ELISA after a booster shot - (Feb/22/2012 )

Hi there,

I recently did an ELISA to detect if my rabbit was producing antibodies...I got postive absorbance readings and sent away for a booster shot...again positive readings buut my question is, I diluted my antibody 1:200, 1:400, 1:800 etc etc and found that the post booster gave a lower reading than pre booster..if a booster shot was given, does this not mean MORE antibodies would have been produced thus a higher ab reading shld have been given the second time around for the same dilution?...It is like this for the first few dilutions and nearing the end as it gets more dilute, the readings are higher for post booster than pre....hope this makes sense...and someone can explain if I have misunderstood!

Thanks,
b_06er

Two things could be going on.

The immunizing antigen may be bound to some of the circulating antibody, so that you have more antibody after the boost, but a proportion of it is complexed to the antigen so is unreactive in the assay. Alternatively, you may have a high dose hook in your assay (prozone effect) , so that the amount of antibody binding to your capture reagent is so high at low dilution, that there simply isn't enough room for the labelled detection antibody to bind. It becomes sterically hindered. The real result is at the high dilution. To remove the high dose hook effect you can reduce the coating concentration. If you are using an anti H+L detection reagent, switch to an anti-Fc and this may also reduce the problem