilized ona plate. That is, $0 ul. of sCD14 (1-307) $286C diluted with O.1-M phosphate buffer (pH 7.4) to I pi was added 10 each well of an immunoplate (Maxisorb, NUNC) and left to sane for I hour at 37°C. After that, the plate was washed with fon-exchanged water times and then 100 of PBS (pH16.4) containing 0.1% BSA was added w each well, followed by Jeaving the plate standing for I hourat oom temperature to effect blocking. Then, the culture supernatant sampled rom the selected hybridomas was added to each well and allowed to react at 37°C. for I hour. After that, the plate was washed 3 times with physiological saline containing 0.05% Tween 20, Subsoquenty, 50 ul. ofa solution obtained by diluting peroxidase-labeled anti-rat immunoglobulin antibody (DAKO) with PBS containing 10% rabbit serum 1000-fokd was.added o each wel, fle reaction at 37°C. for 1 our, the plate was washed 5 times in the same manner as above and a {etramethyTbenzidine solution (TMB, BioFix) was added t0 cach well After a reation for 10 minutes ot rom tempera ture, the reaction was stopped with a 0.5 M sulfuric acid solution and an absorbance at 450 am was measured using & plate spectrophotometer (NI-2100, Japan Intermed). A aUS 7,465,547 B2 29 result, well containing hybridoma capable of producing a antibody binding to sCD14 (1-307) S286C was selected. ‘Next, from the Selected well, cloning was performed by 2 Fimiting dilution method aceording to Tamie Ando and Takeshi Chiba: “Tatraduction to Monocloasal Antibody Experimental Manipulation”, page 83, 1991 (Kodansha) After 10 days, likewise, sereening was performed using as an index thereacivty withsCDI4 (1-307)S286C and 6kindsof hybridomas were televed. The selected hybridomas were cultivated in « 10% FCSIRPMI1640 medium (Sigma) and then cultivated in Hybridoma-SFM medium (Invitrogen) t0 produce an antibody. The antibody was purified using a pro- ‘ein G column (Prosep-G column, Millipore). The subtype of the purified F1146-17-2 antibody was determined to be rat 1gG2b- by using a rat typing kit (ZYMED), By the way, SCDI4 (1-307) S286C was prepared using the ‘method described in Example 9 of WO 01/72993. Example 3 Study of Assay System for Human TLow-Molectlar- Weight CD14 Using the antibodies described ia Examples | and 2, the sssay system for human low-molecular-weight CDI4 with & Sandwich EIA method was studied, 3-(1) Preparation of Recombinant Human CD14 First, for preparing a monocfonal antibody against SCD14 (1-285) to be used as a second antibody in the sandwich ELISA method, sCDI¢ (1-285) a6 an immunogen was pre- pared in Ecol In order to express CD14 (1-285) in E.coli, sn expression plasmid prp1659 was constructed by the fle Towing method, Firs oligomer’, inks (Sage ta ggaatt -¥) (SEQID NO: 15) and oligomer 8 linkA (S-eta gaa att ect a3!) (SEQ ID NO: 16) were synthosized “Those oligomers were mixed in equivalent amounts and heated at 99° C. Tor I minute, and the mixture was thea annealed by gradually cooling it down to room temperature. Furthermore, S-ierminal hereof was phosphorylated by T4 Polymuelootide Kinase to prepate a Finke. "Next, sense primer (S-ace tet ag ign cea ege cag ate ot 3) (SEQ ID NO: 17) and antisense primer (St gga te acta agate pag eae tet-3) (SEQ ID NO: 18) were synthesized and PCR was performed vsing Pyrobest DNA Polymerase and plasmid pM 1689 described in Fsample Sof WO01/72993 as template, ‘Afler a reaction solution had eva heated for 2 minutes t 0° C., the eycle of 98" C. for 10 scconds, $5° C. for 30 seconds, and 72° C. for 1 minute was repeated 30 times. ‘The resulting amplified product of about 90° bp was “double-digested with Xbal and Bamlil to collect DNA Frage ments, The vector pMP10 described in Example 10 of IP (06-025289 A was double-digesed with Hind and Bam and then subjected to agarose gel electrophoresis and col- lected. After three-ligation ofthe linker already phosphory= lated, PCR-amplified DNA fragmentXbal+Banall digested fragment, and veetor/Hindlll+ BamHI fragment, which were described hove, he resultant was transformed into E. coli ‘compotent cells (IMI09 (TOYOBO) to obtain a clone con- taining the target plasmid, Plasmid DNA was prepared by routine procedures. Suibsequently, 3E7924 transformant strain forthe produ tion of sC1D14 (1-285) was prepared using an electroporation method. 0 o 30 Firs £:colf3:7924 J. Bacteriol 173, 4799,(1991)) was restored from a glycerol stock and incubated in an LB ‘medium at 37°C overnight, Furthermore, the bacteria were inoculated to 50 ml ofa fresh LB medium and eontinaously ‘incubated until the absorbance at 600 nm reached 0.5 10 06, followed by diretlyiee-cooling a culture Ask for 30 min- tes. Next, coli cells were collected and washed twice with jce-cooled sterilized distilled water and once with an ice- cooled 10% glycerol solution, followed by being suspended in 100 ul ofan ice-cooled 10% glycerol solution. The sus- pension was dispensed into two tubes with 50 aliquots and (quickly frozen in liquid nitrogen to prepare competent cells (E7924), which were saved at ~80° C. until the time of se. ‘Next, 50 ul of 127924 competent cells was transformed ‘with 30 ng of by electroporation device, Gene Pulser of BIO- RAD Co, Ltd. In aditon, the settings a this time were a vollageof 2.5 KV anda resistance of 2000, and.acapacitance of 2S uF. After that, the resultant was incubated inan LB agar plate containing 50 g/ml of ampicillin overnight to obtain a clone transformed with pTrpi6S9, The clone thereof was jnctibated at 37° C. overnight in an LB medium and was then ‘noeulated int afresh modi, followed by being incubated {or adtionalS hours. OD at GO0 nm of eeltare sspension reached to 2 10 3, 3P-indole serylie acid (Sigma CO., Ltd) ‘was added in 2 final concentration of 100 jig/ml. and the ture was incubated at 37° C. for 4 hours, resulting in induetion expression of sCDI4 (1-288). Next, E.coli was collected and then an inclusion body was prepared using Bug Buster Protcin Extraction Reagent (Novagen, Co. Ld) After that, the inclusion body was dissolved inan SDS-PAGE bur and an SDS-PAGE was carried out to identify the ‘expression of CD14 (1-285) by Westemt blotting by an ant CD1s antibody Similarly, sCD14 (1-285) o be used san immunogen was prepared by incubating JE7924 transformant strain in | Lof ‘an LB medium, Firs, the culture solution was centifuge. ‘Aer F col cell had been collected, the bacteria ells were ‘washed with D-PBS and 50 ml_of Bug Buster Protein Extrc~ tion Reagent (Novagen, hereinafter desribed as Pug Buster) was aida to the collected bacteria cals, The baeterial cols were suspended and left standing for 30 mingles at roam ‘emperature. After lysing, the bacterial cells were subjected to 10-minute sonication teatmeat (US-3, luc Seieido) and vatrifqged at 10000xg at 4” C. for 20 minutes to remove & Supernatant. Likewise, an additional sonication treatment ‘was performed on the cells and the resulting precipitate was suspendedin SOmL of Bug Buster. Thesuspension wasadded with I mi of a 10-mg/mt, lysozyme (Seikagaku Corpora. tion), and the whole was gently sired and left standing fr 10 ‘minutes at room temperature. Subsequently, 200 ml. of Yio volume of high-concentration Bug Buster was added to the ‘mixture and the whole was sired, followed by being su similarly to remove a supernatant ‘The resulting provpitate was suspended by the altion of 200 ml. of Yn concentration of Bug buster and then the jon was centrifged similarly, followed by repeating suchan operation several ies. 100 ml. of D-PBS wasadded inthe finally obisined precipitate, resulting in an inclusion body. For the preparation of sCD14 (1-285), the inclusion body was dissolved ina TE butler (pH1 80, Nippon Gene) contain- ‘ng 196 Triton-X100 andthe solution was then subjected t0 freeze and thawing 3 times, following by collecting a precipi- tate by centrfgation. The precipitate was dssolvedinthe TP buffer (pET 8.0, Nippon Gene) containing 1% Teton-X100 ‘gain, and the solution was ie-cooled and then subjected toa 1 2eminte uliasonie treatment with 250 A at intervals of 10US 7,465,547 B2 31 seconds and centrifuged, fllowed by collecting a precipitate. “The precipitate was dissolved in a TE bullee (pH8.0, Nippoa. Gene) containing 1% Trton-X100 and0.2M NaOH, and then, treated at 37°C. for 10 minutes, cenrfsged, and re-dissolved three times, fllowed by collecting a precipitate. The es 32 precipitate was dissolved in an aquccus solution cor ing 6 M guanidine hydrochloric acd to prepare purified SCD (1-288). The concentration thereof was calculated by a protein assay of Bradford using BSA as a standand prepa ration. 