VIROLOGY VIROLOGY MANUAL OF THE DEPARTMENT OF MANUAL OF THE DEPARTMENT OF

NEUROVIROLOGY, NIMHANS, BANGALORE NEUROVIROLOGY, NIMHANS, BANGALORE

560029 560029
OF OF
INFECTIONS OF THE NERVOUS INFECTIONS OF THE NERVOUS
SYSTEM SYSTEM
Dr.V.Ravi, Dr.Anita Desai and Dr.S.N.Madhusudana
Department of Neurovirology
TECHNIQUES
Cell cultures
Virus growth and titration
Methods for viral antigen detection
Methods for viral antiody detection
I CELL CULTURE TECHNIQUES
Subcu!u"# $% & c# '(#
1. Aedes alopictus C6/36 ( mosquito gut epithelium ) cell line
. !isc"#$ supe#n"t"nt %lui$ %#om " con%luent monol"&e# o% C6/36 ('e$es
"l(opictus ) (ottle cultu#es.
3. '$$ 3 to ) ml t#&psin (*#e+,"#me$ "t 3- C ) to e"ch cultu#e (ottle.
.. /e"0e the (ottle "t #oom tempe#"tu#e %o# 3+) mins.
). !isc"#$ the t#&psin.
6. '$$ )ml o% g#o,th me$ium to the (ottle "n$ gentl& $et"ch the cells %#om the
su#%"ce ,ith the help o% " pipette.
-. '$$ the #equi#e$ "mount o% me$ium (split #"tio %o# this cell line is 11 )) "n$
$ist#i(ute the $ilute$ cell suspension to %#esh %l"s2s/(ottles.
3. Incu("te "t #oom tempe#"tu#e %o# 3+- $"&s.
P"')&"* c# cu!u"# !#c+(',u#
C+'c- #)b"*$ %'b"$b&.!'c cu!u"# /
The $e0elopment o% #outine cell cultu#e metho$s h"s #e$uce$ the impo#t"nce o% eggs
(ut the& "#e still 0"lu"(le %o# the isol"tion o% m"n& impo#t"nt 0i#uses "n$ %o# the
p#o$uction o% 0"ccines. 4e#tile eggs must (e o(t"ine$ i$e"ll& %#om " speci%ic p"thogen
%#ee %loc25 shoul$ (e cle"n5 p#e%e#"(l& un,"she$ "n$ p"le shelle$ to simpli%&
c"n$ling. '%te# l"&ing the& h"0e to (e incu("te$ %o# 16 $"&s "t 3- C 7ith .6+-68
humi$it& "n$ goo$ "e#"tion "n$ tu#ne$ t,ice $"il&. '%te# 6 $"&s the& "#e c"n$le$5
in%e#tile "n$ $e"$ eggs "#e $isc"#$e$. 9n the $"& o% cultu#e those ,ith s"tis%"cto#&
$e0elopment o% cho#io"ll"ntioic (loo$ 0essels "n$ sho,ing em(#&onic mo0ement "#e
m"#2e$ ,ith pencil to in$ic"te the limits o% the "i# s"c.
M&!#"'&. "#,u'"#0
1. Em(#&on"te$ eggs p#e%e#"(l& 16+1 $"&s ol$.
. *hosph"te (u%%e# s"line (pH -.) .
3. 6.)8 t#&psin in *:S.
.. ;#o,th me$ium (<E<5 supplemente$ ,ith 168 (o0ine se#um)
). 4o#ceps5 scisso#s5 egg cup5 pet#i $ishes5 %ilt#"tion unit5 silicone/te%lon
6. Co"te$ m"gnet5 solution (ottles "n$ cultu#e %l"s2s.
P"$c#0u"#/
1. C"n$le "n$ select 16+11 $"& ol$ eggs.
. *l"ce the egg in "n egg cup5 "i# s"c up,"#$s "n$ ,ipe cle"n ,ith spi#it.
3. :#e"2 the shell ,ith the sh"#p en$ o% " ste#ile %o#ceps5 "n$ li%t the mem(#"ne.
7ith " (ent %o#ceps pic2 up the em(#&o "n$ pl"ce it in " pet#i $ish cont"ining
*:S.
.. !issect the em(#&o in the $ish5 #emo0e "n$ $isc"#$ the he"$5 lim(s "n$ 0isce#".
*ic2 up the %i(#o(l"stic tissue "n$ t#"ns%e# into " ,i$e nec2 (ottle cont"ining
*:S.
). <ince the tissue %inel& ,ith scisso#s5 "n$ ,"sh mince$ tissue se0e#"l times in
*:S to #emo0e (loo$ cells "n$ $e(#is.
6. T#"ns%e# tissue to t#"ns%usion (ottle cont"ining ste#ile silicone co0e#e$ m"gnet "n$
)6ml o% 6.) 8 t#&psin solution "n$ stoppe# secu#el&.
-. T#&psini=e on m"gnetic sti##e# unit 3- C %o# 36 mins 1 "0oi$ %#othing o%
contents.
3. The tissue ,ill $isinteg#"te5 %o#ming " tu#(i$ suspension o% cells. 4ilte# the
suspension th#ough ste#ile g"u=e "n$ cent#i%uge %ilt#"te %o# 16min "t 1666 #e0/min.
>. !isc"#$ supe#n"t"nt "n$ #esuspen$ cells in 166 ml g#o,th me$ium.
16. Cent#i%uge once "g"in "n$ #esuspen$ the cells in %#esh me$ium.
11. !ilute 6.>ml suspension ,ith 6.1ml t#&p"n (lue solution "n$ count cells in
h"emoc&tomete#.
1. '$?ust concent#"tion to 1 16 cells / ml g#o,th me$ium "n$ $ispense into tu(es
o# (ottles.
13. Incu("te "t 3- C until monol"&e# is %o#me$ (+3 $"&s).
1.. 7hen cells h"0e %o#me$ " monol"&e#5 #emo0e g#o,th me$ium5 inocul"te 0i#us5 "$$
m"inten"nce me$ium (<E<5 E"gles ("se ,ith 1+8 (o0ine se#um) "n$ incu("te.
M#0'& 1"#1&"&!'$( %$" c# cu!u"#
D#+*0"&!#0 T'..u# Cu!u"# M#0'&
1. T"2e >66ml o% t#iple $istille$ ,"te#.
. '$$ the contents o% one unit 0i"l o% $eh&$#"te$ me$i" to the ,"te# "t #oom
tempe#"tu#e ,ith sti##ing until $issol0e$.
3. @inse the 0i"l ,ith " sm"ll "mount o% t#iple $istille$ ,"te# to #emo0e t#"ces o%
po,$e# "n$ "$$ to the "(o0e solution.
.. '$$ . g#"ms o% so$ium (ic"#(on"te o# HE*ES :u%%e#.
). '$?ust the pH i% #equi#e$ (et,een -.1 to -.. using 1N HC1 o# 1N N"9H o# (&
(u((ling c"#(on $ioAi$e. Note th"t pH ten$s to #ise $u#ing %ilt#"tion "n$ hence
"$?ust it 6. to 6.3 units (elo, the %in"l $esi#e$ pH.
6. <"2e up %in"l 0olume to 1666ml ,ith t#iple $istille$ ,"te#.
S!#"''2&!'$( $% !'..u# cu!u"# M#0'&
1. Ste#ili=e the me$i" (& %ilte#ing th#ough ste#ile mem(#"ne %ilte#(ste#ili=e$ (&
"utocl"0ing "t 1) l(s %o# 1) min B 11 C ) o% 6. mic#on o# less po#osit& using
positi0e p#essu#e to minimise loss o% c"#(on $ioAi$e.
A(!'b'$!'c.
. The %ollo,ing "nti(iotics c"n (e "septic"ll& "$$e$ to lit#e o% me$i" 1
3. 'mphote#icin .) mg
.. ;ent"m&cin ()6mg/ml. Solution) 1.6 ml
). :en=&l *enicillin 16666 units
6. St#eptom&cin 166 mg
S!#"''!* c+#c-
'$$ 6.) + 1.6 ml o% %ilte#e$ me$i" to " tu(e cont"ining ste#ile thiogl&col"te (#oth
"n$ incu("te "t 3- C %o# .3 hou#s. I% the (#oth is cle"# "%te# .3 hou#s the me$i" is
ste#ile.
TVG 3T"*1.'( V#".#(# Guc$.#4
1. TC; consists o% the %ollo,ing components in 1D *:S1
. T#&psin 6.18
3. Ce#sene 6.8
.. ;lucose 6.6)8
S!$c- .$u!'$(. %$" TVG
1). 16A *:S
N"Cl 36.66 gms
ECl .66 gms
N"H *6. 1... gms
EH *9. .66 gms
!ist.H9 upto 1 lit#e
To p#ep"#e 1D *:S + "$$ 166 ml o% 1D *:S to >66 ml o% !/7.
) 8 T@F*SIN
T#&psin .66 gms
!/7 upto 166ml
Sti# the "(o0e solution on " m"gnetic sti##e# %o# .h o# 9/N "t .C. Ste#ili=e (&
%ilte#ing th#ough ste#ile mem(#"ne %ilte# o% 6.u po#e si=e. ' ste#ilit& chec2 c"n (e
$one (e%o#e using the solution.
3) 6. 8 CE@SENE
E!T' 66 mg
!/7 166 ml
Ste#ili=e (& "utocl"0ing "t 1) l(s B 11C %o# 1) minutes.
1) 16 8 ;/UC9SE
;lucose 16.66 gms
!/7 upto 166 ml
Ste#ili=e (& "utocl"0ing "t 1) l(s B 11 C %o# 1) minutes.
) 1 8 *HEN9/ @E!
*henol @e$ 1.66 gm
!/7 upto 166ml
P"#1&"&!'$( $% 5$"-'(6 .$u!'$( $% TVG
*#ep"#e 3.6ml o% 1D *:S "n$ to this "$$ 1.6 ml o% 18 phenol #e$ (in$ic"to#).
Ste#ili=e (& "utocl"0ing "t 1)l(s "n$ 11C %o# 1) minutes. Cool this ste#ile solution
"n$ then "$$ the %ollo,ing ste#ili=e$ solutions to it 1
T#&psin .68 )6 ml
Ce#sene 6.8 166 ml
;lucose 16.68 ) ml
!o " ste#ilit& test "s mentione$ "(o0e (e%o#e using this TC;.
II 7ASHING OF GLASS7ARE FOR TISSUE CULTURE 7OR8
1) 'utocl"0e "ll gl"ss,"#e (e%o#e ,"shing5 "t 16C5 1)psi %o# 6min.
) The outsi$e su#%"ce o% gl"ss,"#e c"n (e cle"ne$ ,ith Cim "n$ insi$e ,ith onl&
Teepol "n$ sc#u((e$ ,ith " cle"n (#ush 5 ,hich is onl& me"nt %o# (#ushing
gl"ss,"#e. 7"sh in t"p ,"te# "n$ le"0e in )8 Teepol solution o0e#night (c"n (e
(oile$)
3) NeAt mo#ning ,"sh in t"p ,"te# "t le"st 6 times "n$ le"0e in 168 HC1
o0e#night.
.) NeAt $"& ,"sh in t"p ,"te# "n$ #inse in ch"nges o% $emine#"lise$ $istille$
,"te# "t le"st 16 times in e"ch5 le"0e in thi#$ (uc2et o% $emine#"lise$ $istille$
,"te# o0e#night.
)) T"2e out neAt $"&5 $#& it in hot "i# o0en5 p"c2 "n$ ste#ili=e.
P&c-'(6 &(0 .!#"''2&!'$(
*ipettes1 7#"p the pipette ,ith (#o,n p"pe# "n$ ste#ili=e in "
hot "i# o0en "t 166C %o# t,o hou#s.
*et#i$ishes 1 7#"p the *et#i $ishes in (#o,n p"pe# "n$ tie ,ith
T,ine "n$ ste#ili=e (& hot "i# o0en "t 166C %o#
t,o hou#s.
Cultu#e B me$i" sto#"ge (ottles5 me"su#ing c&lin$e#s "n$ (e"2e#s 1
Co0e# mouth ,ith "luminum %oil5 o0e# ,hich (#o,n p"pe# is
tie$ ,ith t,ine "t the nec2 "n$ ste#ili=e in " hot
o0en "t 166C. *l"stic me"su#ing c&lin$e#s "n$ (e"2e#s
"#e "utocl"0e$ "t 16C5 psi %o# 36min.
*l"stic cent#i%uge tu(es5 Eppen$o#% tu(es5 sc#e, c"p 0i"ls B tu(es5 1
'##"nge ne"tl& the %ollo,ing items in " pl"stic o#
gl"ss (e"2e# "n$ co0e# the mouth ,ith "luminum %oil "n$
o0e# th"t ,#"p (#o,n p"pe# tie$ ,ith t,ine "t the nec2.
Ste#ili=e (& "utocl"0ing "t 16C5 1) psi %o# 36min.
Co0e#slips1 Co0e#slips "#e put into " *et#i $ish 5 ,hich is co0e#e$ ,ith (#o,n
p"pe# "n$ tie$ ,ith t,ine. Ste#ili=e in " hot "i# o0en "t 166C %o#
t,o hou#s.
4ilte# 'pp"#"tus1 7#"p %ilte# "pp"#"tus %i#st ,ith "luminum %oil "n$ then ,ith (#o,n
p"pe#5 tie it ,ith t,ine tightl&. Ste#ili=e (& "utocl"0ing "t 16 C5
1) psi %o# 36min.
Solutions1 *lug the mouth o% the cont"ine# ,ith cotton5 ,#"p it ,ith (#o,n
p"pe# "n$ tie it ,ith t,ine. Ste#ili=e (& "utocl"0ing "t 11 C 5 1)
psi %o# 6 min.

