Methods Comparison of Purifying Anti-Rat TR Beta Delta Antibodies from the Antiserum

The aim of the study was to develop a procedure of purifying rabbit anti-rat TRβΔ polyclonal antibody from the antiserum. TRβΔ was a newly reported thyroid hormone receptor beta isoform. To gain better understanding of this new receptor, such as its tissue distribution, its specific antibody was needed. In this study, chemically synthesized peptide, which is uniquely existed in the TRβΔ, was used as immunogen to generate antibody in the rabbit. Then rabbit anti-TRβΔ antiserum was purified by means of saturated ammonium sulfate precipitation or protein A affinity chromatography respectively. To compare the effectiveness of these two purification methods, the purity, yield ratio and antigen binding ability of the purifications were analyzed by SDS-PAGE, protein content determination and ELISA seperately. The results showed that 98% purity, yield of 7.47mg IgG per milliliter serum was reached via protein A affinity chromatography and 87% purity, yield of 5.58 mg IgG per milliliter serum could be obtained by saturated ammonium sulfate precipitation. Results of ELISA analysis showed that IgG purified by either method could bind to recombinant TRβΔ protein with same affinity. So, we drew the conclusion that the protein A affinity chromatography method was more suitable for the purification of the anti-TRβΔ polyclonal antibody from the antiserum.