Please... ELISA background...

Graham Shepherd muhero at globalnet.co.uk
Wed Sep 30 16:30:11 EST 1998


Hang-Rae Kim wrote in message <3611B943.900120F4 at plaza1.snu.ac.kr>...

>We are developing an antigen capture ELISA using monoclonal antibodies
>(Mab).
<SNIP>
>the capture Mabs coated on the wells. The capture and detector Mab are
>the same Mab purified using Protein A column from ascitic fluids. Does
>anyone have an explanation for this? Even better, can anyone suggest a
>way to overcome it?
>

A couple of thoughts -

(1) Residues of protein A contaminating the Ab preparation and allowing
cross-linking between capture Ab and conjugate?

If so, you could try using Fab fragments in the conjugate.

(2) BSA binding the conjugate? (I know it's in the conjugate diluent but
there could still be a reaction - solid phase reaction kinetics are
different from free-phase in solution.)

Try a different blocking agent - casein, gelatine, polyvinylpyrrolidone.

GS





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