3-(2) Preparation of Anti-CD14 Monoclonal Antibody [1] Preparation of PL106-13-3 Antibody ‘Using sCD14 (1-285) derived from E- col described above ‘san antigen to be administered, a monoctonsl antibody was prepared. Firs, 20 ug of purified vCDI4 (1-285) was mined with Freund's’ complete adjuvant (DIFCO) in equivalent amounts, followed by intrzabdominally administering 200, UL ofthe mixture 6-week-old female ddy mouse, Ater 2 ‘weeks, 20 jg of purified sCDI4 (1-285) was mined sith Freund's incomplete adjuvant (DIFCO) in equivalent amounts, followed by intrasbdominally administering 200 UML of the mixture, 50 pi. of antigen was inraabdominally administered fo the mouse 3 days before cell fusion, After days spleen was aseptically excised. Lymphocytes were iso- lated rom the spleen and mixed with myeloma cells P3x63- Ag. 8. U.1) in ratio of 10:1 and fusion was performed with polyethylene glycol according to a method described on ‘Tame Ando and Takeshi Chiba “Introduction to Monoclonal Antibody Experimental Manipulation”, page 88, 1991 (Kodansha). After hybridomas had been selected using an HEAT medium, sereening ofhybridomas producing antibodies biding to sCDI4 (1-285) sas performed by an ELISA method Fin, sCD14 (1-285) was diluted with PBS (pH 6.) 00.4 igi and 50 jl. of the resultant solution was then aed to ‘each well ofan immunoplate (Maxisarb, NUNC) and reacted °C. overnight, Aller that, the plate was washes! with ‘on-exchanged water times and then 100 1 of PBS (pH 6.4) ‘containing 0.5% BSA was added to each well for blocking, ‘Thea, the sampled culture supernatant was added to eoeh well, ‘and allowed to reget at 37° C. for I hour. After that, the plate ‘was washed 3 times with physiological saline containing (0.05% Tween 20. Subsequently, 50 pl. ofa solution obtained by diluting peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO) with PRS containing” 10% rabbit seram 1000-fold was added to each well. After a reaction at 37°C. {or 1 our, the plate was wasbed 5 times inthe same manner as above and atetramethiylbenzidine solution (IMB, BioFix) Was added to each well. After a reaction for 10 mimes at oom temperature, the rsaetion was stopped with a 0.5 M sulfrie acid solution and an absorhance at 450 am was mea- sured using @ plate spectrophotometer (NI-2100, Japan Intermed). On the basis of the result, Well containing hybri- ‘dom producing an antibody binding to sCD14 (1-285) was sclcted, Nex, fom the selected well cloning was performed by a limiting dilution method according to Tamie Ando and ‘Takeshi Chiba: “Introduction 10 Monoclonal Antibody Experimental Manipulation”, page 83, 1991 (Kodansha) Aer 10 days, likewise, seréning was performed using the reactivity with sCDIL4 (1-285) as an index to select ybro- mas. AS a result, 12 types of hybridomas producing ant SCD14 (1-285) monoclonal antibody were selected. “The selected hybridomas were cultivated in a 109% PCS! RPMI1640 medium (Sigma) and then culated in Hybri- 0 o 32 {doma-SFM medium (lavitrogen) to produee an antibody. The antibody Was purifed using a protein A column (Prosep-A, Millipore). ‘The subtype of P1106-13-3 antihody, which was an ant body having a particularly high sensitivity, was determined.as IgG2bx« using s0Strip Mouse Monoclonal antibody Isoryp- ing Kit (Roche). (2) Preparation of F1031-8-3 Antibody FIO31-8-3 antibody was prepared using the method scribed in Example 7 of WO 01/22085, Brclly describing. 20 ua of CDI4 protein derived from in human blood was ‘issolved in physiological saline and the solution was mixed with Freund's complete adjuvant (DIFCO) in equivalent ‘amounts, Then, aller | week from each of the initial atraab- ‘dominal administration and the second thereot 2 wooks ater ‘the inital, a increased level of antibody tite in serum was confirmed by an ELISA method! onthe reactivity with recom binant human CDI4 protein asin the ese of Example 5 of WO 01/22085. A 100-18. antigen was intrsabdominally administered to a mouse asa ial ad ‘and after 3 {days the spleen was surgically excised rom the mouse. phocytes were isolated from the spleen and mixed With ‘iyeloma cells (P3463-Ag. 8. U1) ina ratio of 10:1 and cell {sion was performed with polyethylene glycol. Hybridomas were selected ting an HAT meviim and after one week sereening of hybridomas producing antibodies was per formed by the BLISA method described above. The hybri- sdoma that had reacted with the immobilized soluble CD14 protein was cloned by a limiting dilution method. After 10 ‘ays, similarly, screening was performed t0 oblain an ant (CD14 monoclonal antibody, F10S1-8-3 antibody having the subtype of TG2b-« determined using IsoSteip Mouse Mono- clonal antibody Isotyping Kit (Roche) was obtained at a ‘ypical antibody. 34(3) Study of Assay System for Hu Weight CD1a For prepating a system capable of specifically detecting ‘human Jow-molecular-weight CD14, sandwich ELA syste ‘was prepared using the antibodies deseribed in Examples 1 and 3.2), [1] Preparation of Peroxidase abeled Antibody A peroxidase-labeled antibody was prepared according to the method of Nakane et al (J. Histochem. Cytochem., el 22, p. 1084, 1974), That is, 4 my of peroxidase (Toyobo) was ‘iscolved in distilled water andthe sofution was then reacted 125°C for 20 minutesby the addition of 100mM of periodic acid. Aer the completion of the reaction, 1.5% ethylene tlyool was added to the reaction product and the whole was reacted at 25° C. for 10 minutes, followed by dialyzing ‘against a L-mM acetate bufler (pH 4.4). Fach of the purified 1031-83 antibody and P1106+13-3 antibody was dialyzed ‘witha 10-mM bicarbonate butler (pH 9.5), and then 4 mgof peroxidase activated by the addition of 70 jl of a 0.2-M bicarbonate butfer (pH 9.5) per 4 mg was mixed with the antigen inequivalent amounts to allow reaction at 25°C. for hours. Next, 4 mg/ml. of sodium borohydride was added ‘and thon the reaction was continued for additional 2 hours at 4°C. The reaction solution was dialyzed with PBS, resulting in ® peroxidase-labeled F1031-8-3 antibody’ (hereinafter, i may be described a5 F1031-8-3-HRP) and peroxidase-a- belod F1106-13-3 antibody (hereinafter, it may be deseribod ‘as F1106-133-1IRP). The concentration oF antibody was cal- cilated from the amount of antibody used and the volume of the labeled antibody solution, Low-MolecularUS 7,465,547 B2 33 [2] Preparation of Sandwich BIA System <1> Prepared was a2-sep sandivich FLA systom using the S68. antibody prepared as an immobilized antibody in Example | and antibodies prepared in Example 3(2)(1] and [2] as Tabeled antibodies, That is, S68 antibody was diluted with D-PBS (pH 7-4) w 10 ygimL and $0 yl of the resultant solution was then added to each well Of an immunoplate (Maxisorb, NUNC) and reacted at 4° C. overnight. After that, the plate was washed with ion-exchanged water 5 times and then 100 pL of D-PRS containing 0.1% StabilGuand (Sur- Modies, Ine) and 0.1% Tween 20 was added to each well t0 effect blocking. Using asa diluent PBS (pl 7.4) containing 1% normal individval serum (serum ffom which soluble CD14 was removed using 3C10, hereinafter, described a5, ‘CD14absorbing serum) and 0.1% BSA, diluted specimens ‘of human sera of nommal individuals and human sora of patients suffering from sepsis were prepared by diluting the Sora 20-fold, respectively. diluted specimen was added in 2 ‘concentration of 50 per well and reacted at 37° C. foe 2 hours, After the completion of the revcton, the specimen was ‘washed three times with physiological saline containing (0.05% Tween 20 nd SOl-of F1031-8-3-LIRP or FL106-13- S-HRP dlfuted to 0.6 gil. with 76 mM PBS (pH 8.0) ‘containing 5% rat serum, 1% mouse serum and 0.1% Tween, 20 was added to cach well, After a reaction at 37°C. for 2 hours, the plate vas washed 5 times in the same manner as above and a tetramethybenzidine solution (IMB, BioFix) ‘was added to each well. After reaction for 20 mites at room temperature, the rsaetion was stopped with a 0.5 M sulfirie acid solution and an absorbance at 450 nm was mea- sured using # plate spectrophotometer (NI-2100, Japan Intermed), As a result, as shown in Table 2, 9 soluble protein in blood, ic. the low-molecular- weight CD14 being defined ‘nthe present invention, which could not nerease ins normal individual but increase inpatient suffering from sepsis inthe system in which antibody derived {rom S68 peptide was used, Was able to be assayed [3] Preparation of Sandwich BIA System <2> 1) Prepared was a 2-slep sandwich ELA system using the 1146-17-2 antibody prepared as an immobilized antibody in Example 2 antibody prepared in Fxample 3(2) and [2] as a labeled antibody. FI 1467-2 antibody was cited with PBS (pH16.4)t© 120ug/ml-and 0 ul ofthe resultant solution was then added to each well of an. immunoplate (Maxisorb, NUNC) and reacted at $6° C. for 30 minutes. Aller tht, the plate was washed with ion-exchanged water 5 times and then 100 jl of PBS containing 0.19% StabilGuard (SurModies, Ine} and 0.1% Tween 20 (Wako Pure Chomical Industries, ic.) was added 1 each well to effect blocking. Using a8 8 diluent PBS (pH16.4) containing 1% BSA, diluted specimens ‘of human sera of normal individuals and human sera of Patiats suffering from sepsis were prepared by diluting the sera 10-fold, respectively A diluted specimen was added in a concentration of 50 ul. per well and reacted at 25° C. for 2 hours After the completion ofthe reaction, the pate was washed three times with physiological saline containing 0.0 Tween 20 and 50 ul of peroxidase-labeled F1031-8-3 ant body diluted 0.5 ygim. by 76 mM phosphate buler (pH 80) containing 5% rat serum, 1% mouse serum, and 0.1% “Tween 20 was added to each Well After a reaction at 25° C. for 2 hours, the plate was washed 5 times in the same manner asabove and a tetramethylbenzidine solution (TMB, BioFix) ‘wat added to each well. ARer a reaction for 20 minutes at room temperature, the reaction Was stopped with @ 0.5 M 0 o 34 sulfuric acid solution and an absorbance at 450 am was mea- sured using plate spectrophotometer (NI-2100, Japan Intermed). Asa result, similarly to the S68 antibody, in the case of S68-peptide specific monoclonal antibody as shown in Table 2, low-molecular-weight CD14, which was almost ‘ot found inthe sera of normal individuals bat found ina igh level inthe sera of patients sulfering, rom sepsis, was able t0 ‘be assayed. Thais, the result confirmed that an antibody that bind to $68 peptide can prepare a sandwich system irespec- tive of whether the antibody is polyclonal or monoclonal. 2).A tworstep sandwich EIA system, where an immobi- lized antibody used was the polyclonal antibody prepared using the synthetic peptide as an antigen in Example 1-(6), \was prepared. An assay was conducted using as specimens sera. of human normal individuals and human patients suffer- ing from sepsis by the same way as that of 3-[2}, but POOL polyclonal antibody, PO02 polyclonal antibody, orPO12 poly- clonal antibody was use in place of $68 antibody. Asa result, asshown in Table 2, similarly to the S68 antibody, in the case fof the polyclonal aatibody using the syathetc peptide as an n, low-molecular-weight CD14, which was almost not Tuo in the serum of lnm normal individual but found ia ‘high level in the serum ofa patient suffering from sepsis, \wasableto be assayed. The results confirmed thata sandwich system can be perfoemed even in a system using an antibody prepared using peptide san antigen, the peptide having St 16 amino acid residues selected from the amino acid sequences at postions 1 t0 285 of human high-moleca ‘weight CD14 In Table 2, “++” represents. a 4-fold or more absorbance at «450m compared with theabcorbanceot the diluent itself and “0° represents a 2-fold or more absorbance, and *-" repre sents an absorbance equal tothe absorbance ofthe dient TABLE 2 Mesusiie onbiton saa Pac Iediing — Labling tering som Norma setabogy —FIlbe 33 ” : sued prvelon Athy . paloma . [4] Propration of Sandwich BIA System <> ‘A 3step sandwich FLA system using P-1031-8-3 antibody as an immobilized antibody and 368 antibody as a labeled antibody was prepared. The present FIA system was per {formed by biotinylating the $8 antibody as follows. That 50 ul of D-Biotinoyl-e-Aminocaproie Acid N-Lydroxysu «inimide Este (Roche) prepared to 300 un. by dissolving in DMSO vas added 10 0.5 mL of S68 antibody peepared 0 8 concentration of 0.98 mgimil. by substituting with a 0.05-M phosphate bulfer (pH 8.0) containing 0.15 M NaCl and the Iixtire was reacted while being str for 2 howrs at room {emperature-Afterthe completion ofthe reaction, the reaction product was substituted with PBS (pH 7.4) by a desalting column (NAP-S, Amersham Bioscience). The concentrationUS 7,465,547 B2 35 ‘ofthe prepared biotinylated S68 antibody (hereinafter, it may be described as Bio-S68 antibody) was calculated using an absompton coeicent of 4 on the basis of absorbance at 280, ‘The sandwich BIA system immobilized F1031-8.3 anti- body of an immunoplate (NUNC) and blocked. A blocking solution was removed. Then, 500 ng/mL of SCDI4 (1-307) S28%e (hereinafter, it may be described as a standard prepa ration) dissolved in 0.1% BSA/PBS and 2 solution with no Standard preparation added were added to wells as negative ‘contol, respectively, The plate was washedafier a reaction 37°C. for | hour, andsubsequertTy SO of biotinylated S68 antibody prepared to 1 ug/ml. by diluting with PBS (pH 7.4) ‘containing 6 rat serum, 1% mouse serum, 1% rabbit serum, and 0.1% Tween 20 was added and reacted at 37° C. for 1 hour. Afr the completion of the reaction, the plate was ‘washed andl then a 10,000-fld diluted peroxidase-labeled streptavidin (Which may be described as SA-HRP. Invtro- en) was added. The plate was washed after a reaction for 1 hour. Aller color had been developed with 2 TMB solution (BioFix), the restion was terminated by a terminating liquid ‘and an absorbance at 450 nm was messured using a plate spectrophotometer E-Max (Molecular Device, Co.,Ltd) -Asshown in Table3, in the present system, a sandwich FIA, system was able to be prepared. In other wor, the inventors ‘confirmed that a sandwich assay sytem can be prepared eve ian antibody that binds to S68 peptide is used as an immo- bilized antibody or used as free antibody or labeled ant body. In Table 3,44" represents that the absorbance differ ‘ence with 0 to 500 ng/mL. ofthe standard p Abs or more, "4" represenis 0.1 or more, and Jess than 0.1 [5] Preparation of Sandwich BIA System <4> ‘A L-step ELA system was prepared such that immobilized and labeled antibodies were o the same system that of 2}, ‘anda specimen and the labeled antibody were simultaneously added. Tha is, 25 ul of each of O- and 500-ng/ml, standard preparations was add toa $68-antibody immobilized plate, followed by the addition of 25 pl. of F10S-8-3-1IRP pre= pared (01 mL. by dilution with PBS (pH 7.4) containing 29% rat serom, 1% mouse serum, 1% rabbit seram, and 0.1% Toween20,A reaction wascatried out for | hourat 37. Ate the completion of the reaction, the plate was washed and ‘colored by a TMB solution (BioFix). Then the reaction was terminatod by a terminating liquid, followed by measuring an absorbance at 450 nm using a plate absorbance meter F-Max, (Molecular Device, Co, 1d.) As shown in Table 3, a sand- Wick ELA system was also made in the present system, That js, the inventors confirmed that a sandwich assay system using an antibody that biads to S68 peptide ean conduct an assay without any elation to the reation sequence, [6] Preparation of Sendwich BIA System <5» Immobilized and labeled antibodies were ofthe same sys tem as that of [2], and a specimen and the labeled antibody ‘were simltancously reacted Then, a 2-tep FIA system to react wit the immobilized antibody was prepared. Thats, 2S UNL of each of O- and 500-ng/ml. standard preparations was mixed with 25 pl. of FIOBI-8-HIRD prepared to 2 gl. with PBS (pH 7-4) containing 2% rat serum, 1% mouse serum, 1% rabbit serum, and 0.1% Tween 20, After the ‘completion othe reaetion, the reaction solution was added to ‘an $68-antibody-immobilizad plate, and the whole was, reacted at 37C.