I I I DEMONSTRATION OF CYTOPATHIC EFFECT 3 CPE 4
1) HSC + !emonst#"tion o% c&top"thic e%%ect (C*E) in Ce#o cell line.
) ;#o, Ce#o cells to con%luence in Nunc %l"s2s / . ,ell $ishes.
3) !isc"#$ the g#o,th me$ium.
.) In%ect the cell line ,ith HSC+1 suspension "t "n <9I (multiplicit& o% in%ection)
o% 1.
)) '$so#( the 0i#us %o# 36 minutes "t 3- C .
6) '$$ the m"inten"nce me$ium + )ml %o# Nunc %l"s2/16+1)ml %o# <il2 $ilution
(ottle/166ml %o# " @ouA (ottle.
-) Incu("te the (ottles "t 3-C in C9 incu("to#.
3) 9(se#0e $"il& %o# C*E (oth in the mo#ning "n$ in the e0ening.
>) HSC p#o$uces C*E (& .+.3 h. T,o %o#ms o% C*E "#e o(se#0e$. The most
common (egins ,ith c&topl"smic g#"nul"tion "%te# ,hich the cells (ecome
enl"#ge$ o# ("lloone$. These m"c#oc&tes then (ecome #oun$e$5 t"2e on "
#e%#"ctile "ppe"#"nce5 "n$ un$e#go l&tic $egene#"tion. The secon$ t&pe o% C*E is
%o#m"tion o% multinucle"te$ gi"nt cells.
M#&.#. 9'"u. / D#)$(.!"&!'$( $% CPE '( V#"$ c# '(#
1) ;#o, Ce#o cells to %o#m " monol"&e# in Nunc %l"s2s/ <il2 $ilution (ottles/@ouA (ottle.
) !isc"#$ the g#o,th me$ium.
3) In%ect the cell line ,ith me"sles 0i#us suspension "t "n <9I(multiplicit& o% in%ection) o% 1.
.) '$so#( the 0i#us %o# 36 minutes "t 3- C.
)) '$$ the m"inten"nce me$ium + )ml %o# Nunc %l"s2/16+1)ml %o# <il2 $ilution
(ottle/166ml %o# @ouA (ottle.
6) Incu("te (ottles "t 3-C in C9 incu("to#.
-) 9(se#0e $"il& %o# C*E (oth in the mo#ning "n$ in the e0ening.
3) <e"sles 0i#us p#o$uces C*E "%te# .+) $"&s. T,o t&pes o% C*E "#e $esc#i(e$. 9ne t&pe
seen "%te# in%ection ,ith ,il$ 0i#us "n$ $ilute inoccul" is gi"nt cell t#"ns%o#m"tion.
<ultinucle"te$ gi"nt cells cont"ining 16+166 nuclei %o#m "s " #esult o% cell %usion
in$uce$ (& 0i#us. The secon$ t&pe o% C*E5 spin$le cell %o#m"tion5 is "ssoci"te$ ,ith
me"sles 0i#us 5 ,hich h"s (een p"sse$ #epe"te$l& in tissue cultu#e5 o# ,ith #epe"te$ p"ss"ge
o% un$ilute$ inoccul".
:&1&(#.# E(c#1+&'!'. 9'"u. / D#)$(.!"&!'$( $% CPE '( 1$"c'(# .!&b# -'0(#* c# '(#
3PS4
1) ;#o, *S cells to %o#m " monol"&e# in Nunc %l"s2s/<il2 $ilution (ottles/@ouA (ottle.
) !isc"#$ the g#o,th me$ium.
3) In%ect the cell line ,ith me"sles 0i#us suspension "t "n <9I (multiplicit& o% in%ection)
o% 1.
.) '$so#( the 0i#us %o# 36 minutes "t 3- C.
)) '$$ the m"inten"nce me$ium + )ml %o# Nunc %l"s2/16+1)ml %o# <il2 $ilution
(ottle/166ml %o# " @ouA (ottle.
6) Incu("te the (ottles "t 3- C in C9 incu("to#.
-) 9(se#0e $"il& %o# C*E (oth in the mo#ning "n$ in the e0ening.
3) GEC p#o$uces C*E "%te# 13+6 h#s. C*E is seen in the %o#m o% #oun$ing o% cells
%ollo,e$ (& cell l&sis.
IV VIRUS TITRATION
T'!"&!'$( $% R&b'#. 9'"u. in 0i0o b* '(!"&c#"#b"& '($cu&!'$(
'%te# " 0i#us is p#opog"te$ in eithe# cell cultu#e o# in " suit"(le "nim"l5 ,e nee$
to 2no, the in%ecti0it& tit#e o% the 0i#us m"te#i"l o(t"ine$. This c"n (e $ete#mine$ in
0i0o (& inocul"ting inc#e"sing $ilutions o% the 0i#us m"te#i"l to " suscepti(le host
"nim"l such "s l"(o#"to#& mice "n$ ("se$ on mo#t"lit& seen in $i%%e#ent $ilutions5 the
in%ecti0it& tit#e ,hich is the #ecip#oc"l o% highest $ilution sho,ing )68 mo#t"lit& in
the inocul"te$ mice "n$ eAp#esse$ "s /!
)6
/ml "n$ c"n c"lcul"te$ (& suing eithe#
@ee$+<uench o# E"#(e# %o#mul". 's "n eA"mple5 tit#"tion o% #"(ies 0i#us is illust#"te$
in this section.
M&!#"'&. /
1. <ic#o(iologic"l s"%et& c"(inet.
. Ste#ile *ipettes5 16ml "n$ 1ml
3. ' t#"& ,ith ice %l"s2s. @"(ies 0i#us suspension (cell cultu#e supe#n"t"nt o%
in%ecte$ (#"in emulsion)
.. <ice (S,iss "l(ino o# /"2") .+6 ,ee2s ol$
). 9ne ml s&#inge "n$ nee$le %o# I/c inocul"tion
6. Test tu(es.
-. Ste#ile *:S pH -..
3. Ste#ile (o0ine o# equine se#um
>. C"ges %o# housing mice.
P"$c#0u"#/
1. 7o#2ing in mic#o(iologic"l s"%et& c"(inet5 p#ep"#e the $iluting %lui$ ,hich is *:S
cont"ining 8 se#um "n$ $ispense >ml in test tu(es l"(elle$ 16
+1
to 16
+-
"n$ 2eep the
test tu(es in #"c2 imme#se$ in plent& o% ice.
. To m"2e 16 %ol$ (log) $ilutions o% the 0i#us m"te#i"l5 $ilute 1ml o% 0i#us in >ml o%
$iluent to get the initi"l $ilution i.e. 16
+1.
Su(sequentl& t#"ns%e# 1ml o% p#e0ious 0i#us
$ilution to neAt $ilution (& using "t e"ch step " %#esh pipette5 to "chie0e se#i"l
ten%ol$ $ilutions.
3.Inocult"te 6.63 ml o% e"ch 0i#us $ilution int#"ce#e(#"ll& into mice5 st"#ting %#om the
highest $ilution (in this c"se 16
+-).
Use "t le"st 6 mice pe# $ilution "n$ t#"ns%e# these
into c"ges "pp#op#i"tel& l"(elle$.
..9(se#0e the mice %o# 1. $"&s. 'n& $e"th occu##ing ,ithin %i#st ) $"&s shoul$ (e
consi$e#e$ non+speci%ic. 9(se#0e %o# speci%ic signs "n$ s&mptoms o% #"(ies i.e.5 #u%%ling
o% h"i#5 incoo#$in"tion5 t#emo#s "n$ p"#"l&sis o% hin$ "n$ %o#e lim(s "n$ %in"ll&
$e"th. Note $o,n tot"l num(e# o% speci%ic $e"ths in e"ch $ilution "n$ c"lcul"te the
0i#us tit#e (& using @ee$+<uench o# E"#(e# %o#mul".
M'c"$!'!"&!'$( $% P$'$9'"u. u.'(6 V#"$ c# '(#
<ost o% the commonl& encounte#e$ hum"n 0i#uses p#o$uce ch"#"cte#istic c&top"thic
e%%ect in one o# the othe# cell lines #outinel& use$ in 0i#olog& l"(o#"to#ies. The
in%ecti0it& tit#es o% these 0i#uses c"n con0enientl& (e $ete#mine$ (& in%ecting "
p"#ticul"# cell line ,ith inc#e"sing $ilutions o% the 0i#us m"te#i"l "n$ $ete#mining the
highest $ilution p#o$ucing c&top"thic e%%ect in )68 o% the inocul"te$ cells. The )68
en$ point $ilution ,hich in this c"se is eAp#esse$ "s TCI! )6/ml c"n (e c"lcul"te$
(& using eithe# @ee$+<uench o# E"#(e# %o#mul"e. 's "n eA"mple5 tit#"tion o% polio
0i#us I is illust#"te$ in this section.
M&!#"'&. /
1) <ic#o(iologic"l s"%et& c"(inet
) Ste#ile test tu(es o# *encillin 0i"ls
3) <ic#o pippettes5 66 B 1666 mic#ons
.) Co incu("to#.
)) >6 ,ell %l"t (ottom tissue cultu#e pl"tes(Nunc)
6) <inimum Essenti"l <e$ium(<E<5Sigm"5HI) supplemente$ ,ith "nti(iotics
-) 4et"l c"l% se#um(4CS)
3) T#&psin 0e#sene glucose coln.(TC;)
>) CE@9 cells.
16) 4lui$ cont"ining *I.
11) In0e#te$ mic#oscope
P"$c#0u"# /
1.<"2e 16 %ol$ se#i"l $ilutions5 s"& %#om 16
+3
to 16
+-
o% the 0i#us m"te#i"l in <E<
cont"ining 8 4CS5 $ispense$ in eithe# test tu(es o# penicillin 0i"ls5 ch"nging pipettes
o# tips ,ith e"ch $ilution. Eeep the tu(es in #"c2 imme#se$ in ice.
. T#"ns%e# 166 ul o% e"ch $ilution to . ,ells o% the mic#o+tit#e pl"te5 st"#ting %#om
highest $ilution to the lo,est.
3. T#&psinise one <! (ottle cont"ining " con%luent monol"&e# o% Ce#o cells "n$
count the cells "n$ $ilute to .A 16
)
cells pe# ml. In <E< cont"ining 168 4CS.
!ispense 166 ul o% cell suspension in to e"ch o% the ,ell cont"ining 0i#us $ilution
"n$ "lso inclu$e . ,ells "s cell cont#ol in ,hich 166Ul o% cell suspension is
miAe$ ,ith 166 U/ o% <E< ,ith 8 4CS. 7hile $ispensing the 0i#us $ilutions
"n$ cell suspension it is necess"#& to 2eep the pl"te on ice t#"&.
.. Co0e# the pl"te "n$ 2eep it in C9 incu("to# "n$ "$?ust the tempe#"tu#e to 3- C.
). @e"$ the pl"te un$e# "n in0e#te$ mic#oscope "%te# 3 to . $"&s ,hen " con%luent
monol"&e# o% Ce#o cells c"n (e seen in cont#ol ,ells. /oo2 %o# the c&top"thic
e%%ect in the ,ells inocul"te$ ,ith 0i#us $ilutions. This consists o% #oun$ing o%
cells5 i.e. i% o% . ,ells inocul"te$ ,ith 16
+3
$ilution sho,s c&top"thic e%%ect then
the tit#e is 16
+3
pe# 6.1 ml. The tit#e c"n "lso (e c"lcul"te$ (& the metho$ o%
@ee$ "n$ <uench.
C&cu&!'$( $% 50; #(01$'(!.
In "n& (iologic"l qu"ntit"tion5 the most $esi#"(le en$point is one #ep#esenting "
situ"tion in ,hich h"l% o% the inocul"te$ "nim"ls o# cells sho, the #e"ction ($e"th in
the c"se o% "nim"ls "n$ in C*E c"se o% cells) "n$ the othe# h"l% $o not. In othe#
,o#$s5 the en$point is t"2en "s the highest $ilution o% the (iologic"l m"te#i"l 5 ,hich
p#o$uces $esi#e$ #e"ction in )68 o% the "nim"ls o# cells. The )68 en$point c"n (e
("se$ on se0e#"l t&pes o% #e"ctions. The most ,i$el& use$ en$point5 ("se$ on
mo#t"lit&5 is the /!
)6
()68 leth"l $ose). This te#minolog& c"n "lso (e "pplie$ to
othe# host s&stems+%o# eA"mple5 tissue cultu#es + in ,hich the TCI!
)6
#ep#esents the
$ose th"t gi0es #ise to c&top"thic e%%ect in )68 o% inocul"te$ cultu#es. 7hen
computing5 i% closel&+pl"ce$ $ilutions "#e use$ "n$ in e"ch $ilution l"#ge num(e# o%
"nim"ls o# cells "#e use$5 it m"& (e possi(le to inte#pol"te " co##ect )68 en$ point
$ilution5 (ut it is neithe# p#"ctic"l no# economic"l. @ee$ "n$ <uench $e0ise$ "
simple metho$ %o# estim"tion o% )68 en$points ("se$ on the l"#ge tot"l num(e# o%
"nim"ls5 ,hich gi0es the e%%ect o% using "t the c#itic"l $ilutions (et,een ,hich the
en$point lies5 l"#ge# g#oups o% "nim"ls th"n ,e#e "ctu"ll& inclu$e$ in these $ilutions.
C&cu&!'$( $% !+# LD 50 !'!"# b* !+# R##0<Mu#(c+ )#!+$0
/et us p#esume th"t ,e h"0e tit#"te$ " 0i#us suspension (& inocul"ting mice "n$
$e"th is the #e"ction. The num(e# o% $e"ths "n$ su#0i0"ls in e"ch $ilution is
t"(ul"te$ "s gi0en in t"(le I (elo,1
T A B L E I
A""&(6#)#(! $% 0&!& u.#0 '( c$)1u!&!'$( $% LD
50
!'!"# b* !+# )#!+$0 $% R##0<
Mu#(c+
===========================================================================
ACCUMULATED VALUES
=======================================
MORTALITY
===========================
VIRUS DIED SURVIVIED DIED SURVIVED RATIO
;
DILUTION
===========================================================================
16
+3
6 6 1- 6 1-/1- 166


16
+.
6 6 11 6 11/11 166

16
+)
. ) )/- -1

16
+6
1 ) 1 - 1/3 13

16
+-
6 6 6 13 6/13 6
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
'ccumul"te$ 0"lues %o# the tot"l num(e# o% "nim"ls th"t $ies o# su#0i0"l "#e o(t"ine$
(& "$$ing in the $i#ection o% lo,est to the highest 0"lues. The "ccumul"te$ mo#t"lit&
#"tios "n$ the pe#cent"ge mo#t"lit& %o# e"ch $ilution is c"lcul"te$.
In the eA"mple $epicte$ in the t"(le it c"n (e seen th"t mo#t"lit& in the 16
+)
5 is
highe# th"n )68 "n$ in the neAt highe# $ilution5 16
+6
it is onl& 138. So5 ,e nee$ to %in$
the )68 en$point $ilution 5 ,hich o(0iousl& lies (et,een these $ilutions. 4i#st5 ,e h"0e
to c"lcul"te the p#opo#tion"te $ist"nce (*!) o% the )68 en$point %#om these $ilutions
(& using " simple %o#mul".
*@9*9@TI9N'TE
!IST'NCE I (8 mo#t"lit& "t $ilution neAt "(o0e )68) + )68
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
(8 mo#t"lit& "t neAt
HH
(8 mo#t"lit& "t neAt
$ilution "(o0e )68) $ilution (elo, )68)
ED. 1 (-1 + )6) 1
++++++++++++ I +++++ I 6.36 o# 6..
(-1 + 13) )3
The p#opo#tion"te $ist"nce o(t"ine$ thus h"s to (e co##ecte$ (& the $ilution %"cto#5
,hich is the log"#ithm o% the $ilution step emplo&e$ 5 ,hich in this c"se is 1 (log o%
16). Then the )68 en$point $ilution c"n (e c"lcul"te$ thus1
Neg"ti0e log"#ithm o% /!
)6
tit#e I (neg"ti0e log"#ithm o% the neAt $ilution
"(o0e )68 mo#t"lit& J *!) A $ilution %"cto# i.e. in this eA"mple5 (+) J 6..) A 1 I
+).. o# log /!
)6
tit#e I 16
+)..
pe# 6.63 ml ,hich is the "mount o% m"te#i"l inocul"te$
I/C. The s"me metho$olog& c"n "lso (e "pplie$ to compute TCI!
)6
,hen cell cultu#e
s&stem is use$ %o# 0i#us tit#"tion. E"ch $ilution is inocul"te$ into " minimum o% . to
67ells in " >6 ,ell tissue cultu#e pl"te "n$ o(se#0e$ %o# C*E 5 ,hich in this c"se
#epl"ces mo#t"lit&.
V'"u. !'!"&!'$( b* u.'(6 !+# 1&,u# &..&*
1. *#ep"#e con%luent monol"&e#s o% cells (*o#cine 2i$ne& %o# GEC "n$ Hep+ %o#
*olio 0i#us) in . ,ell pl"tes (Nunc).
. *#ep"#e se#i"l 16+%ol$ $ilutions (16
1
to 16
-
) o% 0i#us in chille$ m"inten"nce
me$ium (<E<5 ,ith 18 se#um).
3. @emo0e cultu#e me$ium "n$ "$$ 6.ml o% 0i#us inoculum5 st"#ting %#om the
highest $ilution. Ensu#e th"t " %ilm o% me$ium completel& co0e#s the cell sheet.
.. Incu("te the pl"te "t 3- C %o# 1 hou# ,ith inte#mittent #oc2ing o% the pl"te.
). @emo0e the inoculum5 p#e%e#"(l& ,ith " pipette "n$ then "$$ 1.) ml o% "g"#ose
o0e#l"& me$ium (g#o,th me$ium ,ith 6.38 "g"#ose "n$ .)8 4CS).
6. Ensu#e th"t the o0e#l"& me$ium h"s sp#e"$ e0enl& o0e# the monol"&e#5 le"0e "t
#oom tempe#"tu#e %o# 16 mins "n$ then incu("te "t 3- C .
-. EA"mine the monol"&e#s $"il&5 st"#ting %#om secon$ $"& o% incu("tion.
3. 9nce the pl"ques h"0e $e0elope$5 usu"ll& (& the %ou#th $"& post inocul"tion5
count the num(e# o% pl"ques "t e"ch $ilution5 #emo0e the "g"#ose o0e#l"& "n$
gentl& ,"sh the monol"&e# ,ith *:S.
>. St"in the pl"te ,ith 6.18 c#&st"l 0iolet solution "n$ count the pl"ques "g"in.
16. Estim"te the 0i#us tit#e "s " pl"que %o#ming units pe# ml (p%u /ml) "s %ollo,s (&
counting the num(e# o% pl"ques "t "n "pp#op#i"te $ilution.
4o# eA"mple 1
Num(e# o% pl"ques p#o$uce$ >
!ilution o% 0i#us 1 A 16
)
Colume o% inoculum 6. ml
Ci#us tit#e I > A 1A16
)
A 1/) p%u pe# ml
I ..) A 16
6
V ISOLATION AND IDENTIFICATION OF ENTEROVIRUSES FROM
CLINICAL SPECIMENS
Ente#o0i#uses inclu$ing polio 0i#uses c"use " 0"#iet& o% CNS m"ni%est"tions inclu$ing
"septic m"ningitis5 enceph"litis "n$ p"#"l&sis. These 0i#uses "#e n"tu#"l inh"(it"nts o%
hum"n intestin"l t#"ct "n$ the& c"n (e e"sil& isol"te$ %#om s"mples o% %eces o# %#om
#ect"l s,"(s. :ut me#e isol"tion o% " ente#o0i#us %#om stool s"mple m"& not p#o0e
the $i"gnosis "s these m"& (e p#esent in no#m"l stool specimens "lso. The#e%o#e
isol"tion shoul$ "lso %ollo, " $emonst#"tion o% " se#ologic"l #esponse to co##o(o#"te
the $i"gnosis. Ho,e0e# isol"tion o% 0i#us %#om CS45 o# %#om (#"in postmo#tem c"ses
is $i"gnostic.
I.$&!'$( $% E(!#"$9'"u.#. %"$) %#c& .1#c')#(./
M&!#"'&./
1. .+3 gms o% %eces5
. . Cent#i%uge tu(es.
3. 3.C#&o cent#i%uge
.. .. Test tu(es
). ).@u((e# co#2s si=e 53 ).<ic#o pipettes
6. 6.C9

incu("to#
-. -. 7"te# ("th
3. 3.<E< ,ith "nti(iotics "n$ 4CS 168 "n$ 8
>. >.Hep cells ('TCC5 CC/ 3)
16. 16. @! cells ( 'TCCK CC/ 136)
11. TC; soln.
1. >6 ,ell %l"t (ottom tissue cultu#e pl"tes.
13. 13./:< pools o% 'ntise#".
P"$c#..'(6 $% %#c& .1#c')#(. /
'(out .+3 gms o% %eces is #equi#e$. The specimens "#e 2ept %#o=en o# in col$ till
p#ocesse$. ' 168 o# 68 %ec"l suspension is m"$e in col$ ste#ile *:S in " tightl&
stoppe#e$ (ottle. '%te# 0igo#ous sh"2ing to emulsi%& the %eces5 the specimen is
"llo,e$ to settle in the col$ %o# 16min. The supe#n"t"nt %lui$ is then pou#e$ in to "
cent#i%uge tu(e "n$ cl"#i%ie$ "t 3666 #pm %o# 16min. The supe#n"t"nt %lui$ is #emo0e$
"n$ to this is "$$e$ "nti(iotics to get " %in"l concent#"tion o% 1666 units o% penicillin
"n$ 1666 ug o% st#eptom&cin pe# ml "n$ the s"mple is hel$ "t #oom tempe#"tu#e %o#
36min. It is then cent#i%uge$ in col$ "t spee$ 165666 #pm %o# 6 min. The cle"#
supe#n"t"nt is sep"#"te$ "n$ use$ imme$i"tel& o# sto#e$ %#o=en.
I($cu&!'$( $% .1#c')#(. '(!$ c# cu!u"#/
4o# isol"tion o% most ente#o0i#uses " com(in"tion o% cell lines li2e Hep "n$ @!
,ill su%%ice. These cells "#e g#o,n sep"#"tel& in test tu(es "n$ monol"&e#s "#e
o(t"ine$ (& emplo&ing usu"l cell cultu#e techniques5 $esc#i(e$ else,he#e. The s"mple
p#ocesse$ is inocul"te$ in 66 Ul qu"ntities to e"ch o% . tu(es o% (oth Hep "n$ @!
cell lines cont"ining 1 ml m"inten"nce me$ium "n$ incu("te$ in "n "lmost ho#i=ont"l
position "t 3- C . Cultu#es sho,ing e"#l& $egene#"tion5 less th"n .h#s "#e
su(p"ss"ge$. The tu(es "#e o(se#0e$ $"il& %o# e0i$ence o% t&pic"l ente#o0i#us C*E
"n$ $isc"#$e$ "t the en$ o% -+16 $"&s i% %oun$ neg"ti0e.
I0#(!'%'c&!'$( $% E(!#"$9'"u. b* N#u!"&'.&!'$( / E>/ P$'$9'"u.
I% t&pic"l C*E is seen ,ith eithe# Hep o# @! o# (oth cell lines5 it is "lmost
ce#t"in th"t "n ente#o0i#us h"s (een isol"te$. The isol"te m"& (e i$enti%ie$ "s one o%
the polio "nti(o$ies. The isol"te shoul$ (e tit#"te$ "n$ " $ilution cont"ining 166
TCI! )6 pe# 6.1 ml is p#ep"#e$. E"ch o% the polio "ntise#" *15 * "n$ *3 is $ilute$
to cont"in )6 "nti(o$& units5 i.e.)6 times th"t necess"#& to neut#"lise 166 TCI! )6 o%
0i#us. Equ"l qu"ntities o% 0i#us "n$ se#um "#e miAe$ "n$ incu("te$ in ,"te# ("th "t
3- C %o# h#s. The miAtu#e is then inocul"te$ on to monol"&e#s o% HE* o# @!
cell lines in test tu(es o# mic#otit#e pl"tes. The #e"$ing is t"2en on $"& 3 "n$ - "n$
i% the isol"te is one o% the polio0i#us it ,ill (e neut#"lise$ (& the co##espon$ing
"ntise#" "n$ hence the#e ,ill (e no C*E in the inocul"te$ cultu#es. Ci#us cont#ols not
t#e"te$ ,ith "ntise#" shoul$ sho, t&pic"l C*E "t the en$ o% 3
#$
$"&. Ente#o0i#uses5
,hich p#o$uce C*E5 c"n (e con0enientl& t&pe$ (& neut#"li="tion using /:< pool (/im+
:en&esh+<elnic2) o% "ntise#". 4o# %u#the# $et"ils "(out this technique "s ,ell "s
inte#p#et"tion o% #esults ple"se #e%e# to <elnic2 et "l (1>->). Ente#o0i#uses. In 1 !i"gnostic
p#oce$u#es %o# 0i#"l5 @ic2etsi"l "n$ Chl"m&$i"l in%ections (E.H./ennette5 N.G.Schmitt5 E$s)
)
th
e$ition5 pp .-1 + ).. 'me#ic"n *u(lic He"lth 'ssoci"tion5 7"shington5US'.
I.$&!'$( $% 9'"u. %"$) CSF /
Ente#o0i#uses c"n sometimes (e isol"te$ %#om CS4 $u#ing e"#l& st"ges o% the illness.
4o# isol"tion %#om CS4 66 Ul o% CS4 is inocul"te$ on to the monol"&e#s o% (oth
Hep "n$ @! cells "s $esc#i(e$ p#e0iousl&. No p#e+t#e"tment o# "$$ition o%
"nti(iotics is #equi#e$. The tu(es "#e incu("te$ "n$ o(se#0e$ %o# C*E "s in c"se o%
%ec"l specimens. I% "n isol"te is o(t"ine$ it is i$enti%ie$ "s $esc#i(e$ e"#lie#.
VI IMMUNOASSAYS FOR DETECTION OF ANTI<VIRAL ANTIBODIES


