WORKPLAN MIKROBIOLOGI HEMAGGLUTINATION INHIBITION TEST AND DENGUE BLOT

I.

TUJUAN To establish the presence of dengue virus infection in clinically suspect patients.

II. PENDAHULUAN Test to measure antibodies such as hemagglutination inhibition (HI), complement fixation (CF), and neutralization test exploit biological markers of dengue viruses. There are reliable tests for measuring antibodies, but they do not distinguish IgM from IgG. For this reason these test usually require paired samples (acute and convalescent) to make a diagnosis. More recent serologic techniques such as ELISA, fluorescent antibody and dot blot tests have been developed to measure antibody class individually. HI test has become the World Health Organization standard test for the serologic confirmation and serologic classification of dengue infection. The assay depends on the ability of antibodies to inhibit viral glycoprotein-dependent agglutination of goose or human type O red blood cells. Serum specimens for testing must be treated to remove non-spesific inhibitors of agglutination by acetone or kaolin extraction followed by red blood cell adsorption. The endpoint of the titration is the highest dilution of serum that inhibits agglutination of a standard amount of antigen. Four fold or greater changes in HI titer of paired serum are considered diagnostic for recent infection. Interpretation of the HI test is based on the titer and the time after onset of symptoms. In primary infections, detectable HI antibody generally appears after the fifth day and rises slowly over a period of weeks, the titer generally not exceeding 1/640. In the case of secondary or tertiary infections, there is an anamnestic response, which results in a rapid elevation of the titer within a few days of onset. Titers of 1/2560 to 1/20480 are frequently attained in convalescent samples and may persist for several weeks. Dengue blot (dot blot) tests, which do not require the equipment necessary for conventional serology, are simple and rapid. A limitation of these tests, however, is that they are not useful for testing large number of samples, since individual strips of paper or membrane must be handled for each serum tested. In addition they are expensive. The advantage of these tests is that they can be used to distinguish IgG from IgM and to measure titer of IgG antibodies semi quantitatively. Dot blot tests specific for anti dengueIgM have been modeled after IgM-capture ELISA in order to avoid the interfering effect of IgG antibody from previous infections. This can be accomplished by the use of nitrocellulose membrane coated with anti-human IgM antibodies.

A dot blot test for IgG antibody has potential to replace more cumbersome serologic test such as the HI and IgG ELISA tests. The problem with this type of test is the subjective nature of its quantification, since it requires a visual judgment of color intensity. At a dilution of 1:1000 the test is not sensitive enough to detect low tittered IgG antibody from previous infection, so single serum sample is sufficient for identification of recent infection.

III. METODE PERCOBAAN 3.1 AlatdanBahan 3.1.1 Hemagglutination inhibition test: 1) 96-well microtiter plate. 2) Micropipette. 3) Incubator. 4) Kaolin. 5) Serum. 6) Dengue antigen. 7) Goose erythrocyte. 3.1.2 Dengue blot: 1) Plate. 2) Towel paper. 3) Membrane with dot-blotted dengue antigen. 4) Conjugate. 5) Substrate. 6) Aquadest. 7) Washing buffer. 8) Diluent buffer. 3.2 Prosedur 3.2.1 Hemagglutination inhibition test: 1) Extract serum sample with kaolin to remove non-spesific inhibitor. 2) Absorb the sample with goose erythrocyte to remove agglutinin. 3) Make twofold dilution of the sample starting at 1/10 until 1/10240 dilution. 4) Put diluted sample into well nos. 1-11. Well no.12 is filled with serum diluted 1:1 and serves as a control. 5) Add 25 ul (4 units) of dengue antigen to each well. Incubate at 4oC overnight. 6) On the next day add 25 ul of goose erythrocyte suspended in VAD (pH 6,2). Incubate the plate at 36oC for 45 minutes.