* fr T hour, The plate was washed and then ‘oloredby a TMB solution (BioFix) and then the eaetion was, terminated by a terminating liquid, followed by measuring an absorbance at 450 nm using a plate absorbance meter E-Max, 0 o 36 (Molecular Deviee, Co, Ltd). As shown in Table 3, a sand- ‘wich ELA system was also made in the present system. That is, the iaventors confirmed that a sandwich assay system ‘using an antibody that binds to S48 peptide ean conduct the assay without any elation tothe reaetion sequence, 7] Prparation of Sandwich BIA System <6> ‘A sandvich ELA system using the specific biding of biotin streptavidin was prepared 1) Assay system using streptavidin on the immobilizing side Streptavidin (PIERCE) diluted to 10 ng/mL with PBS (pH 7.4} was dispensed into immunoplates (NUNC) with 50 yl. aliquots and immobilized by treating it at 4° C. overnight, respectively Aer blocking, the liquid was discarded from ‘them and 25 lL ofeach of biotinylated S68 antibodies pre- pared o 2gimi with PBS (pH74) containing 2% rat serum, 196 mouse serum, 1% rabbit serum, and 0.12% Tween 20 and (0 and 500-ng/ml. standard preparations dissolved in 0.1% a Ata Ucar (ewas washed and subsequently SOpL of F1031-8-3-11RP Gite Im was added, fellowed by erections 37°C Tor { hour, Aller the completion ofthe evotion, the plate was ‘washed and colored by a TMB solution (BioFix) and then the reaction was terminated by a terminating liquid, fllowed by ‘measuring an absorbance at 430 amusing a plate spectronho- tometer F-Max (Molecular Device, Co., Lid). The present systems were teste similarly even if the standard prepara jon, biotnated $68 antibody, and peroxidase-labeled F103: 83 antibody were simultaneously added. As shown in ‘Table 3, sundWich ELA systems were able to prepared in both 5 ‘ems 2) Assay system using peroxidase-labeled streptavidin ‘The present system was prepared by the method shown in [4], Furthermore, 2-tep method was studied, where a reae- tion was curied out at 37°C, for 1 hour alter simultaneous addition of a standard preparation and biotnated F1031-83 anuibody (which may be described as Bio-F1031-8-3) pre- pared sceording to [4] and about 1,000-fold diluted peeo {dase- labeled streptavidin (Invitrogen) was added after wash- ing. After the completion of the reaction, the plate was ‘washed and colored by a TMB solution (BioPix) and then the reaction as terminated by a terminating liquid, followed by ‘measuring an absorbance at 450 am using a plate spectropho- fomiter E-Max (Molecular Device, Co.,Ltd). As shown in ‘Table 3, inthe present system, a sandivich EIA system was ablebe also prepared. Thats, the inventors confirmed thatthe assay can be attined even ifan immobilized or labeled sub- stance is prepared usinga second spocfi binding such asthe biding of biotin and streptavidin as far as low-molecular- ‘weight CD14 is sandwiched between an antibody that binds {068 peptideand an antibody thatbinds fo theassay analyte, Jow-molecular-weight CD14 in human serum. By the way. Sie" represents streptavidin and“Bio” represents bitin) ing. TABLE 3 —____ se sample Pate 3_Resctvty 1) HUIS Sanaa BeSen SA 1 Sandee eoUS 7,465,547 B2 37 ‘TABLE 3-continved ees Brple Pie 2 3 Resa ao Srl rome Srl ike Bosra se ya) se mos = + ya) se me) sie Example 4 Preparation of Immunochromatogrophie Assay System 41) Immunochromatographie Method Using Gold-Colloid Labeled Antibody “Anassay system which could be easily used in laboratory ‘or by bedside was prepared. The outline ofthe assay systema was shown in FIG, (4), First, gold collo-labeled FL106- 13-3 antibody was prepared by mixing | mi. of gold colloid (40 nm in panicle diameter, B.B. International) with 9g of 1106-13-3 antibody: Next, a conjugate pad as prepared. That is, the gold coloid-labeled F1106-13-3 antibody was diluted with 2 conjugate-applying buffer so that an absor- bbancest520nm would beabout I'S and I mL of theresultant solution was then applied on a 33-Class stip of 10x150 am, {allowed by drying under educed pressure overnight. Atthis time, the antibody titer of gold collod-labeled F1106-13-3 antibody ina reagent pe test was about SO units (1 unit equals Tl. of gold. colloidlabeled F1106-13-3 antibody at (0520=1.0), Theantibody-immobilized membrane was pre pared as follows. S68 antibody was diluted to I mg/ml. with PBS (pH 7.4) and the solution was linearly applied on 2 nitrocellulose membrane (FF8S/100, Schleicher & Schuell) ‘nan amount of 0.75 u/em using an inkjet coating machine ‘manufactured by BioDot Co.,Ltd. At this time, a control line (anti-mouse. polyclonal antibody, DAKO) was. simlta- neously applied. After drying, the membrane was immersed ina blocking liquid containing 0.5% casein for 30 minutes ‘and then an excess par of the liquid was removed, followed, by dying again. Next, an immunochromatographic reagent as formulated using each ofthe prepared materials, That i, aconjugate pad, immobilized membrane, an upper-absorbing pad (#900 filter paper, Schleicher & Schuell), or sample- ‘dropping pod (33-Giass_ glass fiber filter, Schleicher Schuell) was attached on a PBOZO plastic-hacking sheet (BioDot) and then cut in S mm in width by a strip cuter ‘manufacniredby BioDot Co, Lid. The et strip was housed ia ‘housing case (NIPPN Technoclaster, Inc) nd provided as mmunoehromatographicreageat 38 An assay was performed as described below using the prcpared reagent, A standard preparation diluted 10” folds ‘within terange of 10,0000 1 ng/ml with 1% BSA-PBS was provided as a sample. Then, 100 il of the sample was 5 dropped into the regent to determine the presenev or absence 0 o fof a line after the mixture had been let to stad at room temperature for 20 minutes. The criteria for the judgment were i fallow: (G4): Level at which a thick line is developed so thatthe line can be clearly judged as positive: (4): Level at which color development can be judged as 2 line even though the color development is pale; (2): Level at which what looks like color development is slightly observed but dificult to be recognized as fine; and ‘Level at which no color development is recognized. ‘Asa result, ax shovin in FIG. 2 and Table 4 the sensitivity of "+" or more was obtained at a sample concentration of 10 ‘yim. or more, Therefore, the result confirmed tha the assay can be performed simply and quickly by an immunoehro- ‘alograpic system, 4-(2) immunochromatographie Method Using Gold Colloid- Labeled Antibody <2> “The assay was conducted while the immobilized antigen and gold colloidlabeled antibody of the immunochromate- graphic assay system prepared in 4-(1) were arranged inversely. The gold-colloid marker of S68 antibody and jmmunochromatographic system were able tobe prepared by the same way a that of4(1), Asa reslt as shown in Table, the sensitivity of "¥" oF more wes obtained ata sample con: centration of 100 nla or more, TABLE 4 ois seo —Suuplecomsmaton and) Label —__Inmobied a) 10 4.(3) Preparation of mmunochromatographie Method Using Streptavidin-Biotin System In addition, an immunochromstographic assay using a streptavidin biotin system was prepared. The outline of the assay was shown in FIG. 1(B) First, according to Fxample 53.2[4], FIOBI-8-3 antibody was biotnated, Then, gold col- Ioid-labeled streptavidin was prepared by mixing 1 mL of gold colloid (40 nm in particle diameter, B. B. Intemational) ‘ith 10 yg of streptavidin. The gold collok-labeled strep vidin was dilated with a conjuzatessppying buffer so tha an absorbance at $20 nm would be about 1.8 and 1 ml. of the resultant solution Was thea applied on a 33-Glass stip of 10150 nm, followed by drying under raced pressure over- sight. At this time, the antibody tter of gold eollid-labeled streptavidin in @ reagent per test was about 50 units (Init equals 1 pL of gold. colloid-abeled streptavidin at (0520-1 0). Theantibody-immobilized membrane was pre- pared as follows, S68 antibody was diluted to 1 mn. with PBS (pH 7.4) and the solution was linearly applied on aiteooellulose membrane (FF85/100, Schleicher & Sehvell) in an amount of 0.75 ul fem using an inkjet coating machine ‘manufactured by BioDot Co.,Ltd. At this time, a contr Tine (ent-mouse polyclonal antibody, DAKO) was. simulta- neously applied. After drying, the membrane was immersed in blocking liquid containing 0.5% eascin for 30 minutes thea an excess part ofthe liquid was removed, followedUS 7,465,547 B2 39 by dying again, Next, an immunochromatographic reagent vas formulated using each ofthe prepared materials, That is, a conjugate pad, immobilized membrane, an uupper-absorbing pad (#900 fite, Schleicher & Schuell), oF sample-dropping pad (33-Gilass glass fier filer, Schleicher ‘& Schuell) was attached on a PB020 plastic-backing sheet (GBioDot) and then cut ia 5 mm in width by a strip cuter ‘manufactiredby BioDot Co, Lid. The et strip was housed in housing case (NIPPN Technocluser, Ine) and provided as fn immusochromslographic reagent. An assay was per Jormed as deseribed below using the prepared reagent. A standard preparation diluted 10" fos Within the range of 10,000 16 | ngimL. with 19% BSA-PSS. was provided as a sample. Then, 100 -of the sample was dropped into 100 pl ‘ofreagent containing 0.1 of biotinized FIOS1-8-3, and the hole was mixed. Then, 100. ofthe mixture was dropped nto a sample-dropping pad ofthe hovsing case to determine the presence or absence of line after the mixture had been Jeft to stand atroom temperature for20 minutes. Therefore, in the present system, the sensitivity of "4" oF more was also 2 ‘obianed at a concentration of 100 ng/ml. o more just asin theease of (1). Example 5 Proparation of Flow-Through Assay System A flow-through assay system is prepared according to IP 06-273419 A. That is, I g of a disperse dye (Red Violet, Kayaron, Co. Ld.) is suspended in 10 mi of distilled water, ‘and then resuspended in 5 ml of disiled water ater being ‘washed with distilled water. 