H&#)&66u!'(&!'$( '(+'b'!'$( 3HI4 !#.!
' ,i$e 0"#iet& o% "nim"l 0i#uses h"0e (een sho,n to possess the c"p"cit& o%
"$so#(ing to #e$ (loo$ cells "n$ in m"n& c"ses c"using "gglutin"tion o% these cells.
This h"em"gglutin"tion (H') test %u#nishes " #el"ti0el& simple5 quic25 con0enient "n$
%"i#l& qu"ntit"ti0e ,"& o% $etecting 5 i$enti%&ing "n$ tit#"ting 0i#uses.
Ci#us h"em"gglutin"tion is #e"$il& inhi(ite$ (& speci%ic "nti(o$& "n$ the
h"em"gglutin"tion inhi(ition test %u#nishes " $elic"te ,"& o% $etecting 0i#"l "nti(o$&.
It m"& (e st#"in+speci%ic "s in the c"se o% the in%luen=" 0i#uses o# g#oup+speci%ic "s
,ith most o% the "#(o0i#uses ,he#e the test is m"inl& use$ %o# "ntigenic g#oup
sc#eening.
M&!#"'&. "#,u'"#0 /
R#&6#(!./
1. )8 2"olin (o#"te s"line pH >.6.
. 6..8 (o0ine "l(umin in (o#"te s"line
pH >.6 (6..8 :':S pH >.6).
3. Ci#us "$?usting $iluents o% 0"#&ing pH (C'!s)
.. ;oose e#&th#oc&tes (@:C) in !;C
). 'ntise#"
6. 'ntigens
-. No#m"l se#um
3. 'ci$ cit#"te $eAt#ose ('C!)
>. !eAt#ose gel"tin 0e#on"l (!;C)
16. :o#"te s"line pH >.6
11. 6.>8 N"Cl
1. 'cetone
13. +<e#c"ptoeth"nol
P"#1&"&!'$( $% R#&6#(!.
1. :o#"te s"line pH >.6
1.) < N"Cl 166 ml
6.) < H
3
:9
.
(:o#ic "ci$) 66 ml
1.6 < N"9H .- ml
<"2e it to lit#es ,ith !/75 pH "$?uste$ to > ,ith 1< N"9H o# 6.)< H
3
:9
3.
. 6..8 :o0ine "l(umin in 1666ml (o#"te s"line pH >.6 2ept 9/N in the col$.
pHLLLLLLL..356
'$?ust pH >.6 ,ith 1< N"9H.
3. )8 2"olin in 166 ml (o#"te s"line pH >.6 (Use "ci$+,"she$ 2"olin)
:o#"te s"line is sti##e$ on " m"gnetic sti##e# "n$ 2"olin po,$e# is "$$e$ slo,l&
to m"2e " uni%o#m suspension.
.. !eAt#ose+gel"tin+0e#on"l (!;C) %o# p#ese#0"tion o% goose cells1
'. ;el"tin 6.666 g
!ieth&l ("#(itu#ic "ci$ (Ce#on"l) 6.)36 g
:. So$ium ("#(itone 6.336 g
C"Cl

('nh&$#ous) 6.66 g
N"Cl 3.)66 g
!eAt#ose 16.666 g
<e#thiol"te (Thiome#os"l po,$e#) 6.166 g
<gS9
..
-H

9 6.16 g
'$$ )6 ml o% hot !/7 to ' "n$ -)6 ml o% !/7 to :. <iA ' "n$ :.
'utocl"0e "t 16 l(s %o# 16 minutes.
). 6.>8 N"Cl
> g N"Cl in 166 ml !/7. 'utocl"0e "t 1) l(s %o# 1) minutes.
6. *#ep"#"tion o% stoc2 solutions
'. 1.)< so$ium chlo#i$e (N"Cl) + 3-.6-) g N"Cl m"$e to 1 lit#e ,ith $istille$ ,"te#.
:. 6.)< (o#ic "ci$ (H
3
:9
3
) + 36.> g H
3
:9
3
"#e $issol0e$ in "(out -66 ml o% hot
!/7. The solution is "llo,e$ to cool to #oom tempe#"tu#e "n$ then m"$e to 1
lit#e ,ith !/7.
C. Concent#"te$ so$ium h&$#oAi$e (N"9H) + "pp#oAim"tel& )66 ml o% $istille$ ,"te#
is "$$e$ to "pp#oAim"tel& )66g o% N"9H pellets. The concent#"te$ N"9H is "(out
13< "n$ is 0e#& st"(le.
!. 6.)< $i("sic so$ium phosph"te (N"

H*9
.
) + -6.>> g N"

H*9
.
("nh&$#ous) m"$e
to 1 lit#e ,ith $istille$ ,"te#.
E. 1< mono("sic so$ium phosph"te (N"H

*9
.
) + 133.61 g N"H

*9
.
.H

9 m"$e to 1
lit#e ,ith $istille$ ,"te#.
'$?usting $iluents
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

Stoc2 6.6 6. 6.3 6..
Solutions ml ml ml ml
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
1.) < N"Cl 166 166 166 166
6.) < N"

H*9
.
3 6. > 11
1.6 < N"

H*9
.
13. 16> 1). 1..

<"2e up %in"l 0olume to 1/ using $ou(le $istille$ ,"te#.
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
-. 'ci$ Cit#"te !eAt#ose ('C!)
So$ium Cit#"te 11.6 g
Cit#ic "ci$ ..66 g
!eAt#ose 11.66 g
!/7 )66 ml
'utocl"0e "t 16 l(s %o# 16 minutes.
C$#c!'$(?5&.+'(6 $% 6$$.# RBC
". Collection o% @:C
1. ;oose @:C "#e collecte$ un$e# ste#ile con$ition.
T"2e 1) ml o% !;C in " )6 ml %l"s2.
. T"2e 16 ml 'C! in " ste#ile tu(e.
3. 7ith$#", -.) ml 'C! into )6 ml s&#inge %itte$ ,ith " 6 g"uge nee$le.
.. Collect )6 ml goose (loo$ %#om the ,ing o% ?ugul"# 0ein.
). @emo0e nee$le %#om s&#inge "n$ t#"ns%e# (loo$ %#om s&#inge to %l"s2 ,ith !;C.
6. <iA ,ell.
-. Sto#e "t .C (c"n (e 2ept %o# up to ,ee2s)
(. 7"shing o% @:C
1. Cent#i%uge #equi#e$ qu"ntit& o% @:C suspension in " g#"$u"te$ cent#i%uge tu(e %o# siA
<inutes "t 666 #pm.
. 'spi#"te o%% the pipette "tt"che$ to " #u((e# (ul( ,ithout $istu#(ing the @:C.
3. '$$ ml o% no#m"l s"line (NS).
.. <iA gentl& ,ith *"steu# pipette to #esuspen$ cells "n$ "$$ "n eAt#" qu"ntit& o% NS to
(#ing to the o#igin"l 0olume.
). Cent#i%uge "t #oom tempe#"tu#e %o# 3 minutes "t 66 #pm.
6. @epe"t steps 535. B ).
-. Cent#i%uge "t 666 #pm %o# 6 minutes.
3. @e"$ 0olume o% p"c2e$ @:C.
>. @emo0e supe#n"t"nt %lui$.
16. '$$ 6.>8 N"Cl to m"2e " 168 suspension o% @:C "n$ miA. The cells "#e #e"$& %o# use
in IC (use ,"she$ "n$ p"c2e$ cells %o# "$so#ption o% se#").
T'!"&!'$( $% A(!'6#(
1. 7ith " ,"A pencil m"#2 " MUM mic#otit#e pl"te "s sho,n in %igu#e1
6 .6 36 166 36 6.6 136 )66 )16 16.6 ccNN cc
6.6
6.
6..
6.6
6.3
-.6
-.
cc + cell cont#ol.
. 7ith " 6.6) ml $#oppe#5 "$$ one $#op o% 6..8 :':S pH >.6 to e"ch ,ell.
3. Eeep pl"te in #e%#ige#"to# %o# chilling.
.. Eeep se0en 6.6) ml mic#o $ilute#s ,ith tips imme#se$ in 6.>8 N"Cl in " #e%#ige#"to# %o#
chilling.
). !ispense 6.> ml o% 6..8 :':S in " tu(e in "n ice ("th.
6. '$$ 6.1 ml o% "ntigen "n$ miA tho#oughl& (1116 $ilution o% "ntigen).
-. @emo0e pl"te "n$ mic#o $iluto# %#om #e%#ige#"to#.
3. :lot 6.6) ml mic#o $iluto#s on " (lotting p"pe#5 $ip the tips in tu(e ,ith 1116 "ntigen "n$
pl"ce in e"ch o% the ,ells m"#2e$ 116 in the pl"te.
>. 7ith the mic#o $iluto#s5 p#ep"#e se#i"l + %ol$ $ilution o% the "ntigen upto ,ell 16
(11165.6) (& s,i#ling the mic#o $iluto#s 16 to 1) times.
16. '$$ .. ml o% C'!s 6.65 6.5 6..5 6.65 6.35 -.65 B -. to l"(elle$ tu(es.
11. '$$ 6.1 ml o% 168 @:C to e"ch o% the tu(es "n$ miA ,ell (this gi0es " $ilution o% 6..8
@:C).
1. 7ith " 6.6) ml $#oppe# pipette "$$ one $#op o% @:C to the co##espon$ing #o,.
13. T"p pl"te gentl& to miA cells "n$ the "ntigen.
1.. Co0e# the pl"te "n$ incu("te "t #oom tempe#"tu#e until cells in the ,ells o% the cell
cont#ol #o, settle $o,n "n$ %o#m " (utton (usu"ll& this t"2es 6 + 36 minutes)
1). @e"$ "n$ #eco#$ the tit#"tion.
16. Complete "gglutin"tion consists o% " uni%o#m l"&e# on the lo,e# su#%"ce o% the ,ell. '
neg"ti0e consists o% " comp"ct5 sh"#pl& $em"#c"te$ (utton o% se$imente$ cells in the
cent#e o% the ,ell5 the p"tte#n (eing i$entic"l to th"t seen in the cont#ol ,ells. The #esults
"#e co$e$ "s %ollo,s1
6 .6 36 166 36 6.6 136 )66
J J J J J J 6 6
then the "ntigen tit#e is the "0e#"ge o% the "ntigen $ilutions 6.6 "n$ 136 i.e.
(6.6 J 136) / I >66.
1-. The C'! ,ith ,hich the "ntigen sho,s the highest tit#e is the optimum one "n$ shoul$
(e use$ %o# th"t p"#ticul"# "ntigen.
13. !ete#mine the $ilution5 ,hich ,ill cont"in 3 units pe# 6.6) ml (& $i0i$ing the en$point
(& 16. 4o# e.g.5 en$ point 136 I (136/16) I 36. The#e%o#e " 1136 $ilution o% the
"ntigen ,ill cont"in 3 H' units pe# 6.6) ml.
1>. C"lcul"te the qu"ntit& o% "ntigen #equi#e$ %o# the test "n$ $ilute the "ntigen "cco#$ingl&.
6. Sto#e "ntigen "t .C.
T"#&!)#(! $% .#"& %$" HI !#.!
The se#" m"& (e eithe# eAt#"cte$ ,ith "cetone o# t#e"te$ ,ith 2"olin. These se#" "#e then
"$so#(e$
7ith goose e#&th#oc&tes to #emo0e he"m"gglutinins %o# goose e#&th#oc&tes.
& Ac#!$(# #>!"&c!'$( $% .#"&
1. /"(el 16A16 mm tu(es ,ith specimen num(e# o% se#" (this shoul$ inclu$e neg"ti0e "n$
positi0e se#").
. '$$ 6.> ml no#m"l s"line to e"ch tu(e.
3. '$$ 6.1 ml se#um to e"ch co##espon$ingl& l"(elle$ tu(e.
.. Eeep se#" in "n ice ("th.
). '$$ 1 ml chille$ "cetone to e"ch tu(e.
6. Stoppe# e"ch tu(e "n$ miA (& in0e#ting tu(es se0e#"l times.
-. Incu("te %o# ) minutes miAing occ"sion"ll&.
3. Cent#i%uge "t 3666 #pm %o# ) minutes "t .C.
>. !ec"nt supe#n"t"nt %lui$.
16. '$$ 1 ml %#esh "cetone to e"ch tu(e.
11. <iA tho#oughl&.
1. Incu("te in col$ %o# 1 hou#. <iA the m"te#i"l occ"sion"ll&.
13. Cent#i%uge "t .C %o# ) minutes "t 666 #pm.
1.. 'spi#"te o%% the supe#n"t"nt.
1). T"p e"ch tu(e "g"inst p"lm o% h"n$ so th"t se$iment %o#ms " uni%o#ml& thin l"&e#.
16. !esicc"te in " $esicc"to# "tt"che$ to " 0"cuum pump %o# 1+ hou#s.
1-. '$$ 6.) ml (o#"te s"line pH >.6 to e"ch tu(e "n$ miA until completel& #esuspen$e$ (2eep
%o# minimum o% 1 hou# in col$).
13. '$so#( ,ith goose cells.
". 8&$'( !"#&!)#(! $% .#"&
1. /"(el 1A-) mm tu(e ,ith specimen num(e#s o% se#".
. '$$ 6.1 ml se#um to e"ch co##espon$ingl& l"(elle$ tu(e.
3. '$$ 6.. ml o% 2"olin solution ()8 in (o#"te s"line5 pH >.6)5 to e"ch tu(e "n$ miA (&
sh"2ing tu(e #"c2.
.. Incu("te %o# 6 minutes "t #oom temp. sh"2ing e0e#& ) minutes.
). Cent#i%uge "t )66 #pm %o# 36 minutes "t #oom temp.
6. '$so#( ,ith goose cells.
(. A0.$"1!'$( $% .#"& 5'!+ 6$$.# c#.
1. Eeep se#" ("cetone eAt#"cte$ o# 2"olin t#e"te$) "n ice ("th.
. '$$ 6.1 ml o% ,"she$ "n$ p"c2e$ goose @:C to e"ch tu(e (It m"& (e necess"#& to "$$
mo#e
cells %o# pig "n$ #"((it se#")
3. <iA ,ell "n$ incu("te %o# 6 minutes in "n ice ("th5 miAing e0e#& ) minutes.
.. Cent#i%uge "t 666 #pm %o# 1) minutes "t .C.
). T#"ns%e# supe#n"t"nt %lui$ to %#esh tu(e (& $ec"nting (t#"ns%e# l"(els "t the s"me time).
The se#" "#e #e"$& to (e teste$ in HI.
P"#')'(&"* !'!"&!'$( $% &(!'6#(
The $ilute$ "ntigens "#e tit#"te$ "g"in (e%o#e using them in the test.
1. <"#2 " MUM t&pe mic#otit#e pl"te "s sho,n (elo,.
'ntigen !ilution Cell
Num(e# ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ cont#ol
1 3 . ) 6 -
1

