特色ELISA试剂盒

E L I S A w i t h p l a t e l e t s

OUTLINEThismodificationofqualitativeELISA(Enzyme-LinkedImmunosorbentAssay)isusedforeitherscreeningdetectionofanti-plateletantibodiesorfordetectionofplatelet-associatedIg(PAIg)(shownhere).

PROTOCOL

  1. prepareplateletsfromcontrolandimmunizedmice
  2. distribute2.5x106plateletsperwellonF-bottomedMaxiSorpNunc-Immunomicroplates(GibcoBRL,Roskilde,Denmark),centrifugeat3000rpm,7min,10ѓC.
  3. fixwithPBS-PF2%for5min,RT
  4. wash3timesinPBS-tween20,dry
  5. blockhalfoftheplate(withplatelets)byPBS-BSA1%andcoatanotherhalfbyglycine(x1)-BSAfor1hourat37ѓC
  6. wash3timesinPBS-tween20,dry
  7. incubatedat37ѓcfor1hourwitha1:1000dilutionofratIgG2aanti-mousekappachainmonoclonalantibodyconjugatedtohorse-rADIshperoxidase(LO-MK1,LO/IMEX)
  8. revealwitho-phenylenediaminedihydrochloride(OPD)inrevelationsolution.

SOLUTIONS

  1. PBS-PF(paraformaldehyde),2%
  2. PBS-tween20,0.05%
  3. glycine(x1)-BSA,1mg/ml
  4. revelationsolution(citrate/phosphatebuffer),pH5,1L,watersolution=citriqueacid,5.1065g+Na2HPO4x2H20,9.08g
  5. glycinebuffer(coatingsolution),pH9.2,watersolution=glycine1M+NaCl1.5M
ADDITIONALINFOThisprotocolmaybeusedforscreeningofhybridomasupernatantsforanti-plateletAb.Inthiscase,theextrastepsuchasanincubationwithacertainSNandadditionalwashingshouldbeaddedbeforethestepNo7.

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