Montanide adjuvant

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I would like to ask your advice about the use of Montanide in rabbits.

We use rabbits for antibody production and some of the inocula contain Montanide as an adjuvant. After subcutaneous administration, a few rabbits get swellings on the injection site, frequently accompanied with bruising. I've treated the affected animals with Carprofen but it doesn't seem to decrease the size of the swelling.

The swellings are not ulcerated and don't seem painful to the touch or to be causing any discomfort but it's hard to tell in rabbits. We use the weight gain as a guide to help us decide if the rabbits are suffering.

The severity of the protocol is mild and one of the endpoints is an inflammation bigger than 1cm diameter but that is a value inherited from past studies.

My question is: do you think a swelling of 1cm is a reasonable endpoint or it would make more sense if it's accompanied by other signs or pain/discomfort that we can observe in rabbits? How have you measured the pain in similar studies?

Have you considered other alternatives to the use of rabbits for this purpose? The use of chicken egg yolk is non-invasive.

The EU created a document about the non-use of animals for antibody production: The EURL ECVAM Recommendation on Non-Animal-Derived Antibodies.

I wouldn’t regard a 1cm swelling alone with no other signs as approaching a mild severity limit. But that is a decision for your PPLH and for your AWERB to advise on. The information we don’t have is the relation between occurrence of swellings and their size, and the efficacy of antibody production.

We discussed this issue at the last AWERB meeting when the study was presented and the group evaluated the use of recombinant antibody technology. Unfortunately for these particular antibodies, it was not feasible because the recombinant antibodies did not perform as expected in the statutory tests. Also, the cost and time of producing recombinant antibodies for the range required would be unrealistically high without a guarantee of successful outcome. But we'll keep trying and hopefully we can find viable alternatives soon.

Thanks for your reply. With Montanide we usually observe swellings in most animals but smaller than 1cm that don't seem to cause any pain. The last couple of times we have seen a small percentage of animals (2/18) getting larger swellings. The researcher is evaluating the possibility of using a different adjuvant that absorbs better subcutaneously.

As the UK is no longer a member state of the European Union, I don't know what attitude the UK Competent Authority will take to an application for the use of animals for antibody development or production.  In Ireland, the Competent Authority has made it clear that they don't expect that anybody will even bother submitting an application for a Project Authorisation that includes a proposal to use animals for antibody production (although they will, of course, continue to judge each application on its own merits).