3
.
. '$$ " $#op o% 6..8 :':S pH >.6 to e"ch ,ell %#om $ilutions 1+- "n$ cell cont#ols in
#o,s 1+)
,ith " 6.6) ml $#oppe#.
3. Eeep pl"te in the #e%#ige#"to# %o# chilling.
.. Eeep ) mic#o$ilute#s (6.6) ml) ,ith tips imme#se$ in 6.>8 N"Cl %o# chilling.
).:lot out the tip o% e"ch mic#o$ilute#. !ip the tip in e"ch o% the "ntigens "n$ pl"ce in e"ch o%
the co##espon$ing ,ell $ilution no.1.
6. *#ep"#e se#i"l t,o+%ol$ $ilutions o% e"ch "ntigen %#om ,ell No.1 to ,ell NoL
-. '$$ one $#op o% 6..8 cells in #equi#e$ C'! ,ith " 6.6) ml $#oppe# pipette to
co##espon$ing
#o,s o% "ntigens inclu$ing cell cont#ol ,ells.
3. Sh"2e the pl"te gentl& "n$ incu("te "t #oom tempe#"tu#e till cell cont#ols settle $o,n
("(out 36 minutes)
>. @e"$ "n$ #eco#$ the tit#"tion. '$?ust i% necess"#& "n$ #e+tit#"te.
P#"%$")&(c# $% HI !#.!
1. Select "ntigens to ,hich se#" "#e to (e teste$.
. T#e"t "ll test se#"5 "ntise#" homologous to "ntigens selecte$ "n$ " neg"ti0e cont#ol
se#um5 "s sho,n in t#e"tment o% se#".
3. <"#2 the MUM t&pe mic#otit#e pl"tes to "ccommo$"te the num(e# o% se#" to (e teste$
%o# e"ch "ntigen. 'lso m"#2 the cont#ol pl"te (see (elo,)
.. 7ith " 6.6) ml $#oppe# "$$ " $#op o% 6..8 :':S pH >.6 to e"ch ,ell.
). !ip p#eteste$ 6.6) ml mic#o $ilute#s into e"ch t#e"te$ se#um "n$ pl"ce in ,ell No.1
(1116 $ilution).
S#.No. Se#um $ilutions S#.No. se#um $ilutions
++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++++++++++++++
16 6 .6 166 36 6.6 136 16 6
1

3
.
)
6
-
3
>
16
Neg"ti0e Neg"ti0e
S#. cont#ol S#. cont#ol
@e%. J0e Cont#ol @e%.J0e Cont#ol
6. *#ep"#e se#i"l t,o+%ol$ $ilutions ,ith the mic#o$ilute#s %o# e"ch "ntigen "n$ %o# se#um
cont#ols.
-. 7ith " 6.6) ml $#oppe# "$$ " $#op o% test 0i#us "ntigens cont"ining 3 HI units5 to "ll
7ells o% the se#um $ilutions in e"ch test pl"te.
3. 7ith " 6.6) ml $#oppe# "$$ " $#op o% 6..8 :':S pH >.6 to "ll ,ells o% the se#um
$ilution se#ies in the cont#ols pl"te. Sh"2e pl"tes to miA5 co0e# "n$ 2eep in the
#e%#ige#"to#.
>. <"#2 the pl"te %o# post incu("tion tit#"tion.
16. '$$ 6.6) ml o% 6..8 :':S pH >.6 to e"ch ,ell ,ith $#oppe#.
11. '$$ 6.6) ml o% e"ch "ntigen to the %i#st ,ell "n$ miA.
1. Incu("te "ll pl"tes "t . C o0e#night ("(out 13 hou#s)
13. *l"ce " 6.6) ml mic#o$ilute# in e"ch o% the sells cont"ining "ntigen $iluent miAtu#e in
The post incu("ting tit#"tion pl"te (,ell No.1).
1.. *#ep"#e se#i"l t,o+%ol$ $ilutions th#ough ,ell No.-.
1). '$$ one $#op o% 6..8 goose @:C in #especti0e C'! to e"ch ,ell o% tit#"tion pl"te
,ith 6.6)ml $#oppe#.
16. Sh"2e "n$ incu("te "t #oom tempe#"tu#e.
1-. '$$ one $#op 6..8 goose @:C (in #especti0e C'!) to e"ch ,ell o% test pl"tes "n$
cont#ol pl"tes ,ith " 6.6) ml $#oppe#.
13. Sh"2e pl"tes "n$ incu("te "t #oom tempe#"tu#e till the cells in cont#ols settle $o,n.
1>. @e"$ "n$ #eco#$ the cell p"tte#ns.
". Chec2 @:C cont#ols. These shoul$ settle $o,n into " (utton.
(. Chec2 the tit#"tion o% "ntigens. E"ch "ntigen shoul$ cont"in .+3 H' units.
c. Chec2 the se#um cont#ols. I% no "gglutin"tion occu#s in "n& $ilution o% " se#um the
HI #esults in test pl"tes "#e consi$e#e$ 0"li$.
$. Chec2 the neg"ti0e cont#ol se#um. It shoul$ sho, complete "gglutin"tion in "ll
$ilutions. I% not5 the test is in0"li$.
e. Chec2 the positi0e #e%e#ence se#um. The se#um must sho, complete inhi(ition o%
h"em"gglutin"tion.
%. Chec2 the test se#". The en$ point use$ to $esign"te the tit#e o% HI "nti(o$&
p#esent is the highest $ilution o% se#um th"t inhi(its he"m"gglutin"tion.

