Monoclonal antibodies were generated against a toxin. But in vitro neutralization assay has shown the antibodies not able to neutralize the toxin.
Biological effects of toxins depend upon binding to certain receptors or body components that we can call targets (depending on the toxin). When thy bind such targets inhibit important process or cause other deletereous effects to the organism (or to the cells if we think in an in vitro cellular assay). Antibodies bind small regions on the antigens, called epitopes. Each monoclonal antiboy bind a given epitope. If the epitope recognized by an anti-toxin antibody does not overlap with the toxin regions that are responsible for its binding to the target, the toxin can still bind to its target and cause toxic effects despite the presence of the antibody bound to the toxin. Only antibodies that block binding of the toxin to its target are neutralizing.
Perhaps the antibody binds the correct epitope to block toxin function, but its binding affinity is too low compared to the affinity of the interaction between the toxin and the target, so when all of them are together the toxin binds to its target and the antibody cannot bind. This antibody cannot neutralize toxic effects. Another antibody recognizing the same epitope in the toxin, but with higher affinity, is able to bind strongly to the toxin and inhibit its binding to the target.
You also have to consider the relative amounts of antibody, toxin and target in your assay. If the toxin is in excess over the antibody, the antibody will neutralize part of the toxin, but the remaining free toxin will cause the toxic effects that you detect. Depending on the antibody affinity, the amount of antibody required to achieve neutralization can vary. With lower affinity, higher concentrations are needed. At the end, you have a competition between the antibody and the target to bind the toxin. Depending on the winner you have neutralization or toxicitxy. On the other hand, if the antibody does not bind the right epitope, as described above, it will not neutralize even at very high concentrations.
Yes, I agree with Gertrudis comments. In addition, I would recommend to determine the affinity constant by a physicochemical method, such as SPR. In addition, it has to be proven that the antibody really binds to the native toxin. Antibody quality is an issue for about 50% of all monoclonals.
http://insights.sagepub.com/quality-issues-of-research-antibodies-article-a5474
I would like to add one observation to Gertrudis perfect explanation:
It's the logical way to screen antibodies first for neutralization in in vitro assays before going on with in vivo protection assays. In our research on antibodies against some viruses (viruses are somehow a more complex kind of toxin), we found antibodies which are protective in vivo, but are not neutralizing in vitro (We assume, that the protection is based on opsonization of virus particles and uptake by immune cells)!
Im summary: a neutralization assay is essential, but the desired protection in vivo is more complex and sometimes you loose protective antibodies when focusing on in vitro neutralization.
Thank you Michael H. That is an interesting observation you have added. But in our research, we are dealing with a chemical toxin not an organism. Your observation with viruses may not apply in our own case. However, we shall consider your observation. I wish you supplied us with a reference that we can consult and appreciate the finding better and to also quote accordingly.
Hi Hamza,
you are right a chemical toxin would be completely different. For protein toxins the situation is different. Here the observation we made for viruses are perhaps transferable.
I attached a VEEV paper showing in vitro neutralization only for the vaccine strain not for the wildtype virus, but protection against both.
We published neutralizing antibodies against WEEV (also attached). Here, we have a neutralizing, two "partially-neutralizing" and one non-neutralizing antibody. In an ongoing unpublished in vivo protection study, the neutralizing antibody (which was expected) and the non-neutralizing antibody were protective.
Article Isolation and Characterisation of a Human-Like Antibody Frag...
Article Human- like antibodies neutralizing Western equine encephalitis virus
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays