We shall be immunizing rabbits with peptide and the blood so obtained post collection will have polyclonal antibodies. How shall we purify monoclonal antibodies from it? Please share the protocol or kit details.
No chance, sorry. You can (and should) perform antigen affinity purification, but you won't be able to distinguish antibodies from two clones.
Maybe someone else can suggest how to isolate genetic information from immune cells and to make clonal cell lines producing rabbit monoclonals. It's quite a big endeavor, though, like making hybridomas.
Edit: By fine/tuning the elution conditions, you might be able to obtain several fractions, where the binding characteristics of the antibodies differ, but still (unless you're sequencing the proteins), you won't be able to demonstrate (mono-)clonality
In follow-up to what Wolfgang Schechinger wrote, if I recall correctly, rabbit hybridomas have been generated in the past. I believe the lead scientist on that was Kay Knight of Loyola University. I suspect she has long since retired, but the publication of her procedure should be out there in the literature.
Monoclonal antibodies are only produced by hybridomas. Most are made by fusing immunized mouse B lymphocytes with mouse myeloma cells. I know that rat:mouse heterohybridomas can be produced. I do not recall if Kay made rabbit:mouse heterohybridomas or if she had a rabbit myeloma cell line as the fusion partner.
Hi Shubhita Mathur,
Certainly, separating monoclonal antibodies from polyclonal antibodies is difficult, but not impossible.
The basic procedure assumes operation in a protein fusion model. Rabbits are immunized and the resulting spleen cells are fused with partner cells to make an immortal cell line that expresses antibodies. As Gary mentioned you can use mouse myeloma cells. Such technology, moreover, is mentioned in a major review by Hilal Parray:
Article Hybridoma technology a versatile method for isolation of mon...
Sanchita Mitra's article may also be helpful at this stage:
Article Hybridoma technology; advancements, clinical significance, a...
The resulting hybridoma is then subjected to selection. The cheapest and simplest option for antibody differentiation is screening clone candidates by monoclonal ELISA. An example protocol is described in:
Article A cell surface ELISA for the screening of monoclonal antibod...
The next step will be small-scale production and characterization of the resulting monoclonal antibody. Of course, it is necessary to confirm that the obtained product is indeed monoclonal and has the right characteristics. But that's a topic for another meeting.
It is possible that in your case the preparation of hybrids will not be possible, therefore I also recommend reviewing the literature on alternative antibody purification, which I attach below:
Article Evaluation of immunoglobulin purification methods and their ...
Article Method development for the separation of monoclonal antibody...
Article Recovery and purification process development for monoclonal...
Article Separation of antigens and antibodies by immunoaffinity chromatography
Chapter Separation of Monoclonal Antibodies by Analytical Size Exclu...
Chapter Methods in Molecular Biology
https://www.sciencedirect.com/science/article/abs/pii/S0731708515001284
https://www.sciencedirect.com/science/article/abs/pii/B9780128223185000089
https://www.seracare.com/globalassets/seracare-resources/tg-guide-to-purification-of-polyclonal-antibodies.pdf
You could also check Standard Immunization of Rabbits protocol: https://cshprotocols.cshlp.org/content/2020/9/pdb.prot100305.long
Remember about humane, and ethical principles by following Guide for the Care and Use of Laboratory Animals available in: https://www.ncbi.nlm.nih.gov/books/NBK54050/
Good luck
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