0.2 ml of 0S-mg/ml FIOSI-8-3 antibody diluted with physiological saline isadded to 0.2m ‘ofthe disperse dye and the wholes incubated at 45° C. for 30, minutes. te the resultant has been cooled oa ie, centri tal separation is performed. Te resulting precipita is resus- pended in PBS (pH 7.4) containing 0.3% BSA and 10% Ietoseto prepares disperse dye-labeled1031-8-Santibody. Next, the disperse dye-laboled FIO31-8-3 antibody is dis- pensed with O.1 mil aliquots and immersed into fier paper (No, 63, Advantee Toyo)eut into 14mm indiameter, followed by freeze-drying to prepare a porous body adhered with @ soluble reagent Immobilization on a membrane is performed as follows int, 2 mg/ml. of S68 antibody diluted with physiological saline is applied on a nitocelinlose membrane (Advantec Toyo) af S microns in pore diameter and deed a 37°C. Next, blocking is performed using PBS (pH 74) containing, 1% BSA to prepare an antibody-immobilized membrane. The prepared materials are assembled in a housing ease in the following order. An assay reagent is prepared by assembling the porous body adhered with a soluble reagent, antibody= ‘immobilized “membrane. polypropylene-laminated fer paper (No. 28, Advantee Toyo), anda transparent plate made ‘of polycarbonste of 0.5 mm in thickness i onder. An assay is initiated by the addition of 05 mL ofa sample to the assay reagent and a judgments performed by observing coor from the back side by the naked eyes after the sample has been ‘completely absorbed Example 6 Specificity of $68 Antibody For confirming the specificity of S68 antibody prepared in [Example I the inventors studied whether blocking occurs by peptide by the same assay a that of Bxample 33). Thais, o 40 ‘S68 peptide (amino acid sequence at positions $3 10 68), syallictic polypeptide prepared by the same way as that of Example I (amino aeid sequence at positions 531058, amino acid saquence at postions 57 to 62, and amino acid sequence at positions 59 to 64), or negative eontrl peptide (SEQ ID NO: 19)(Cys Glu Gly Asn Gly Asn Asn Phe Glu Ser Arg Gio ‘Ala Cys) was diluted to 0, 0.1, 1, and 10 ug/L and 25 iL of ‘ach dilated solution was added to 25 i Of each of 50-fold luted soltions of the sera obtained from patients suffering rom sepsis and the sera of normal individuals to initiate ‘competitive reaction by mixing with S68 antibody. After that, the levels of low -molecular-weight CD14 ound to S68 ant body without inhibition by any peptide were determined. As ‘rest, 2s showa in FIG, 3, in both the sera of the normal individuals showing low levels and of patients suffering form sepsis showing high level, the binding between S68 antibody and the low-molecular-weight proeinin blood was inhibited inthe case of S68 peptide but not inhibited in the eave of other partial peptides (each containing 6 amino seis) and a negs- ‘iveconteol peptide, Theabove result confianed that protein being detected in blood by S68 antibody is specifically ee- ognized by S68 antibody. In addition, the result also eon- firmed thal the saquence recognized bythe antibody requires ‘length of at least 7 amino acids because the inhibition ean ‘not be attained by tree kinds of synthetic peptides (the num berof amino eid: 6) corresponding othe patil peptides of S68 peptide, Example? Reaction Rate Constant of Prepared Antibody ‘The specificities and reaction rate constants of S68 an body prepared in Example | and F'1146-17-2 antibody pre- pared in Example 2 were analyzed wsing Biscore 3000 (Bia- core), respectively. First, S68 peptide-BSA tobe immobilized was prepare by the same way as one deseribed in Example 1 Using malcimidated BSA Cimject Maleimes! Activated BSA, PIERCE), Nex, the S68 peptce-BSA was immobilized on 3 ‘censor tip CMS (Biacore) using an ansine-coupling it (Bia- core), An assay was performed such that HBS-FP (Biacore) ‘Nas used as a running buffer and a dilution series (50, 100, 150, 200, and 300 aM) of F1146-17-2 antibody was injected into low cells. The data analysis was performed using Biae- valuation software version 3.0 (Biacore) by subtracting re crence-cell data from flow cell measurement data of S68 peptide-BSA. As. result of analyzing adissociation constant (KD), the F1146-17-2 antibody showed allinity as high as 48x10. M, By the way, the KD value of specifcally-purifid rubbit S68 peptide polyclonal antibody measured similarly was 2.24109 M, Example 8 ‘Specificity of AntiCD14 Monoclonal Antibody 8-(1) Analysis of P1106-13-3 Antibody For clarifying 2 binding region (epitope) of F1106-13 antibody, a peptide brary membrave (Custom SPOTS, Sigma Genosys} on which the amino acid sequence of CD14 Was synthesize from the N-terminal thereo! 10 amino aeids ata time was used for analysis. That is, the membrane was ‘locked based on the instruction manual thereof and thea Was reacted with F1106-13-3 antibod, washed, and then reacted ‘with F-galacosidase-bound anticmouse antibody, After the ‘membrane had been washed, peptide sequence on which the ‘antibody ws bound was detected using X-pal, By the way,US 7,465,547 B2 4 the peptide sequences on the peptide library membrane were analyzed using 19 peptides which were syntbesized suc that 10.amino acids were subjected tothe synthesis ata time sos tooveriap two amino acidso the respective C terminals ofthe sequences of amino acids at positions 1 10 154, The peptides ‘vere prepared by the same way’as that of Example 1-1). “The result found that F1106-13-3 antibody’ binds to the rogion corresponding to an amino ac sequence at positions 17 to 26 of SEQ ID NO: 5(CNFSEPQPDW) fromm the N-er- minal of high-molectlar-weight CDIM. 8.2) Analysis of FIO31-8-3 Antibody <1> Por confining the specificity of F1031-8-3 antibody, SCDI4 (1-285) derived from B. coli described in Example 3-(1) and sCD1- (1-356) and sCD14 (1-307) S286C prepared from COS cells using methods described ia Examples 8 and 9 of WO 01/72993, the binding activity was determined. First, sCD14 (1-386), sCDI4 (1-807) S286C, sCDIS (1-285), or BSA was immobilized 250 ng/spot on @ men- brane, Hybond-C extra (Amersham Bioscience), and after dying it was blocked by 0.05% Tiwoen 20 containing 008 im. of skim milk (Meiji Milk Produets), PBS (pH 6.4) ‘After the resultant had been left to stand for | hour st room temperature, F1031-8-3 antibody diluted to 3 yim. with (0.05% Twoen 20 containing 0.5% BSA, PBS (pH 6.4) was ‘adked and reacted for | our at room temperature, followed by washing with 0.059% Tween 20, PRS (pl 6.4) Nett, peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO) diluted $00 folds with 0.05% Tween 20 ‘containing 10% rabbit serum, PBS (pl 6.8) was added and reactod for 30 minutes at 37° C. Thea, the membrane was ‘washed similarly followed by confirming the binding ativity ‘of the antibody with ECL kit (Amersham Bioscience). As a result, as shown in Table S, F1031-8-3 antibody bound to SCDI4 (1-285), sCD14 (1-307) S286C, and sCD14 (1-356) rived from E.coli not to BSA. Thus, the resul found tat the F1051-8-3 antibody specifically recognized all types of ‘CD14 proteins. In Table 5." represents situation in whieh spot was detected ona film and" represents a sitation ia which no spot was detected, TABLES Bingo = 7 ea 8.3) Analysis of PO31-8-3 Antibody <2> For clarifying a binding region (epitope) of F1031-8-3, sntihody, the spots analysis was performed as in the case of (1) However, nthe spots method, no recognition region of F1O31-8-3 antibody could be specified. For the purpose