7) Positive result will show intact erythrocyte accumulating of the bottom of the wells. 3.2.2 Dengue Blot: 1) Washing buffer preparation: 10 ml of 20 x concentration washing buffer + 190 ml aquadest (can be stored for a week). 2) Diluent buffer: 20 ml of washing buffer + 1 gram of non-fat skimmed milk. Mix well (should be freshly made). 3) Sample preparation: 5 ul of serum buffer + 500 ul of diluent buffer. 4) Put the membrane into the wells. 5) Fill the wells with 450 ul of diluent buffer. 6) Add 50 ul of diluted sample, cover the plate with paper, and stand it at room temperature for 60 minutes. 7) Suck the fluid from the wells, wash the membrane with washing buffer 3 times (3 minutes each time). 8) Conjugate preparation : 6 ul conjugate + 3 ml of diluent buffer. 9) Fill the wells with 250 ul of conjugate, incubate at room temperature for 60 minutes. 10) Take the fluid out of wells, wash the membrane with washing buffer as described above. 11) Substrate preparation: 1,5 ml of substrate A + 1,5 ml of substrate B (prepare it just before use). 12) Add 250 ul of substrate solution into the wells. 13) Cover the plate, let it stand at room temperature for 30 minutes. 14) Suck the fluid and add aquadest into the wells. 15) Examine the development of colored dot on the membrane.

WORKPLAN MIKROBIOLOGI UJI INHIBISI HEMAGGLUTINATION DAN DENGUE BLOT

I.

TUJUAN Untukmenentukanadanyainfeksi virus dengue secaraklinispadapasienterduga.

II. PENDAHULUAN Tesuntukmengukurantibodisepertihemaglutinasiinhibisi (HI), fiksasikomplemen (CF), danujinetralisasimengeksploitasitanda-tandabiologisdari virus dengue.Ada tes yang dapatdiandalkanuntukmengukurantibodi, tetapimerekatidakmembedakanIgMdariIgG.Untukalasaninitesinibiasanyamembutuhkans ampelberpasangan (akutdankonvalesen) untukmembuat diagnosis.Lebihteknikserologibaru-baruiniseperti ELISA, antibodi fluorescent dantes dot blot telahdikembangkanuntukmengukurkelasantibodisecara individual. HI testelahmenjaditesstandarOrganisasiKesehatanDuniauntukkonfirmasiserologisdanklasifi kasiserologiinfeksi dengue. Assay tergantungpadakemampuanantibodiuntukmenghambat virus tergantungglikoproteinaglutinasiangsaataujenismanusia O seldarahmerah. Spesimen serum untukpengujianharusdiperlakukanuntukmenghilangkan inhibitor nonspesifikaglutinasidenganasetonatauekstraksi kaolin diikutiolehadsorpsiseldarahmerah.Titikakhirtitrasiadalahpengencerantertinggi serum yang menghambataglutinasidarijumlahstandar antigen.Empatperubahan kali lipatataulebihdalam titer HI daripasangan serum dianggapdiagnostikuntukinfeksibaru. Interpretasites HI didasarkanpada titer danwaktusetelahtimbulnyagejala.Padainfeksi primer, antibodi HI terdeteksiumumnyamunculsetelahharikelimadannaikperlahanlahanselamabeberapaminggu, titer umumnyatidakmelebihi 1/640.Dalamkasusinfeksisekunderatautersier, adarespon anamnestic, yang menghasilkanelevasicepat titer dalamwaktubeberapaharidari onset.Titer 1/2560 to 1/20480 seringdicapaidalamsampelsembuhdandapatbertahanselamabeberapaminggu. Dengue blot (dot blot) tes, yang tidakmemerlukanperalatan yang diperlukanuntukserologikonvensional, sederhanadancepat. Keterbatasantesini, bagaimanapun, adalahbahwamerekatidakbergunauntukmengujisejumlahbesarsampel, karena strip individukertasataumembranharusditanganiuntuksetiap serum diuji.Selainitumerekamahal.Keuntungandaritesiniadalahbahwamerekadapatdigunakanun tukmembedakanIgGdariIgMdanuntukmengukur titer antibodiIgGsecara semi kuantitatif. Dot blot teskhususuntuk anti dengue IgMtelahdimodelkansetelahIgM-capture ELISA untukmenghindariefekmenggangguantibodiIgGdariinfeksisebelumnya.Hal

