INDIRECT ELISA

AIM: To determine the alpha tubulin IgG titers in the given set o sera !RINCI!LE:
ELISA is a po"er ul te#hni$ue used or dete#ting and

measuring antigens and antibodies% It #ombines the spe#i i#it& o antibodies "ith the sensitivit& o simple en'&me assa&s( b& using antibodies or antigens #oupled to an easil& assa&ed en'&me that possesses a high turnover number% ELISAs #an provide a use ul measurement o antigen or antibod& #on#entration Indirect ELISA In this t&pe o ELISA the plate is #oated "ith the antigen "hi#h is then probed "ith an antibod& that is spe#i i# to it )usuall& #alled the primar& antibod&*% Another antibod&( the se#ondar& antibod& that is spe#i i# to the primar& antibod& is then used% The se#ondar& antibod& is usuall& #on+ugated to an en'&me that enables the rea#tion to be dete#ted% Thus it is re erred to as indire#t be#ause the se#ondar& antibod& that is used to viasualise the rea#tion does not bind dire#tl& to the antigen%

!ro#edure: ,% -sing the given sto#. o alpha tubulin prepare /0ml o a "or.ing #on#entration o /ug1ml o alpha tubulin in !2S% Coat the plate "ith alpha tubulin b& dispensing ,003l o the "or.ing #on#entration in ea#h o the "ells o the given ELISA plate% /% Cover the plate "ith saline "rap and in#ubate it at 40C overnight% 5% !our o the #oating a material% 4% 6ash the plate "ith /003l o !2ST1"ell "ith sha.ing or three minutes% 6ash three times% 7% 2lo#. or t"o hours( at room temperature( using 0%78 I92L:C; in !2ST)/003l1"ell* <% During the last hour o blo#.ing prepare the samples to be used% Dilute ea#h set o sera "ith a dilution o ,:700 in I92L:C;% =% A ter t"o hours o blo#.ing( pour o the blo#.ing bu er% >% Add ,703l o ea#h sample in dupli#ate in the "ells o ro" A ?% Add ,003l o I92L:C; to the rest o the "ells% ,0% Ma.e a serial dilution rom Ro" A to Ro" G b& ta.ing 703l rom "ells o Ro" A and pla#ing it in the "ells o Ro" 2)mi@ "ell* and ta.e it to the "ells o Ro" C and so on up to Ro" G%

,,% In#ubate or ,hour at 5=0C% ,/% 6ash as des#ribed in step 4 but "ash si@ times% ,5% Add ,003l o Se#ondar& antibod& diluted at a dilution o ,:7000 to ea#h o the "ells% ,4% In#ubate at 5=0C or ,hr% ,7% 6ash as des#ribed in step 4% ,<% Add ,003l o the substrate to ea#h o the "ells and in#ubate in the dar. or ten minutes ,=% Stop the rea#tion "ith ,00ul o ,M A/S:4% ,>% Measure the :%D at 4?/nm ,?% !lot a graph o :%D against dilution% -se the immune and pre9immune sera to do this% -sing the graph determine the #ut9o then use the #ut9o to determine the antibod& titer o the immune sera%

Assignment: ,% 6hat se#ondar& antibod& is needed in this assa&B /% Mention the substrates that #an also be used in this assa&% 5% To prepare 0%07 M phosphate9#itrate bu er( pA 7%0 the ollo"ing reagents are needed: 0%/ M dibasi# sodium phosphateCCCCCC /7%=ml 0%, M #itri# a#idC%%CCCCCCCCCCCCCC%%/4%5ml ddA/:%CCCCCCCCCCCCCCCCCCCCCC70 ml Give a detailed outline o ho" &ou "ould practically prepare 0%07M !hosphate #itrate bu er% 4% Give the prin#iple in brie o the three other t&pes o ELISA