Question

You have purified a novel protein and have a small aliquot of it. Unfortunately, there’s no commercially available antibodies to this new novel protein of yours. What can you do to obtain monoclonal antibodies to your new, novel protein so that you can use an antibody to screen sets of tissue samples to determine if your protein is expressed? You also want to have antibodies to use for a later time for other possible experiments.

Answer 1

ANSWER )

Through hybridoma technology we can produce monoclonal antibodies against the protein we have.

In hybridoma, injection of a specific protien will be injected  into a mouse, making mouse immunized against the protein procuring the antigen-specific plasma cells (antibody-producing cell) from the mouse's spleen. The protein specific antibodies producing splenocytes from mouse will be isolated for invitro production. Once splenocytes are isolated from the mouse, the B cells are fused with immortalized myeloma cells - which lack the HGPRT(hypoxanthine-guanine phosphoribosyltransferase) gene - using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin in the myeloma cells die, as they cannot produce nucleotides by the de novo or salvage medium blocks the pathway that allows for nucleotide synthesis. Hence, unfused D cell die. Unfused B cells die as they have a short life span. Only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and foetal bovine serum) and produce antibody. Larger tissue culture flasks will be used to culture the hybridoma. This maintains the well being of the hybridomas and provides enough cells for cryopreservation and supernatant yield 1 to 60 ug/ml of monoclonal antibody speicfic to protein, which is maintained at 20°C or lower until required. By using culture supernatant or a purified immunoglobulin preparation, further analysis of a potential monoclonal antibody producing hybridoma can be made in terms of reactivity, specificity, and crossreactivity.