The use of recombinant human thyrotropin produced by Chinese hamster ovary cells for the preparation of immunoassay reagents.
Recombinant human TSH (rec-hTSH; Thyrogen, lot M-17073) obtained from transformed Chinese hamster ovary cells was tested for both radioiodination and preparation of a secondary standard used in RIA and immunoradiometric assay (IRMA) for routine clinical investigation. Results were compared to those obtained with high quality pituitary TSH (pit-hTSH; Dr. P. Torjesen, Oslo, Norway; and NIDDK, Rockville, MD), traditionally used in these assays. After extensive characterization and testing, it was found that [125I]rec-hTSH matched all binding and chromatographic criteria usually obtained with [125I]pit-hTSH, including Stokes' radius, labeling, and storage stability, and did not introduce any significant bias when used in the measurement of unknown serum samples. A preparation of rec-hTSH was calibrated against a local secondary standard as well as against two well known international reference preparations (NIDDK hTSH RP-1 and WHO International Reference Preparation 80/558) by IRMA and RIA. In the RIA, NIDDK anti-hTSH-3 polyclonal antibody was used, whereas in the IRMA, two commercial preparations were used: a monoclonal antibody as the detecting antibody, and a polyclonal antibody as the capture antibody. In both assays, the recombinant standard preparation yielded good fit displacement curves, showing significant parallelism compared to pit-hTSH and therefore allowing an unbiased measurement of unknown serum samples. The specific activity of the rec-hTSH preparation calibrated against the WHO International Reference Preparation was 7.7 IU/mg protein when measured by IRMA and 7.1 IU/mg when measured by RIA. In conclusion, these results indicate for the first time that rec-hTSH can fully replace pit-hTSH as both standard and tracer in diagnostic in vitro systems such as RIA and IRMA, suggesting that other recombinant glycosylated hormones might also serve for immunoassay reagent preparation.
Ribela
M T
MT
Department of Application of Nuclear Techniques in Biological Sciences, University of São Paulo, Brazil.
Bianco
A C
AC
Bartolini
P
P
D000818:Animals; D016466:CHO Cells; D002138:Calibration; D006224:Cricetinae; D006801:Humans; D015592:Immunoradiometric Assay; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000032:analysis; D012015:Reference Standards; D013972:Thyrotropin / Q000032:analysis*
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013972:Thyrotropin