E"#l& con0"lescent se#" "#e t#e"te$ ,ith +<E to $etect the p#esence o% Ig< "nti(o$ies. The
"cetone eAt#"cte$ se#" "#e t#e"te$ ,ith +<E. *#ep"#e 1< +<E in (o#"te s"line pH >.6 1 (")
1..- ml +<E J13.)3ml o# (() 1.)6 ml J 13... ml5 $epen$ing upon the molecul"# ,eight o%
stoc2 solution. '$$ one p"#t o% +<E (1<) to %ou# p"#ts o% se#um "n$ incu("te the miAtu#e
%o# one hou# "t 3- C "n$ m"2e se#um $ilutions. 4o# post tit#"tion o% "ntigen ,ith +<E5
6.< +<E is miAe$ ,ith equ"l 0olume o% "ntigen "n$ incu("te o0e#night "t . . 'ntigen
$ilution is c"##ie$ out "n$ H' tit#e is $ete#mine$. The +<E t#e"te$ "n$ unt#e"te$ se#" "#e
teste$ in HI "s pe# #outine test.
VIRUS NEUTRALISATION TESTS
' 0i#us ,ill lose its in%ecti0it& ,hen it is miAe$ ,ith speci%ic "ntise#um #"ise$ "g"inst
th"t 0i#us. This is c"lle$ neut#"lis"tion. The neut#"lise$ 0i#us is un"(le to in%ect "n& cell
cultu#e o# eApe#iment"l "nim"l. This p#ope#t& o% the 0i#us c"n (e con0enientl& emplo&e$ %o#
its i$enti%ic"tion "n$ "lso %o# $etection "n$ qu"nti%ic"tion o% speci%ic "nti0i#"l "nti(o$ies. In
the 0i#us neut#"lis"tion tests %o# "nti(o$& "ss"&5 " %iAe$ "mount o% " speci%ic 0i#us is m"$e to
#e"ct ,ith inc#e"sing $ilutions o% " speci%ic "ntise#um un$e# st"n$"#$ con$itions "n$ the
$eg#ee o% 0i#us neut#"lis"tion is $ete#mine$ eithe# in 0i0o (& "nim"l inocul"tion o# in 0it#o (&
in%ecti0it& in cell cultu#e. 9n the othe# h"n$5 %o# i$enti%ic"tion o% " p"#ticul"# 0i#"l isol"te5 "
%iAe$ qu"ntit& o% 0i#us is #e"cte$ ,ith " st"n$"#$ "mount o% speci%ic "ntise#um "n$ "lso ,ith
no#m"l se#um. The i$entit& o% the 0i#us gets est"(lishe$ i% it is neut#"lise$ (& speci%ic
"ntise#um "n$ #em"ins in%ectious ,ith no#m"l se#um.
In this section ,e ,ill illust#"te "ss"& o% "nti+#"(ies se#um (& in 0i0o neut#"lis"tion in mice
"n$ $etection "n$ qu"nti%ic"tion o% polio 0i#us "nti(o$ies (& in 0it#o neut#"lis"tion in cell
cultu#e.
ASSAY OF ANTIRABIES SERUM BY MOUSE NEUTRALISATION TEST 3MNT4
This test is the st"n$"#$ test #ecommen$e$ %o# estim"tion o% "nti+#"(ies "nti(o$ies in hum"n
o# "nim"l se#"5 to 2no, the le0el o% p#otection "%te# 0"ccin"tion o% %o# $i"gnosis o% the
$ise"se. In this test $i%%e#ent $ilutions o% the se#um un$e# test is incu("te$ ,ith " st"n$"#$
"mount o% " %iAe$ #"(ies 0i#us st#"in 2no,n "s CCS (ch"llenge the 0i#us st#"in) "n$ se#um
0i#us miAtu#e is inocul"te$ I/C in to ,e"nling mice "n$ $epen$ing on the su#0i0"l #"te in
mice the tit#e c"n (e eAp#esse$ in inte#n"tion"l units (IU) in comp"#ison to " st"n$"#$ "nti+
#"(ies se#um ,hose unit"ge is 2no,n.
MATERIALS REQUIRED /
1.Ste#ile test tu(es o# penicillin 0i"ls.
.*ipettes5 16 ml5 ) ml "n$ 1 ml
3.7"te# ("th
..1 ml s&#inges
). 7e"nling mice5 . ,ee2s ol$
6. Ste#ile *:S pH -..
-. :o0ine o# ho#se se#um
3. CCS o% 2no,n tit#e. >.@e%e#ence '@S o% 2no,n unit"ge.
PROCEDURE /
1. In"cti0"te se#" un$e# test "t )6 C %o# 36 min in ,"te# ("th.
. *#ep"#e $ilutions o% test "n$ #e%e#ence se#" in *:S cont"ining 8 (o0ine o% ho#se
se#um. !ilutions shoul$ (e so "$?uste$ "s to get )68 en$ points p#e%e#"(l& in the
mi$$le $ilutions. To get " $ilution o% s"& 113 "$$ 6.1 ml o% se#um to 6.- ml o% $iluent
"n$ then continue ,ith $ou(ling $ilutions i.e 165356. etc.
3. *#ep"#e " $ilution o% CCS cont"ining 166 /!
)6
/6.63 ml so th"t ,hen miAe$ ,ith "n
equ"l qu"ntit& o% se#um $ilution the ch"llenge is #e$uce$ to )6 /!
)6.
To c"lcul"te the
tit#e o% CCS gi0ing 166 /! )6 ,e shoul$ 2no, the o#igin"l tit#e o% CCS. 4o# E.g. i% the
tit#e is 16
+-
/6.63 ml then 166 /!
)6
,ill (e p#esent in $ilution 16
+-
/ 6.63 ml then 166
/!
)6
,ill (e p#esent in $ilution 16
+)
.
.. <iA 6.) ml o% se#um $ilution ,ith 6.) ml o% CCS $ilution5 16
+)
"n$ incu("te in " ,"te#
("th "t 3- C %o# 66 min. Simult"neousl& p#ep"#e 3 mo#e $ilutions o% CCS i.e5 16
+6
to
16
+3
"n$ miA 6.) ml o% this $ilution ,ith 6.) ml o% $iluent. This is "lso incu("te$ "long
,ith se#um+0i#us $ilutions.
). '%te# 66 min #emo0e the tu(es %#om ,"te# ("th "n$ chill the tu(es (& 2eeping in
@e%#ige#"to# "t . C o# in " ice t#"&.
6. Inocul"te I/C 6+3 mice ,ith e"ch se#um $ilution "n$ to $ete#mine the EA"ct /!
)6
use$
%o# ch"llenge inocul"te 0i#us $ilutions "lso in simil"# m"nne#. 9(se#0e the mice %o# 1.
$"&s. Igno#e "ll $e"ths (e%o#e ) $"&s post inocul"tion. Note $o,n the speci%ic $e"ths
occu##ing %#om 6 to 1. $"&s.
-. The )68 en$ point tit#e o% se#um c"lle$ e%%ecti0e $ose )6 (E!
)6
) o# neut#"lising $ose )6
(N!
)6
) is c"lcul"te$ (& @ee$ "n$ <uench metho$. 't the s"me time note $o,n the
mo#t"lit& in mice inocul"te$ ,ith CCS $ilutions "n$ c"lcul"te the eA"ct /!
)6
use$ in the
test. This shoul$ (e "pp#oAim"tel& )6 /!
)6.
3. The tit#e o% the test se#um c"n (e eAp#esse$ in IU/ml in comp"#ison to #e%e#ence se#um.
E.g. I% #e%e#ence se#um h"s "n IU o% ) IU "n$ its N!
)6
is 1113 "n$ the test se#um
sho,s N!
)6
o% 116. then the unit"ge o% test se#um is
#ecip#oc"l o%
test se#um N!
)6
HHHHHHHHHHHHHHHHHHHHHHHHHHHHH D unit"ge o% #e%e#ence se#um
#ecip#oc"l o%
#e%e#ence se#um N!
)6
i.e.
6.
HHHHHH D ) I .) IU/ml

13
ASSAY OF POLIO VIRUS ANTIBODIES BY NEUTRALISATION TEST IN CIT@9
BY USING CELL CULTURE TECHNIQUE
'nti(o$ies to polio 0i#uses $e0elop "%te# 0"ccin"tion ,ith polio 0"ccine o# "%te#
in%ection ,ith ,il$ 0i#us. 'nti(o$ies to polio "n$ othe# ente#o0i#uses m"& (e p#esent in
no#m"l popul"tion "n$ hence $emonst#"tion o% %ou# %ol$ #ise in the tit#e o% "nti(o$& is
"(solutel& necess"#& %o# the se#o+ $i"gnosis o% ente#o0i#"l in%ections. 4o# the test5 t,o se#um
s"mples5 one t"2en ?ust "%te# the onset o% the $ise"se "n$ the secon$5 "%te# " g"p o% ,ee2s o#
mo#e is #equi#e$.
In the test p#ope#5 inc#e"sing $ilutions o% p"tientOs se#um is miAe$ ,ith 166 /!
)6.
9%
the 3 t&pes o% polio 0i#uses "n$ incu("te$ in ,"te# ("th %o# h#s. The miAtu#e is then
inocul"te$ to p#e%o#me$ monol"&e#s o% eithe# Ce#o o# Hep cells in %l"t (ottom >6 ,ell
tissue cultu#e pl"tes "n$ incu("te$ %o# - $"&s. @e"$ings "#e t"2en "t 3
#$
"n$ -
th
$"& "n$
$epen$ing on the C*E seen in $i%%e#ent $ilutions5 tit#e o% the se#um s"mple is c"lcul"te$ (&
using @ee$ "n$ <uench metho$.
MATERIALS REQUIRED /
1. >6 ,ell %l"t (ottom tissue cultu#e pl"tes
. <ic#o pipettes5 66 "n$ 1666 Ul
3. <ic#o(iologic"l s"%et& c"(inet (/"min"# %lo,)
.. C9 incu("to#.
). Test tu(es o# penicillin 0i"ls
6. <E< ,ith "$$e$ "nti(iotics "n$ cont"ining 8 4CS
-. 7"te# ("th )6 C "n$ 3- C
PROCEDURE /

E"ch se#um s"mple h"s to (e teste$ sep"#"tel& %o# "nti(o$ies "g"inst 3 polio 0i#uses. :e%o#e
st"#ting the p#oce$u#e " pl"n shoul$ (e $#",n %o# the $ist#i(ution o% s"mples "n$ cont#ols in
the mic#otit#e pl"tes.
1. In"cti0"te the se#um s"mples "t )6 C %o# min.
. !ilute the se#um s"mples %#om 1116 to 116.3 in test tu(es o# penicillin 0i"ls using <E<
,ith 8 4CS.
3. !ilute the 3 t&pes o% polio 0i#uses5 *15 * "n$ *3 in <E< to cont"in 166 TCI! )6 pe#
6.1 ml using <E< ,ith 8 4CS.
.. <iA equ"l se#um qu"ntities o% se#um $ilution "n$ 0i#us $ilution in test tu(es "n$ incu("te
in
,"te# ("th "t 3- C %o# h#s.
). Simult"neousl& p#ep"#e th#ee 16 %ol$ $ilutions o% ch"llenge 0i#us $ose st"#ting %#om the
$ilution cont"ining 166 TCI! )6. <iA equ"l qu"ntities o% these $ilutions "n$ <E< ,ith
8 4CS "n$ incu("te in " simil"# m"nne#.
6. 't the en$ o% incu("tion5 $ispense 166 ul o% se#um 0i#us miAtu#e "n$ 0i#us <E< miAtu#e
to
,ells o% the mic#o tit#e pl"te5 st"#ting %#om lo,e# $ilution o% the se#um to the highe#.
Inclu$e ,ells %o# cell cont#ol "n$ to this "$$ 166 U/ o% <E<. E"ch se#um $ilution "n$
0i#us $ilution shoul$ (e inocul"te$ to "t le"st . ,ells.
-. Incu("te the pl"tes in " C9 incu("to# "n$ t"2e the #e"$ing "t the en$ o% 3
#$
"n$ -
th
$"&.
/oo2
%o# the C*E in ,ells in ,hich 0i#us $ilutions ,e#e "$$e$ "n$ c"lcul"te the "ctu"l TCI!
)6
emplo&e$ in the test. This shoul$ (e "#oun$ 166 TCI!
)6
pe# 6.1 ml. Note $o,n the
highest se#um $ilution sho,ing complete neut#"lis"tion o% C*E. C"lcul"te the )68
en$point $ilution (& using @ee$ +<uench %o#mul". I% the#e is initi"l neut#"lis"tion "t $"&
3 %ollo,e$ (& (#e"2th#ough "t $"& -5 it in$ic"tes p"#ti"l neut#"lis"tion $ue to "ntigenic
#el"tion ,ith othe# ente#o0i#uses.
EN@YME LIN8ED IMMUNOSORBENT ASSAY
P"#1&"&!'$( $% .$u!'$(. &(0 "#&6#(!. %$" ELISA
1. 6.6)< c"#(on"te (u%%e# pH >.6.
'. !issol0e 6.)3 g N"

C9
3
("nh&$#ous) in $istille$ ,"te# "n$ m"2e %in"l 0olume to 166
ml.
:. !issol0e 6.. g N"HC9
3
in $istille$ ,"te# "n$ m"2e %in"l 0olume to 166 ml.
To 3 ml o% solution '5 slo,l& : till pH is >.6 ("pp#oAim"tel& 63 ml).
. *hosph"te cit#"te (u%%e#5 pH ).6.
". !issol0e 11.> g o% N"

H*9
.
5 H

9 in $istille$ ,"te# "n$ m"2e up to 1 lit#e (Solution ').