of ‘analyzing the similacity of the recognition regions of bath antibodies, inthe sandwich EIA system of Example 3-(3)((2] ‘where $8 antboxy was usedas immobilized one and FLOSI- S3-FIRP was used at labeled one, an inbibition test was performed using F1106-1-3 antibody: Firs, 3 inthe ease of Example 343)[2}, 100 ngml ofthe standard preparation was added to and rected with an S68- aniibody-mmobilized pate. After the plate had been washed, before the addition of FLO81-8-3-HIRP, 25. buffer con- taining 6 pg/ml. of F1106-13-3 antibody, mouse laG anti body or no antibody was added. Then, 25 uL of F1031-8-3- TIRP antibody was added, followed by the measurement by the same way as that of Bxample 34@}-(2] 0 o a2 As shown in Table 6,20 inhi IgG antibody addition system while the inhibit between F103} antibody oecurred. This fact indicated that F1 106-1 body may bind to at least one region to be recognized by F1031-8-3 antibody. By the way, an “inhibition rate” was calculated from each absorbance being decreased atthe time ‘of defining the absorbance of the bufler alone as 100%. TABLES: Asie Tahini 0) Nowe 16 aby 2 Frioe 53 aati) * Example 8 Assay Kt for Haman Low-Molecular-CD14 (1) Typical Format of Assay Kit for Sandwich EIA ‘A ypical format of a soluble protein kit using a combina- ‘ion of immobilized and labeled antibodies that show high let mtn es jeats suffering from sepsis and low levels in specimen {fom noma individuals in Example 3-3) will be described below. -<1> Immobilized antibody: Plate on which $68 antibody is ‘immobilized <2» Labeled antibody: Peroxidase labeled body - Substrate solution (tetramethy benzidine solution) Other Accessories Configuration Example ofa Plate System - Plato-washing solution (0.9% NaCl, 0.05% Tween 20 solution < Sample-diiting solution (0.1%-BSA-contsining PAS solution}e -<6> Reaction-trminating liquid (0.5 M H.SO, solution) «<7» Standand preparation (CD14 (1-307) S286C) ‘Measuring Instruments for Perorming an Assay Using the Above Assay Kit ‘<8 Plate spectrophotometer (eg, E-Max (Molecular Deviee, Co, Ltd)) 84{2)t0 (11) Configuration Examples of Assay Kit for Sand wich BIA Sytem In addition to 8-(1), the examples of the asay kit fora sandwich EIA system are shown in Table 7.<1> represents binding substance immobilized on a plate. <2> represents a Jbeled binding substance. The constituent elements of<3>-10 -<7> anda messuring instrument 98a reference example fare identical with 8-(1), <9 represents an antibody. bound ‘witha sevond specific biding substance. 1031-83 ant TABLET 6) Satie rue iss.me © Finnts HiwsarauneUS 7,465,547 B2 43 ‘TABLE 7-continved 1) FIGS Soramg tubo 1) Sssanatoty SAR Boris 8.(12) Standard Curve of Assay Kit for Sandwich FIA Sys- Using the assay kit of (1), an assay was performed by the same way as that of Example 3-3)[2]. Thats, $68 antibody ‘was dfuted to 10 ug/ml. with D-PBS (pH 7.4) and 50 uL of the resulant solution Was then added to each well of ua jmmunoplate (Maxisors, NUNC). After a reaction at 4° C. ‘ovemight, the plate was washed five times with ion-ex- ‘changed water and blocked by the addition of 100 yl of D-PBS containing 0.1% StabilGuard (SurModies, ine.) and (0.1% Tween 20 to each well. Next, 76 mM PBS (pH 7.4) containing 1% CD14-absorbing serum and 0.1% BSA was used as a diluent w prepare a dilution series of 0,3, 25,60, 100, and 150 ng/mL ofCD14 (1-307) S286C prowinstandand preparation. The dilation series ofthe standard preparation was added in an amount of $O ul. per well and reacted for 2 hours at 37°C. After the completion ofthe reaetion, the plate was washed thee times with physiological saline containing 0.05% Tween 20. Then, 50 pL. of cited labeled antibodies prepared by diluting 5% rat serum, 1% mouse serum, and peroidase-labeled 1081-8-3 antibody to 0.6 yim. with 76 1M PBS (pH 8.0) containing 0.1% Tween 20 were added 10 ‘each well. fora reaction 37°C. for? hours, the plate was ‘washed ive times in the same way as above and a tetrameth- ‘lbenzidine solution (TMB, BioF ix) was added to each well, Aer a reaction for 20 minntes at room temperature, the reaction was terminated by 0.5 M sulfuric cid salution and an absorbance at 450 nm was measured using a plate spec- trophotometer (NJ-2100, Jgpan Intermed). A standard curve prepared was shown in FIG. 4, simple assay system with high sensitivity as a measuring sensitivity of 0.6 ngiml. (lank+38D) was realized, 8.(13) Specificity of Sandwich ELA Syste "Pr studying the influence ofhigh-molecula-weight CD14 present in hunian serum ontheassay system prepared, soluble ‘CD14 decived from normal individual serum al concent tion oF 004 g/ml. was added tho standard preparation of ‘CD14 (1-307) S286C to condhet the same assay’ as that of (12)-Asa result, there was no inlvence on the measured level ‘even though the concentration ofthe soluble CD14 derived, from normal individual serum was 4 ug/ml. The result found that the cross-reactivity of the present sandwich FIA system with high-moecular-weight CD14 was 0.3% or les. In other Words, the result confirmed that the preseat system does not dotct aman seram high-molecular-veight CD14 andisspe- ‘ile toa soluble protein showing a high level in serum ofa patient suffering from sepsis 8.(14) Bvaluation on Assay Kit for Sandwich FLA System Reprodieibilty of the assay results of the kit of (1) was ‘evalited, The coefficient of variation (CV) of within-ran reproducibility using 3 samples of specimens asin the ease of (12) was 58, 3.6, and 3.5% and_ reproducibility between measurements ws 6.2, 5.2, and 5.1%, respectively. Thus 0 44 ood results were obigined, hile no influence of an antico- ‘gulant (heparin, citric acid, or EDTA) was observed. The results described above showed thatthe preseat kit as @ sufficient ability for the assay of human low-molecular ‘weight CD14, 8-(15) Example of Imasinochromstographie Assay Ki ‘ Labeled antibody: F1031-8-3 antibody labeled w colloid -<2> Conjugate pad: Glass fiber iter (33-Glass stip, ma trea by Scleicher & Schuell) on which <1> is applied Antbody-immobilized membrane: Nieocellulose mem- ‘brane (PF8S/100, manufactured by Schloicher & Schuell) blocked by 0.5% easeinanal having an immobilizing fine of ‘S68 antibody and s control line (an immobilizing line of anti-mouse polyelonal antibody) onthe downstream of the jmmobilizing line ‘<4> Sample-dropping pad: 33-Glass plas fiber filter (mam factured by Schleicher & Schuell) = <5 Absorbing pad (#900 filler paper (manufactured by ‘Schleicher & Schell) -<6> Sheet: PBO2 plastic backing sheet (manufictured by [BioDot); <2> to <$> are assembled on <6> such that a liquid dropped in <4> is allowed to tow through <2>,<3>, and inthis order. -<7> Housing ease (OEM case available from NIPPN Tech- socluster, Inc) By the way, the outlines of <1> to <5> are represented in FIG. 10). 84(16) t0 (19) Example of Immonochromtographie Assay Kit Table 8 shows, in addition to 8-15), examples ofan assay kit fora sandwich EIA system utilizing the scond specific binding between binding of biotin and streptavidin, and ‘examples of an assay kit fora sandwich FIA system utilizing the fragment ofan antibody that binds to a peptide having 16 amino acid residues described in SEQ TD NO: 2. repre- sents a labeled binding substance. The constituent elements ‘> to <7> are identical. Asa substance to be applied on <>, -<3>-{i) represents binding substance to be immobilized on an immobilized membrane andl <3>-(i) represents a binding ‘substance to be immobilized on acontrol line. <> represents ‘an antibody bound with a second specific binding substance, the substance being a reagent to be applied on <2> oF as in the ease of , of o be added o-a specimen or simul neously added togethor with the spocimen, By the way, the outlines of (16)<1> to ate shown in FIG. 1(3),and (17) to 20) ate similarly understood. old TABLES as Sisoed” sti 19) Godson ‘Savas winston ay Rabjyefge—ruuoni3a. Amie By the way, F(ab), of S68 antibody labeled with gold colloid of (20)<1> is prepared as follows. The preparation ofUS 7,465,547 B2 45 F(ab), from S68 antibody is performed a follows using Immobilized Pepsin PIERCE). That is, $68 antibody is is solved in a20 mM acetate ber (pH 4.5} to be prepared to $ ‘mg/ML. 0.25 mal. of immobilized Pepsin is prepared by suse pension aovonding to the protocol of PIERCE and mised with ‘mi. of the above antibody. Next, the mixture is tired foe 4 hours in an incubator at 37° C., and