Author : Satish Kumar, Ram Kumar Pundir, Meenakshi and G. Lakshmi Kumari

In the present endeavor, monoclonal antibody production against recombinant thyroid stimulating hormone (TSH) was carried out. Two inbred BALB/C mice were immunized with recombinant TSH injection and checked for immune response by ELISA. The TSH antibody response of both immnunized animals as tested in TSH-coated wells using goat anti-mice IgG-HRP for detection. The spleen of mouse No. 2 showed better antibody response and used for fusion of spleen with myeloma cell line (SP2/0). After fusion, hybrid cell confluences were tested in antigen coated wells by antibody capture ELISA. Of 6 clones, three clones were chosen for expansion and further subcloning and limited dilutions were made. Based on ELISA response four subclone were chosen for P6D4 (P6D4C3, P6D4B7, P6D4 B8, P6D4 G8) and one subclone for test two clones (P4B2H3 and P1A10E3) and tested for their binding to other glycoprotein hormones (hCG) and its subunits. All of them showed high binding to alpha- hCG, then to TSH followed by beta hCG. In hCG coated wells, the antibody response was very low, suggesting some overlap in recognition of epitopes. So these hybrid cells were subclones further at least two times to ensure monoclonally of the cells. The antibody seemed to be mainly against alpha subunit of hCG assuming that it was directed at alpha subunit of TSH. A second fusion using TSH (recombinant) mouse was also carried out and a single clone P4B12 was obtained, which was sub-cloned to get monoclonal antibody directed against single epitope. Comparatively to the previous fusion, binding was less in these clones, but directed at the same antigen. Antibody response of ascetic fluid of P1A10E3 was tested at different dilutions of cells. This particular clone showed very high titre showing binding to alpha hCG, followed by TSH, β-TSH and minimum in hCG. The maximum protein was recorded in P4B12B9 (1.869mg/ml) (ascetic fluid) and total protein (6.564mg) in P1A10E3 clone (ascetic fluid). The maximum protein was recorded in P4B2H9D9C3 (1.869mg/ml) and total Protein (6.678mg) in P1A10E3G12 clone (supernatant/culture medium). The present findings suggested that purification and characterization of the monoclonal antibody are needed for further use.

Keywords: Antibody, monoclonal, ELISA, TSH, hCG, hormone, mice.