inidapatdicapaidenganmenggunakanmembrannitroselulosadilapisidenganantibodiIgM anti-manusia. Sebuahtes dot blot IgGantibodimemilikipotensiuntukmenggantikantesserologilebihrumitsepertites HI danIgG ELISA. Masalahdenganjenistesadalahsifatsubjektifdarikuantifikasinya, karenamemerlukanpenilaian visual intensitaswarna.Padapengenceran 1:1000 tesinitidakcukupsensitifuntukmendeteksiantibodiIgGterkikikrendahdariinfeksisebelumny a, sehinggasampel serum tunggalsudahcukupuntukidentifikasiinfeksibaru. III. METODE PERCOBAAN 3.1 AlatdanBahan 3.1.1 Hemagglutination inhibition test: 1) 96-well microtiter plate. 2) Micropipette. 3) Inkubator. 4) Kaolin. 5) Serum. 6) Dengue antigen. 7) Goose eritrosit. 3.1.2 Dengue blot: 1) Piring. 2) Kertas towel. 3) Membrane dengan dot-blotted dengue antigen. 4) Conjugate. 5) Substrate. 6) Aquadest. 7) Bufferpencuci. 8) Bufferpengencer. 3.2 Prosedur 3.2.1 Hemagglutination inhibition test: 1) EkstraLLksampel serum dengan kaolin untukmenghapus non-spesifik inhibitor. 2) Menyerapsampeldengangooseeritrosituntukmenghapus agglutinin. 3) Buatlahpengencerandua kali lipatdarisampelmulaidari 1/10 sampai 1/10240 pengenceran. 4) MasukkansampeldiencerkankeWell no. 1-11. Well no.12 diisidengan serum diencerkan 1:1 danberfungsisebagaikontrol. 5) Tambahkan 25 ul (4 unit) dari antigen dengue padasetiap Well. Inkubasipadasuhu 4oC semalam.

6) Padahariberikutnyatambahkan 25 ulgoose eritrosittergantung di VAD (pH 6,2). Inkubasipiring di 36oC selama 45 menit. 7) Hasilpositifakanmenunjukkaneritrositutuhterakumulasidaribagianb awahWell. 3.2.2 Dengue Blot: 1) Persiapanbuffer pencuci : 10 ml dari 20 x konsentrasicucibuffer + 190 ml aquadest (dapatdisimpanselamaseminggu). 2) Buffer pengencer: 20 ml buffer pencuci+ 1 gram non - lemaksususkim .Aduk rata (harusbarudibuat). 3) Preparasisampel : 5 ul serum buffer + 500 ulbuffer pengencer. 4) MasukkanmembrankedalamWell. 5) Isi Well dengan 450 ulbuffer pengencer. 6) Tambahkan 50 ulsampeldiencerkan, tutupipiringdengankertas, dantempatkanpadasuhukamarselama 60 menit. 7) SedotcairandariWell, mencucimembrandenganbuffer pencuci3 kali (3 menitsetiap kali). 8) PersiapanConjugate : 6 ulkonjugat + 3 ml buffer pengencer. 9) Isi Well dengan 250 ulkonjugat, inkubasipadasuhukamarselama 60 menit. 10) AmbilcairankeluardariWell, mencucimembrandenganbuffer pencucisepertidijelaskan di atas. 11) PersiapanSubstrat: 1,5 ml substrat A + 1,5 ml substrat B (mempersiapkansebelumdigunakan). 12) Tambahkan 250 ullarutansubstratkedalamWell. 13) Tutuppiring, diamkanpadasuhukamarselama 30 menit. 14) MengisapcairandanmenambahkanaquadestkedalamWell. 15) Periksapengembangan dot berwarnapadamembran.