E L I S A w i t h p l a t e l e t s
来自 : 蚂蚁淘
OUTLINEThismodificationofqualitative
ELISA(Enzyme-LinkedImmunosorbentAssay)isusedforeitherscreeningdetectionofanti-plateletantibodiesorfordetectionofplatelet-associatedIg(PAIg)(shownhere).
PROTOCOL
- prepareplateletsfromcontrolandimmunizedmice
- distribute2.5x106plateletsperwellonF-bottomedMaxiSorpNunc-Immunomicroplates(GibcoBRL,Roskilde,Denmark),centrifugeat3000rpm,7min,10ѓC.
- fixwithPBS-PF2%for5min,RT
- wash3timesinPBS-tween20,dry
- blockhalfoftheplate(withplatelets)byPBS-BSA1%andcoatanotherhalfbyglycine(x1)-BSAfor1hourat37ѓC
- wash3timesinPBS-tween20,dry
- incubatedat37ѓcfor1hourwitha1:1000dilutionofratIgG2aanti-mousekappachainmonoclonalantibodyconjugatedtohorse-rADIshperoxidase(LO-MK1,LO/IMEX)
- revealwitho-phenylenediaminedihydrochloride(OPD)inrevelationsolution.
SOLUTIONS
- PBS-PF(paraformaldehyde),2%
- PBS-tween20,0.05%
- glycine(x1)-BSA,1mg/ml
- revelationsolution(citrate/phosphatebuffer),pH5,1L,watersolution=citriqueacid,5.1065g+Na2HPO4x2H20,9.08g
- glycinebuffer(coatingsolution),pH9.2,watersolution=glycine1M+NaCl1.5M
ADDITIONALINFOThisprotocolmaybeusedforscreeningofhybridomasupernatantsforanti-plateletAb.Inthiscase,theextrastepsuchasanincubationwithacertainSNandadditionalwashingshouldbeaddedbeforethestepNo7.
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