Effect of affinity on polyclonal interaction with antigen

[Angelbio]

We are developing in-house assays to measure quantitatively the amount of polyclonal antiserum produced in rabbits against a specific antigen.

To do this we have an elisa where we coat with the antigen, use an affinity purified (in house) preparation of polyclonal antibody from a representative pool of rabbit serum (approx 20 rabbits), to prepare a standard curve using known amounts of antibody determined by A280. Then the amount of antibody bound is measured by an anti rabbit HRP conjugate.

The assay works really well. However I have always maintained that the theory is flawed, but my boss disagrees with me.

If each rabbit makes a seperate population of polyclonal antibodies with different affinities, then each rabbit serum will react differently with the antigen coated on the plate. the only antibody population that can be measured accurately with the standard curve is the one from which the standard curve was made, with each affinity of antibodies in the same ratios. This is because what you measure depends not just on the amount of antibody present but also the affinities of the antibodies present.

We do indeed see a differnece in the quantitation that the most recent batch of affinity purified antibody (from different rabbits to that the standard curve is made up with ) measured in the ELISA and by A280.

So anybody any thoughts? I now have to justify why my ELISA reds different results to the A280 and she does not accept my argument...

[Elisadeveloper]

I am not surprised that the two preparations of affinity purified antibodies don't match up perfectly. However, this does not mean that the first set of rabbits responded differently to the antigen compared to the second set of rabbits.

If the two standard curves are the same shape but shifted slightly from each other, the differences could simply be due to denaturation during storage, preparation or handling. If the two curves are different shapes or are not parallel, then that might indicate that there is some difference between the responses in the two groups of rabbits.

The standard material is somewhat detached from the antibodies within the indvidual rabbit sera. I am assuming that the affinity purification has been performed on an antigen column which will select for high affinity populations. Also consider what acid dissociation (or other procedure) will do to the purified product, also consider freeze thaw and leached antigen etc etc. Another concern will be the timing of bleeds relative to the timings of immunizations as antibody/antigen complexes may circulate for some considerable time and the complexes won't purify.

With all this to consider, I don't think it's surprising that two preparations of affinity purified antibody don't perfectly match up in the assay and by mass and it may be more appropriate to express results from individual rabbits in relative terms or as a titre which is closer to an activity measure.

The questions that you are concerned with are common to classical immunogenicity principles (anti drug antibody or ADA) and the specific issues and ways to deal with them in regulated research are covered by a number of recent white papers on the subject which would be a good source of information for you.

[Angelbio]

Wow! Fabulous answer - can I come and work for you?!!!

I am looking up those white papers now.

I agree with you about how the standard antibody is quite different from each of the rabbit sera, I think we need to regard the assay as semi-quantitative. However I do not think it is without merit, and will give us information as to which rabbits have responded better in comparing immunisation protocols (though I have always sugeested end point titres as a simpler alternative...). I am not very good at arguing my case though and face stiff oppposition.

[Elisadeveloper]

yes the assay is a fit for the purpose of comparing the response of each rabbit, and it is quantitative, but in relative terms. I think the crucial thing here is to work with one reference preparation, maintain stocks at -80 or even consider lyophilizing stocks so you have some confidence that you won't get getting a gradual degradation over time. If you have to change reference preparation, make sure you qualify the new batch against the old and demonstrate that it generates the same rank order of response in a panel of rabbits as the previous batch.

[Angelbio]

I was wondering if it was possible to post the graph of the curves of the two antibody preps mentioned above to see if anyone could see if there was an obvious 'affinity' difference between them?

I couldn't work out how to put in the graph to the post...

[Elisadeveloper]

you can e-mail me the graphs and I'll see if I can figure something out
aurata78athotmaildotcom

[Angelbio]

okay thanks, I have just done this - been really busy for the past week, no time to do it before now - earning my keep!