(. !issol0e -.6 g o% cit#ic "ci$ in $istille$ ,"te# "n$ m"2e up to 1 lit#e (Solution :).
<iA )) ml o% Solution ' ,ith .) ml Solution :.
'$?ust pH to ).6 ,ith eithe# " o# ( i% necess"#&.
3. 6.61 < *hosph"te (u%%e# s"line (*:S)5 pH -..
!issol0e 3.6 g o% N"Cl5 6.g o% EC15 o.g o% EH

*9
.
"n$ 1.) g o% N"

H*9
.
. H

9 in
$istille$ ,"te# "n$ (#ing the %in"l 0olume to 1666ml. '$?ust pH to -. ,ith 6.1 N N"9H
o# 6.1N HC1 i% necess"#&. '$$ 6.) ml// T,een 6 (*ol&oA&eth&lene so#(it"n
monol"u#"te) to 1D *:S to m"2e *:ST.
ANTIGEN PREPARATION OF HERPES SIMPLEA VIRUS
1) 6.)ml o% " stoc2 o% He#pes simpleA 0i#us (HSC+1) is "$$e$ to " @ouA (ottle ,ith "
monol"&e# o% Ce#o cell line "%te# $isc"#$ing the g#o,th me$ium.
) '$so#( the 0i#us %o# 36 mins "t 3- C "n$ "$$ 166 ml o% m"inten"nce me$ium. Incu("te
in " C9

incu("to# "t 3- C.
3) 9(se#0e %o# e0i$ence o% C*E (.+.3 h).
.) @emo0e the supe#n"t"nt me$ium "n$ h"#0est the cells ,ith 16+6 ml o% ste#ile *:S5 ,hen
P>68 o% the cells sho, C*E.
)) Cent#i%uge %o# ) mins "t 1666 #pm "n$ ,"sh the cells th#ice ,ith ste#ile *:S.
6) @esuspen$ the cells in " minim"l "mount o% *:S (eA11ml).
-) Sonic"te the suspension "t 66 c&cles/secon$ continuousl& %o# ) mins "n$ pulse$
sonic"tion %o# 3 mins.
3) Cent#i%uge the sonic"te %o# 16 mins "t 1666 #pm.
>) The supe#n"t"nt is use$ "s the "ntigen "%te# estim"ting the p#otein concent#"tion.
16) Cont#ol Ce#o cell "ntigen is p#ep"#e$ in " simil"# m"nne# ,ith moc2 in%ecte$ Ce#o cells.
(Ste#ile me$ium to (e use$ inste"$ o% the 0i#us suspension).
N9TE 1 The p#otein 0"lues o% the HSC "n$ Ce#o cell "ntigens shoul$ (e m"tche$ (e%o#e it is
use$ in the test.
MEASLES VIRUS ANTIGEN PREPARATION
1) 1 0i"l o% comme#ci"l <e"sles 0"ccine #econstitute$ in 6.) ml o% cell cultu#e me$ium "n$
m"$e up to ) ml ,ith s"me me$ium "n$ inocul"te$ into 1 @ouA (ottle ,ith " monol"&e#
o% Ce#o cell line.
) '$so#ption is c"##ie$ "t 3- C %o# 1 h
3) <"inten"nce me$ium is "$$e$.
.) C&top"thic e%%ect is %i#st seen on thi#$ $"& "n$ it g#"$u"ll& inc#e"ses o0e# neAt %ou#
$"&s. 9n the se0enth $"& eAtensi0e C*E c"n (e seen
)) The %lui$ is then %#o=en "n$ th",e$ t,ice "n$ 2ept "s see$ stoc2.
PREPARATION OF ANTIGEN
1. Ci#us is g#o,n in @ouA (ottle "n$ %lui$ h"#0este$ "septic"ll&.
. Spin "t 1666 #pm %o# 36 min.
3. To supe#n"t"nt "$$ so$ium chlo#i$e (.gms/166ml) "n$ *E; p#ecipit"te "t "
concent#"tion o% 168. (16gms/166ml)Q ("$$ N"cl %i#st "llo, it to $issol0e "n$ then "$$
*E; slo,l&)
.. Sti# %o# h"l% "n hou# in ice ("th "n$ "$$ 6.18 so$ium "=i$e ,hile sti##ing.
). Eeep %o# t,o hou#s o# o0e#night in #e%#ige#"to#.
6. Spin "t 16666 A g %o# 36 min.
-. !isc"#$ supe#n"t"nt.
3. @esuspen$ pellet in 1/166
th
o% the o#igin"l 0olume ,ith no#m"l s"line.
>. !i"l&se "g"inst ste#ile no#m"l s"line cont"ining 18 so$ium "=i$e %o# .h ,ith th#ee
ch"nges. (!i"l&sis ("gs "#e p#et#e"te$ (& (oiling in 18 so$ium c"#(on"te solution in
$istille$ ,"te# cont"ining lm< E!T').
16. Collect the $i"l&se$ "ntigen cent#i%uge "n$ collect supe#n"t"nt )<< E!T' J 66<<
N"HC9
3
in 166 ml !.7. A 6 min. "n$ estim"te the p#otein (e%o#e using it in the test.
11. Cont#ol Ce#o "ntigen is p#ep"#e$ in " simil"# m"nne# ,ith %lui$ collecte$ %#om moc2
in%ecte$ Ce#o cells.
EATRACTION OF :APANESE ENCEPHALITIS VIRUS ANTIGEN 7ITH
SUCROSE AND ACETONE
1. The in%ecte$ suc2ling mouse (#"in is homogenise$ ,ith %ou# 0olumes o% chille$ 3.)8
"queous solution o% suc#ose in homogenise# (7"#ing (len$e#) (eA. I% the tot"l ,eight o% the
mouse (#"in is g then 3ml o% suc#ose solution shoul$ (e "$$e$)
. The homogen"te is "$$e$ $#op ,ise ,ith (#is2 mech"nic"l sti##ing to 6 0olumes o%
chille$ "cetone. The miAtu#e is sh"2en 0igo#ousl& "n$ "llo,e$ to st"n$ %o# 1) min.
The mil2& supe#n"t"nt "cetone is $ec"nte$5 the se$iment "t this st"ge (eing " pin2 gumm&
m"ss ,hich is tightl& "$he#ent to the (ottom o% the cent#i%uge (ottle.
3. ' 0olume o% %#esh "cetone equ"l to th"t o#igin"ll& use$ is "$$e$ to e"ch (ottle "n$ the
p#ep"#"tion "#e "llo,e$ to st"n$ in "n ice ("th %o# "t le"st one hou# to $eh&$#"te the
se$imentQ "%te# su%%icient time the se$iment is #e"$il& #e$uce$ to " %ine suspension (& use o% "
thic2 gl"ss #o$. The (ottles "#e then cent#i%uge$ "s (e%o#e. The supe#n"t"nt %lui$ is
"spi#"te$ out "n$ the se$iments %#om "ll (ottles "#e poole$ into one (ottle ,ith the help o%
sm"ll qu"ntit& o% %#esh "cetone.
.. '%te# most o% the %in"l supe#n"t"nt "cetone h"s (een #emo0e$ (& "spi#"tion the (ottle is
close$ ,ith " #u((e# stoppe# %itte$ ,ith gl"ss tu(ing5 ,hich is connecte$ to " 0"cuum pump
%o# $#&ing. The $#&ing is continue$ %o# one hou# ,ith the (ottle 2ept imme#se$ in "n ice ("th.
The pump is p#otecte$ %#om cont"min"tion (& " t#"p cont"ining " l"#ge qu"ntit& o% non+
"(so#(ent cotton.
).To the $#& po,$e# " 0olume o% 6.>8 N"Cl is "$$e$ ,hich is equ"l to /)
th
o% the tot"l
Colume o% homogen"te o#igin"ll& use$ (EA. I% the homogen"te is 16ml5 .ml o% 6.>8 N"Cl is
"$$e$ %o# #eh&$#"tion). The (ottle ,ith "ntigen is le%t o0e#night in ice ,"te# in the col$ #oom.
The neAt mo#ning it is cent#i%uge$ "t 16666 #pm %o# one hou#. The supe#n"t"nt %lui$ is
"ntigen. 4o# gel $i%%usion test uncent#i%uge$ "ntigen is use$.
N$!# / T#is (u%%e# (6.1<) pH 3 to > c"n (e use$ #eh&$#"tion o% suc#ose "cetone "ntigen
inste"$ o% 6.>8 N"Cl.
P"$c#0u"# %$" b'$!'(*&!'$( $% )$($c$(& &(!'b$0*
R#&6#(!. /
1) <onoclon"l "nti(o$ie s 1 'scitic %lui$s %#om :'/:/c mice inocul"te$ ,ith $esi#e$
clones h"0e to (e p#ecipit"te$ using the so$ium sulph"te technique. '%te# $i"l&sis the
p#otein concent#"tion is me"su#e$ ("(so#("nce "t 36nm) "n$ "$?uste$ to 1mg in 6.1<
so$ium (ic"#(on"te solution (stoc2 solution).
) :iotin+N+h&$#oA& succin"mi$e (:@/ :ethes$" )1 1mg o% :iotin+n+h&$#oA& succin"mi$e is
$issol0e$ in 1ml o% $imeth&lsul%oAi$e (Sigm" o# <e#c2). *#ep"#e it %#esh on the $"& o%
use. !9 N9T ST9@E S9/UTI9N *@E*'@E! E'@/IE@.
3) ;oo$ qu"lit& $i"l&sis ("gs ()666 mol.,t cuto%%)
PROCEDURE
To 1666 ug o% monoclon"l "nti(o$& in 1ml o% so$ium (ic"#(on"te solution5 "$$ 66 ul o%
stoc2 (iotin solution $#op+,ise (1mg/ml !<S95 it c"n (e 1665 66 B .66 "cco#$ingl& chec2
(e%o#e use). '%te# miAing ,ell incu("te "t #oom temp. %o# . hou#s ,ith pe#io$ic miAing. 't
the en$ o% . hou#s $i"l&se the miAtu#e "g"inst ste#ile no#m"l s"line ()66 ml A 3 ch"nges) %o#
13 hou#s "t . C to #emo0e un(oun$ (iotin. '%te# $i"l&sis me"su#e the eA"ct 0olume. I% the
0olume is 1 ml then the p#otein concent#"tion is )66 ug/ml. !ist#i(ute )6 ul 0olumes into 6
Eppen$o#% tu(es ((ullets). '$$ )6 ul o% ste#ile gl&ce#ol to e"ch (ullet (c#&op#ese#0"ti0e).
E"ch (ullet no, cont"ins )6 ug o% (iotin&l"te$ monoclon"l "nti(o$&. Sto#e "t +-6 C.
Estim"te optim"l concent#"tion o% this $iluent %o# this "nti(o$&. !9 N9T USE */'IN *:S
SINCE HI;H :'CE;@9UN! IS 9:T'INE!. The con?ug"te use$ is ST@E*T'CI!IN
*E@9DI!'SE (:@C :ethes$") "t " $ilution o% 111666 ()6 ul/,ell in <'C+E/IS').
Incu("tion ,ith (iotin&l"te$ monoclon"l "nti(o$& is %o# hou#s "t 3- C in " ,"te# ("th
(%lo"t the pl"tes).
DETECTION OF I6G ANTIBODIES TO HSV<B BY ELISA
1. Co"t E/IS' pl"tes ,ith HSC "n$ Ce#o "ntigens "t " p#e$ete#mine$ concent#"tion in
c"#(on"te
(u%%e# pH >.65 166 ul/,ell.
. Incu("te o0e#night "t . C.
3. !isc"#$ "n$ ,"sh the pl"tes th#ice ,ith *:ST.
.. Quench the pl"tes ,ith 18 s2imme$ mil2 po,$e# in *:ST5 1)6 ul/,ell.
). Incu("te %o# h#s "t 3- C.
6. !isc"#$5 ,"sh pl"tes th#ice ,ith *:ST.
-. '$$ 166 ul/,ell se#"5 CS4 "pp#op#i"tel& $ilute$ in *:ST. Inclu$e positi0e5 neg"ti0e
cont#ols "n$ (l"n2s.
3. Incu("te %o# h#s "t 3- C.
>. !isc"#$5 ,"sh th#ice ,ith *:ST.
16. 166 ul/,ell o% go"t/#"((it "nti+hum"n Ig; pe#oAi$"se con?ug"te (:"ng"lo#e ;enei)
($ilution to (e use$1 11666 in *:ST ,ith 18 no#m"l go"t se#um)
11. Incu("te %o# h#s "t 3- C.
1. !isc"#$5 ,"sh th#ice ,ith *:ST.
13. '$$ 166 ul/,ell o% " solution cont"ining 9*! (.mg/16ml o% *C:5 pH )) ,ith 16 ul
o% H

9
.
1.. Incu("te in the !'@E "t #oom tempe#"tu#e %o# 6 minutes
1). Stop the #e"ction ,ith )6 ul/,ell o% .N H

