Rayalam S, Eizenstat LD, Davis RR, et al.
One out of every three hundred cats is now diagnosed with hyperthyroidism. A diagnostic tool that can sensitively detect changes in thyroid status is essential to understand thyroid pathology in the cat and also to diagnose this condition at an earlier stage. Lacking an available source of pituitary-derived fTSH, previously cloned fTSH alpha and beta subunits were ligated into the expression vector PEAKTM and expressed in modified human embryonic kidney cells (PEAKTM). As the alpha subunit has two and beta has one N-linked glycosylation sites, expression in a mammalian cell is critical for appropriate post translational modifications and folding. The fTSH alpha and beta subunits were cloned separately downstream of the EF-1α promoter of the PEAKTM expression vector, and transiently co-transfected into PEAKTM cells. Similarly, a previously cloned and sequenced yoked fTSH (yfTSH) gene was ligated into the PEAKTM vector, transfected into PEAKTM cells with puromycin selection generating a stable cell line expressing yfTSH. The alpha subunit was also stably transfected into PEAK cells to generate a stable cell line expressing the feline pituitary alpha protein. Expression levels of at least 1 µg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity based upon polyacrylamide gel electrophoresis and silver stain. All glycoproteins were standardized by protein assay. The purified alpha-FLAG glycoprotein had a molecular weight of 20.4 kDa and that of yfTSH-FLAG was 45 kDa. Both were recognized by anti-FLAG monoclonal antibody in Western blot. The glycosylated beta subunit had a molecular weight of 16.2kDa. Both heterodimeric and yoked glycoproteins were recognized by an in-house canine TSH ELISA employing a capture monoclonal antibody previously selected for ovine/canine TSH-beta and a polyclonal antibody generated previously against pituitary canine TSH, as well as by the commercially available canine TSH assay (Diagnostic Products Corp.). However, both assays detected only 33% of the recombinant fTSH as standardized by protein assay. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA. The recombinant heterodimeric fTSH exhibited 30% of the potency of a pituitary-source bovine TSH (bTSH) standard at inhibiting 125I-bTSH binding by JP09 cells expressing the human TSH receptor [IC50 (ng/mL; mean +/- SD) fTSH: 43±2; bTSH: 13±3]. Discrepancies in immunological detection and biological potency remain to be clarified. This work constitutes the first report of in vitro expression and purification of recombinant feline thyrotropin. The demonstration of immunological recognition by antibodies generated against pituitary-source TSH, and of bioactivity confirms that the recombinant glycoprotein may be used to standardize and improve clinical assays for feline TSH.