then the reaction is terminated by the addition of 1.5 mL of 10 mM Tris-HCl (pH 7.5). The reaction solution is centei fuged (1000) 10 separate ‘gel and a superntaat. Then, the separated supernatant is faded to | mL of prosep-A (Millipore) allow the binding of Peptides including Fe portion such as Fe fragment and undi iBeved, Likewise, the mixture is centrifged to collect the Supernatant, and then the supernatants dialyzed against PBS (pH16.4).Theabsorbanceof F(ab) at 280m ismeasuredand then the concentration of Faby. is calculated from the absorption coustant (0.533 mp/mnLiem!). The resulting F(ab), is labeled wit gold colloid as inthe case of Example 4, resting in F(ab’) of the gold collo-labeled S68 anti body. 84(21) Configuration Fxample of Flow-Through System Dye-lbeled antibody: RED VIOLET dye-labeled S68 antibody -<2> Conjugate pad: Filter paper (No. 63, manufactured by ‘Advantec Toyo Co. [4 impregnated with the above (I)< -<3>-Antibody-immobilized membrane: Nitrocellulose mem- ‘brane (Advantec Toyo) on which S68 antibody i immobi lized ‘<4> Absorbing pad: Filter paper (No. 28, Advantec Tokyo) laminated with polypropylene <5 Housing ease: Case described in JP 06-273419 A (mame Tctured by Mochida Pharmaceutical), <2> 10 <8 are assembled in <5> such that liguid dropped in <2> is allowed to flow through <2>, <3, and <4 in this onder. Example 9 Detection of Human Low-Molecular-Weight CD14 (1) Ge Filtation Chromatography Por analyzing the substance in serum of a patient sufering from sepsis detected by the assay kit deserbed in Example 8.(1), the serum of the patient suffering from sepsis was fractionated through a gel itration chromatography column Superdex 200PC 3.2130 (Amersham Bioscience). with SMART SYSTEM (Amersham Bioscience) using D-PBS as a elution buile. Then, each fraction was assayed using the assay kit described in Example &(1) and the commercially available CDIELA kit (BL-Hamburg). The molecular ‘weight thereof was etleulated by calibrating the column using aldolase (18 kDa), BSA (67 kDa), ovalbumin (43 Da), and chymotrypsin 25 kDa) from the LMW calibration Kitand FMW calibration kit (Amersham Bioscience), Asa result, a8 shown in FIG. 6, the commercially avilable CDIGEIA kit detected soluble CDI having a molecular weight of about $7 KDa, which was defined as high-molecu- Jar-weight soluble CDI4 of 49 to 35 kDa conventionally reported, On the other hand, in the kt described in Example S-(1), a peak derived from human jow-moleculat-weight ‘CD1d detected inpatient sutfering from sepsis was detected around a molecular weight of 35 to 45 kDa but no peak was ‘detected around S7 kDa. Thus, the result confiemed thatthe kit deseribed in Example 8-(1) specifically detects only a soluble protein present in blood. 46 2) Gel Filtration Chromatography <2> ‘As in the case of 2}-e1>, $0 yl of serum from a patient sulTering from sepsis was ractionated through gelation chromatography colimn Superdex 75 10/30) GL, (Amer sham Bioscience) using 200 mM ammonium acetate (pH 68) fsa elution bufler and was subjected ta the assay sing each it The molecular weight thereof was calculated by calibrat- ing the columa sing BSA (67 kDa), ovalbumin (43 kD), ciymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa} tom the MW calibrationkit and HMW calibration kit (Am- ersham Bioscience), "The results are shown in FIG. 7, In the kit desribed ia Example 8-(1), a peak derived from human low-molecular- weight CD14 was detected around a molecular Weight of 25 1035 kDa, (8) F1025-3.1 Antibody Affinity Column Chromatography ‘When a peaked fraction (¢. faction 12) derived from human Jow-molecular-weight CD14 obtained in (2)-<2> is applied to P1025-3-1 antibody affinity column chromatogr- phy; peak derived from human low-molocular-weight CD14 Js eluted inanafisity column aon-absorbing fraction. By the way, te adjustment and operation ofthe F1025-3-1 antibody affinity column can be performed on the basis ofthe method described in Example 10 of WO 01/22085. “These results show that the human low-molecular-weight CD14 is. soluble protein in blood that specitieally binds to antibodies against a specifi peptide described in SEQ ID NO: 2 having a sequence detected only in human CD14 and also binds to an ant-CD14 unibody recognizing an amino ‘acid sequence at positions 17 to 26 from the N-terminal of hhuman CD14, The gel filtration determines the molecular ‘weight thereof to be 25 to 45 kD. Thus, itean be defined that ‘he lhuman low-moleeular-weight CD14 smallerin molecu- CD14 does not bind o F1025-3-1 antibody that specifically binds vo high-molecular-weight CDI. Example 10 Assay of Low-Molecular-Weight CD14 in Sera of Patients Suffering from Various Kinds of Diseases 10 examples, from which isolates were identified, were sed (Table 9) asthe ser of patients suffering from sepsis. ln addition, the assay was conducted using the assuy it described in Fxample 8-1) on 52 examples of normal ind Viduals (male 31 examples and female 21 examples), and patients suffering from various kinds of diseases (20 diseases, 60 examples. TABLES T Wale al Couple arate bce a 4 "Mile $2 Soro basen 3 Male 37 Exhonohocoll & Femi Eich col ; Mae 4)—_‘Suppforwt eas ‘ Male $1 Fame axtomerne 3 Fammle, MLB Mle Exerc ‘The level of low-molecular-weight CD14 in secum of a ‘normal individual was inthe range of 0.008 to 0.100 g/m. ‘and the average thereof was 0.04 igiml.. In the ease of @US 7,465,547 B2 47 patient suffering fom sepsis, the level of lowmolecular- ‘weight CD14 was in the range of 0.190 to 7.260 jim and theaveraze thereof was 2.0m. The level of ow-molecu- Jar-weight CD14 of the patient suffering from sepsis was higher than those of the normal individuals and patients su {ering from other various kinds of diseases. Among patients suffering fom other various kinds of diseases, there Was no patient showing a high level, compared with that of the or- ‘mal indiviial Example 11 Comparison with Commercially Available ELISA Kit for CD14 Soluble in Blood 11-(1) Assay of Soluble CD14 in Blood of Patients Suffering fiom Varioas Kinds of Diseases ‘Spocimensol Example 10 were assayed using the commer- ly available CDL4-ELISA kt (IBL-Hlamburg). The level, ‘ofsoluble CD14 inblood estimated asatotal oflow-molecu- Jae-weight CD14 and high-molecula-weight CD14) of anor mal individual was inthe range of 5.6 011.2 ng/mL but an ‘example of ahigh evel inthe easeof a patient suffering fom sepsis was obsorved. However, many cases that showed high Jevels of soluble CD14 were found in sera of patients sulfer ing from various kinds of disease, s0 that thee was no dilfer ‘ence withthe patients sufering from sepsis 11-@2) Comparison with Kit Using S68 Antibody ‘The comparison with and investigation of the measured levels of low-molecularweight CD14 determined ia Example 11 were performed As showa in Table 10, the ‘commercially available CDI4-EIA kit showed an almost 17> fold diference at maximum among the normal, various dis- ceases, and sepris, while the assay kit of Example 9-(1) showed a S0fold difference between the normal individuals ‘and the sepsis in spite of no difference between the nonmal individuals and various diseases, Therefore, the result Was ‘lear that the measured level of the assay’ kit of Example 9.