ELISA readings after booster shot

[biology_06er]

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

[Wallyanna]

Could it be that the maximum amount of antibody antigen reaction readable was already there pre booster in the higher dilutions, while there was more antigen available for reaction in the testing at the lower dilutions?
Hope my meaning is clear.
Let me try another way. Does the test have only so much antigen available to react at each level? If the antigen available for reaction was fully utilized at low dilutions pre booster, then there would be no greater reaction post booster even if there were more antibodies available to react.

Antigen all used up, some additional antibodies in there not having antigens available with which to react. If so, then the reaction would not be stronger on your test even if there are more antibodies in vivo. If so, then no greater number of antibodies could give any stronger reactions.

Hope this helps.

[Wallyanna]

Oh yeah, forgot to add, if you are using antigen reagent from a supplier, did you use the same supplier pre and post? Also the same lot number of antigen reagent? Small variability in antigens can sometimes occur with new reagent or between lots of same reagent. Also check your expiration dates on everything to see if you can discover a reason for the variance.
Did you save some immediately frozen serum from the pre to test again alongside the post, using exactly the same reagent same method same equipment same day, to eliminate variables? Or are you comparing results from prior test on pre with current test on post?

[JackBean]

did you quantify added amount of antibody?