S9
..
16. @e"$ the pl"te "t .> nm on "n E/IS' #e"$e#.
1-. ' s"mple is consi$e#e$ positi0e i% the $i%%e#ence in 9! 0"lues is P6.1 (et,een the
HSC "ntigen "n$ Ce#o "ntigen "t th"t p"#ticul"# $ilution. 'ntigen is consi$e#e$
signi%ic"nt "t th"t $ilution.
DETECTION OF I6G ANTIBODIES TO MEASLES VIRUS BY ELISA
1. Co"t E/IS' pl"tes (166 ul /,ell) ,ith me"sles 0i#us "ntigen "n$ Ce#o "ntigen "t "
p#e$ete#mine$ concent#"tion in c"#(on"te (u%%e# pH >.6
. Incu("te o0e#night "t .C.
3. !isc"#$ contents o% the ,ells "n$ ,"sh th#ice ,ith *:ST.
.. Quench ,ith 18 s2imme$ mil2 po,$e# in *:ST (1)6 ul/,ell). Incu("te "t 3-C %o#
h#s.
). !isc"#$ "n$ ,"sh th#ice ,ith *:ST.
6. '$$ the #equi#e$ $ilutions o% CS4/Se#um s"mples (166 ul/,ell) "n$ incu("te "t 3- C %o#
h#s. Inclu$e positi0e 5 neg"ti0e cont#ols "n$ (l"n2s.
-. !isc"#$ "n$ ,"sh th#ice ,ith *:ST.
3. '$$ 166 ul/,ell o% #"((it "nti+hum"n Ig; pe#oAi$"se con?ug"te (:"ng"lo#e ;enei)
$ilute$ 11666 in *:ST ,ith 18 no#m"l go"t se#um "n$ incu("te %o# h#s. "t 3- C.
>. !isc"#$ "n$ ,"sh th#ice ,ith *:ST.
16. '$$ 166 ul/,ell o% " solution cont"ining 9*! (.mg/16ml o% *C:5pH)) "n$ 16 ul o%
H

9
.
Incu("te "t @T %o# 6 mins in the $"#2.
11. Stop the #e"ction ,ith )6 ul/,ell o% .N H

S9
.
.
1. @e"$ the #esults "t .> nm on "n E/IS' #e"$e#.
13. ' s"mple is consi$e#e$ positi0e i% the $i%%e#ence in 9! 0"lues is P 6.1 (et,een the
me"sles 0i#us "ntigen "n$ Ce#o "ntigen "t "n& gi0en $ilution.
DETECTION OF I6M ANTIBODIES TO :EV BY AN IGM ANTIBODY CAPTURE
ELISA 3MACELISA4
1. Co"t E/IS' pl"tes ,ith 166 ul/,ell o% " p#e$ete#mine$ concent#"tion o% #"((it "nti+
hum"n Ig< "nti(o$ies (!'E9*'TTS) in c"#(on"te (u%%e# pH >.6.
. Incu("te "t . C o0e#night.
3. !isc"#$ "n$ ,"sh ,ith *:ST th#ice.
.. Quench ,ith 18 s2imme$ mil2 po,$e# in *:ST (1)6 ul/,ell). Incu("te "t 3-C %o# h#s
). !isc"#$ "n$ ,"sh th#ice ,ith *:ST.
6. '$$ 166 ul/,ell o% CS4 ($ilute$ 11166 in *:ST) in $uplic"tes. Incu("te %o# .h# "t 3- C.
-. !isc"#$ "n$ ,"sh ,ith *:ST th#ice.
3. '$$ 166 ul/,ell o% GEC mouse (#"in "ntigen ()6H' units/166 ul $ilute$ in *:S).
Incu("te "t . C o0e#night.
>. !isc"#$ "n$ ,"sh ,ith *:ST th#ice.
16. '$$ 166ul/,ell o% (iotin&l"te$ NIC c#oss+#e"cti0e monoclon"l "nti(o$& to GEC ()ul/16
ml o% *:ST cont"ining 18 s2imme$ mil2 po,$e# "n$ 18 no#m"l go"t se#um). Incu("te
"t 3- C %o# h#.
11. !isc"#$ "n$ ,"sh ,ith *:ST th#ice.
1. '$$ 166 ul/,ell o% st#ept"0i$in pe#oAi$"se con?ug"te (!'E9*'TTS) $ilute$ 11)666 in
*:ST cont"ining 18 no#m"l g#o,th se#um. Incu("te %o# 1) mins "t @T.
13. '$$ 166 ul/,ell o% " solution cont"ining 9*! (.mg/16ml o% *C:5pH)) "n$ 16 ul o%
H

9
.
Incu("te "t @T %o# 6 mins in the $"#2.
1.. Stop the #e"ction ,ith )6 ul/,ell o% .N HS9..
1). @e"$ the #esults "t .> nm on "n E/IS' #e"$e#.
16. 'pp#op#i"te high positi0e cont#ol5 lo, positi0e cont#ol5 neg"ti0e cont#ol %o# CS4 "n$
se#um "s ,ell "s (l"n2s shoul$ (e inclu$e$ in e0e#& "ss"&.
1-. The #esults "#e eAp#esse$ "s <'CE/IS' units ,hich is c"lcul"te$ using the %ollo,ing
%o#mul" 1
E/IS' units I (9! o% test s"mple + 9! o% neg"ti0e cont#ol)
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++ A 166
(9! o% ,e"2 positi0e + 9! o% neg"ti0e cont#ol)
Inte#p#et"tion o% #esults is "s %ollo,s 1
". 'n& test s"mple ,ith P 166 units is consi$e#e$ positi0e %o# Ig< "nti(o$ies to GEC.
(. ' s"mple ,ith E/IS' units (et,een 36+>> is consi$e#e$ p#o("(l& positi0e %o# Ig<
"nti(o$ies to GEC (ut #equi#es %u#the# testing ,ith !engue "n$ 7est Nile 0i#us "ntigens
"s ,ell "s no#m"l mouse (#"in "ntigen to eAclu$e %"lse positi0e #e"ctions.
c. ' s"mple ,ith E/IS' units K 36 is consi$e#e$ neg"ti0e Ig< "nti(o$ies to GEC.
IMMUNOASSAYS FOR THE DETECTION OF RETROVIRAL ANTIBODIES
7#.!#"( b$! &(&*.'. %$" HTLV<B 1"$!#'(.
&4 P"#1&"&!'$( $% 7#.!#"( b$! .!"'1. / Use solu(ili=e$ 0i#us o(t"ine$ %#om <T o#
HUT16 cells ,hich h"s (een pu#i%ie$ "n$ concent#"te$ 1666 %ol$ (& suc#ose g#"$ient
cent#i%ug"tion .
b4 P$*&c"*&)'0# G# E#c!"$1+$"#.'. /
1) /"&e# the 168 #esol0ing gel "n$ incu("te %o# 1)min. "t @T ,ith $istille$ ,"te# l"&e#e$
o0e# it.
) '%te# this #emo0ing the ,"te# l"&e# .8 st"c2ing gel on to the #esol0ing gel "n$ inse#t
com(s into the st"c2ing gel. Incu("te "t @T %o# 1) min.
3) @emo0e the com( "n$ %lush the ,ells ,ith $istille$ ,"te#.
.) C"#e%ull& #emo0e the cl"mps "n$ mount the gel pl"tes on to the Elect#opho#esis unit "n$
"$$ t"n2 (u%%e# into (oth lo,e# "n$ uppe# t"n2s. Ensu#e th"t the uppe# t"n2 h"s su%%icient
(u%%e# to co0e# the ,ells in the gel.
)) !en"tu#e the p#otein s"mple (HT/C+1 "ntigen) in s"mple (u%%e# (& (oiling %o# 3 min. in "
,"te# ("th. /o"$ ) to )6 ul o% this $en"tu#e$ s"mple into e"ch o% the ,ells c"#e%ull&
"0oi$ing spills "n$ "i# (u((les using " mic#o pipette.
6) @un the gel in the const"nt 0olt"ge mo$e "t " setting o% 66 0olts until the $&e %#ont
#e"ches the (ottom o% the gel pl"te.
-) @emo0e the gels c"#e%ull& "n$ use %o# t#"ns%e##ing o% p#oteins to nit#ocellulose
mem(#"nes (& 7este#n :lotting.
c4 7#.!#"( B$!!'(6 /
1) T#"ns%e# Elect#opho#ese$ gel to t#"& cont"ining 7este#n :lot (u%%e# (68 meth"nol in 1A
t#is+gl&cine (u%%e# ,hich h"s (een $e+"e#"te$ %o# 1) min.)
) *l"ce " p#e+,ette$ nit#ocellulose sheet5 ,hich h"s (een cut to the s"me $imensions "s the
gel on the gel ensu#ing th"t the#e "#e no "i# (u((les t#"ppe$ in (et,een.
3) Eeep %ilte# p"pe# p"$s (elo, the gel "n$ "(o0e the nit#ocellulose p"pe# to m"2e "
s"n$,ich.
.) *l"ce this s"n$,ich in the "pp#op#i"te clip "n$ inse#t into the Elect#o (lotting t"n2
cont"ining (u%%e#.
)) Ensu#e th"t the "no$e is to,"#$s the nit#ocellulose mem(#"ne si$e ,hile the c"tho$e is
to,"#$s the gel.
6) The elect#o(lotting is c"##ie$ out using const"nt cu##ent mo$e "t 1)6 <' %o# h#s.
-) @emo0e the nit#ocellulose mem(#"ne ("l,"&s h"n$le gentl& using " %o#ceps) "n$ pl"ce in
" t#"& cont"ining 18 mil2 po,$e# in *:S. Incu("te "t . C o0e#night.
3) The nit#ocellulose mem(#"ne is #emo0e$ %#om the t#"& "n$ c"#e%ull& cut into thin st#ips.
Sto#e in " =ip loc2 ("g "t . C %o# upto 1 month.
>) *l"ce thin st#ips in " 7este#n :lot immuno "ss"& t#"& "n$ ,"sh ,ith *:S using "
#oc2ing pl"t%o#m.
16) '$$ .) ml o% 11)6 $ilution o% CS4 o# 11166 $ilution o% se#um p#ep"#e$ in *:S
cont"ining 18 s2imme$ mil2 po,$e#.
11) Incu("te "t #oom tempe#"tu#e on the #oc2ing pl"t%o#m %o# h#s. 7"sh th#ice ,ith *:ST
on the #oc2ing pl"t%o#m.
1) '$$ .) ml o% "pp#op#i"te $ilution o% "nti+hum"n Ig; pe#oAi$"se con?ug"te p#ep"#e$ in
*:ST cont"ining 18 mil2 po,$e# "n$ incu("te on the #oc2ing pl"t%o#m "t @T %o# h#s.
13) 7"sh th#ice ,ith *:ST on the #oc2ing pl"t%o#m.
1.) '$$ ml o% su(st#"te solution (!i"mino(en=i$ine . mg /16 ml o% *:S "n$ 16 ul o% H

)
"n$ #oc2 on the pl"t%o#m %o# 1 min. "n$ 2eep the t#"& in the $"#2 %o# 16 min. "t #oom
tempe#"tu#e.
1)) Stop the #e"ction (& "$$ing $istille$ ,"te# to e"ch o% the ,ells.
ELISA 1"$c#0u"# %$" 0#!#c!'$( $% &(!' HTLV<B &(!'b$0'#.
1. Use cell cultu#e pu#i%ie$ "n$ concent#"te$ 0i#us solu(ili=e$ 0i#us %o# E/IS'. The optim"l
concent#"tion shoul$ (e p#e+$ete#mine$ in " chec2e#(o"#$ tit#"tion. Usu"ll& )+16 ug/ml
o% 0i#"l "ntigen gi0es (est #esults.
. Co"t pl"tes ,ith 166 ul o% "ntigen $ilute$ in )6 m< N"HC9
3
(u%%e#5 pH >.6 "n$ incu("te
"t . C o0e#night.
3. !isc"#$ contents "n$ #inse th#ice ,ith *:S. '$$ 66 ul o% .)8 :S' solution in *:S
cont"ining 1.) 8 mil2 po,$e# "n$ )8 suc#ose to e"ch o% the ,ells "n$ incu("te "t 3-
C %o# h#s. 7"sh pl"tes th#ice ,ith *:ST.
). '$$ 166 ul o% CS4/se#um s"mples $ilute$ %i0e %ol$ (11) to 116)) in *:ST ,ith 8 mil2
po,$e# to the ,ells in the E/IS' pl"te.
6. Incu("te "t 3- C %o# h#s. 7"sh pl"tes th#ice ,ith *:ST.
-. '$$ 166 ul o% go"t "nti+hum"n Ig; pe#oAi$"se con?ug"te (:"ng"lo#e ;enei) $ilute$
111666 in *:ST cont"ining 18 no#m"l go"t se#um. Incu("te %o# h#s. "t @T. 7"sh th#ice
in *:ST.
3. '$$ 166 ul o% su(st#"te (. mg 8 o% 9*! in *C: ,ith 6.66)8 H

) "n$ incu("te in the

$"#2 %o# 6 mins. "t @T.
>. Stop the #e"ction ,ith )6 ul o% .N H

S9
.
.
16. <e"su#e the 9! 0"lues "t .>nm in "n E/IS' @e"$e#.