(1) specifically increases in sepsis TABLE 10 (Chis ieelatend ala Nomml seat Sop _Sepss Nomad Astor To 008 nanple 3) ‘uiic CDA The average level43 S.D of the tested normal individuals was provided as acutofflovel (low-molecular-weight CD14- EIA0.134jgiml, commercially available CDI4-E1A: 11.14 HpimL.) and then the analyses were divided into postive ‘amples (sepsis) and negative samples (normal various dis- ‘eases), The resus were shown in Table I. According to the 0 48 results, rate of identieal between both kis (he number of ‘identical for ELA positivetthe number of identical for ELA negative) total 00), sensitivity (the numberof identical for FIA positiveipositive examplesx100), and specificity (the ‘number of dential for ETA positive!negative examplesx100) were calculated. As. rest, as shown in Table 12, in the case of low-molecularsweight CDI4-BIA, the identical rate was 943%, the sensitivity was 100.0%, and the specificity was 93.89%. Thus, it was found that the low-molocular-weight (CDI4-EIA could be usefilin diferential diagnosis on sepsis by defining the eutofflevel, On the other band, inthe ease of the commercially aailable CDI4ELA, there was no seas tivity and specificity which were specific to allow diagnosis of sepsis. TABLE 11 Nowie anal “ase tat vs raphe 0) Comercial 6 8 ‘abe eDLeEIA TABLE 12 ‘Asay tot Commeraly Example walle CDIA-E1A Specie Sue HS INDUSTRIAL APPLICABILITY According tothe present invention, there is provided the antibody prepared using a peptide as an antigen, the peptide having 8 to 30 amino acid resides selected from an amino acid sequence at postions 1 1o 68 of human high-molecular- ‘weight CD14, and abo provided the antibody that binds to 8 peptide having amino acid residues deseribed in SEQ ID NOS: 20 4 “Those antibodies can be used in an assay’ kit for human Jow-molecilar-weight CD14 and the kit is ale to quant tively or qualittvely determine human low-molecl ‘weight CD14 with high sensitivity ina simple manner, sot the Kits useful forthe digunosis ofa patient suffering fro sepsis. Inthe present invention, the assay kt for human low ‘molecular-weight CD14 containing the above antibody and the assay method are provided. Furthermore, the novel diag- nostic method for sepsis in which human low-molecular weight CD14 is dircelly assayed is provided. Furthermore, the peptide useful in the preparation ofthe above antibody and the method of preparing the above antibody are prove segues LISTING 260» mowpea oF $09 1D nos: 18 “20> 689 19 mo 1US 7,465,547 B2 49 ~continued 50 soo suqumee: x HE Ms Pro Ol Bso Cpe GIA Leu Aap ep CLL Aap Phe Arg Cys Val cys ton the Ser Gia Peo Gln feo Aap Esp er Glu Ala Bho Gln Cyo Val Ger 44 Val Glu Val GlM He lke Ala Gly Gly Leu Asn Leu Olu ro the Leu Wye Aig Val Bap Ala Rap Ala Dep fro Aug Gln TYE ALA 210 £50 10 Wo 2 segue: 2 fg Val Aop Ala Aop Ala Aop Pro Arg Gin Tyr Ala Aop Thr val yo 210 589 10 0 3 soo» sgrimce: 3 ‘he The Pro Ghy Pro Gye atu teu Aap Aap Glu Aap Phe Arg Eve Val ove 210» sn9 10 #04 toos Segre: ¢ ug Gye Val oye Aon Phe Ser ai Pro Gin Pro Aap Trp Ser ai Ala Phe gin oye: toes sugumee: 5 he the Pro Glu Pre Cys Gly Lew Aap Rep Glu Rep Phe Arg cys Val cys Aen bhe Ser Glu Pro Gin Fro hop top Ser Glu Als Phe Gin GyeUS 7,465,547 B2 ~continued 52 an Val Pro Aa Gio teu Leu Val Oly Ala Le Aig Val Leu Ata 2yE fer hig Lau We Gia Leu The Leu OLY Rp Le Lye THe The Gly The eg Leg Aig Aan Vek Sor Tap Ala he Gly Ag Soe Txp Lau Ma Ot ‘Ala lie Ser Pro Ala Phe Ser Cyw Glu Gin Val Arg Ala Phe Pro Ala leu Thr Ser teu Rep teu Sex Rep Aap Pro Gly Lau Gty Giu Arg Sty leu Met Ala Ala beu Cye Pro Hie tye Phe Pro Ala Tie Gin Aen Leu 2a Una aeg Aan Te Oly Tie GLA The Feo THE Gly Val Oye Ala ALA Ser tow Arg Ala Thr Val Aon Pro Sor Ala Pro Arg cys Met Trp Ser Pro tye Giy teu Pro Ala Lye teu Arg Val teu Asp Leu sex Cys Aon Hig Gis Giy Sex Met Aan Ser Gly Val Val Pro Ala cys Ala Arg Ser 2102 589 10 0 ‘Arg Yal Aap Alo Aap Ala Aap #0 ‘210 549 10 80 7US 7,465,547 B2 ~continued ie so 1D we 400 suqumce: © 210 suo 10 ¥0 © foo suqumce: 9 210 su 1 ¥0 10 $00 segumice: 10 op Ala Aop Pro Arg Gin Tyr Ala 2102 949 10 wo 11 4000 sngrimce: 11 21> sto 1 80 12 ‘soos sugrimice: 12 dep Pro Arg Oln Tyr Ala Rep Thr 210» sto 1 80 13, Pro arg Gin Tyr Ala Asp Thr val toes sequimier: 14 dog @in tye Als ep the Val tyeUS 7,465,547 B2 56 ~continued 400 suqumiee: 15 <210> 560 1 ¥0 16 SU; okotma” ncestcsan saquene ‘2am OMER TipoRIRATON. oligomer 8 Link $00» segumice: 16 ctagnaatee ct £10 su 1 ¥0 17 ‘oo sugrimice: 17 <210> sg 1 ¥0 18 400» sgrimce: 18 “2109 549 10 ¥0 19 -s00> segumice: 19 cys Gis chy Aan Gly Aan Aan Phe cia Ser seg chu Ais cys The invention claimed is 1. A method of detecting human Jow-molecular-weight CDIA ina specimen, which comprises: contacting the specimen with: ss (@) an aatibody that binds 10 9 peptide consisting of the amino aid Sequence of SEQ ID No: and (6) an antibody that hinds to @ peptide consisting of the ‘amino acid sequence from position 17 to postion 26 of SEQID NOS; ‘wherein sail human low molecular weight CD 14: (1) is not bound by P102S-3-1 (Accession No. FERM 1BP-7296) antibody, (2) has a peak elution ta molecular weight range of 28 to 6s ‘45 kDa as detemnined by gel filtration chromatography. and (3) is obtainable from human blood; and detecting binding ‘of antibodies (a) and (b to said human low-molevular- ‘weight CD14, whereby said method can detect low- molecular-weight CD14 without detecting. high-mo- lecular-weight CD14. 2A method of det ding human low-molecular-weight CD14 without detecting humanhigh-molecular weight CD14 (6 whieh comprises: binding ssi human low-molecularsweight CD14 with () an antibody that binds to a peptide consisting of the ‘amino ail sequence of SEQ TD NO:2; and (©) an antibody that competes with an antibody which binds toa peptide consisting ofthe amino acid sequence from position 17 to position 26 of SEQ ID NOS;US 7,465,547 B2 37 ‘wherein said human low-moleculae weight CD14: (1) is not bound by F102S-3-1 (Accession No. FERM. 'BP.7296) antibody, (2) has peak of elation at a molecular Weight range of 25 0.45 kDa ona gl filtration chromatography, and @) is obtainable from human blood; and etocting binding of antibodies (a) and (b) to sad humaa Tow-tolecula-weight CD14, whereby'said method ean detect low-moleculanweight CD14 without detecting high-molecularaweight CD 1. 3. A metho! for diagnosing sepsis in a patient comprising the steps of: detecting human low-molecular weight CD14 in patient ‘ood by contacting patient blood with () an antibody that binds 10 @ peptide consisting of the amino acd sequence of SEQ ID NO:2; and (©) an antibody that binds to @ peptide eonssting of the ‘amino acid sequence from position 17 to postion 26 of SEQID NOS; ‘wherein sid human low-molocular-weight CD 14 (1) is not bound by F1025-3-1 (Accession No. FERM. 'BP-7296)autibody: 2) hase peak ofeation at a molecular weight range of 25 to 48 KDa as detemained by gel filration ehromatogra- phy, and (@) is obtainable from human blood ‘measuring in sid patient blood the amount of law-moleeu larweight CD14 bound to both of the above described antibody (@) and the above deseribed antibody (b), thereby determining the amount of human loe-molecu- larsveight CD14 in said patient bloods ‘comparing the measured amount of ow-molecular-weight (CD 14 in said patient blood to a standard amount of Jow-molecular-weight CD14 ina normal individual: and valuating whether the meusured amount of buna low= molecular wejght CD14 observed insaid patient bloods higher than te standard amount of human loe-moloeu- Jar weight CD14 observed in a normal individual 58 4.A method for diagnosing sepsis i a patient cx the steps of ‘detecting human low-moleculaeaveight CD14 in patient blood by contacting patient blood with a sandwich immunoassay kit, wherein said kit comprises: (@) an antibody that binds to a peptide consisting ofthe amino acid sequence of SEQ ID NO:2; and (@) an antibody that competes with an antibody which hinds to a peptide consisting of the amino acid sequence from position 17 to postion 26 of SEQ ID Noss: ‘wherein said human low-molecular- weight CDM: (1s not bound by F1025-3-1 (Accession No. FERM. 'BP-7296) antibody. (2) has peak of elution at molecular weight range of 25 to 45 kDa as determined by gel filtration chroma- tography, and (3) is obtainable from human blood. ‘measuring said patient blood the amount oflow-molecu Tae-weight CD14 bound to both of the above described antibody (a) and the above identtiod (b), thereby deter- mining the amount of human low-molecular-weight CDI in said patient lod: ‘comparing the measured amount oflow-molecular-weight CD 14 observed in said patient blood to a standant amount oflow-molecular-Weight CD14 present ina nor- nal individual and evaluating whether the measured amount of low-molect lar-weight CD14 observed in said patient blood is higher than the standard amount of low-moleculae-weight CD14 observed in a normal individ 5. The method for diagnosing sepsis according to claim 3. ‘wherein in sid comparing step, the average +28 of normal individuals is used as eutoft level, (6. The method aevording to claim 1, wherein said detecting binding of antibodies (a) and (b) to said human fow-moleew- Jar-weight CD14 is by sandwich immunoassay. "7 Themethod according wo elaim 2, wherein sad detecting binding of antibodies (a) and (b) to said han low-moloe- Jar-weight CD14 is by sandwich immunoassay. '8. Themethod according to elaim 3, wherein said detocting binding of antibodies (a and (b) to said human fow-molect- Jar-weight CD14 is by sandwich immunosssay uprising