H Wallaschofski1, R Paschke. 1. Department of Internal Medicine III, University of Leipzig, Germany.
Abstract
BACKGROUND: With the aim of assaying TSH-receptor antibodies (TSHR AB) and to distinguish different TSHR AB activities, several in vitro bioassays for the determination of TSHR AB have been developed. The bioassay using the CHO cell line expressing the hTSH-receptor (hTSHR) is the most sensitive and least cumbersome assay system. However, up to now, the CHO cell bioassay has been performed with cells expressing much higher TSHR numbers than normal thyroid epithelial cells and with variable assay conditions. DESIGN AND PATIENTS: To determine the optimal CHO cell bioassay conditions we therefore systematically investigated the influence of different assay parameters on the sensitivity of the CHO cell bioassay for the detection of TSHR antibodies in 197 patients with Graves' disease treated with antithyroid drugs and tried to define optimal assay conditions. RESULTS: Compared to JP09 cells (90,000 hTSHR/cell), JP26 cells (2000 hTSHR/cell) exhibited a higher stimulation index (maximal bTSH-stimulated cAMP production/cAMP production stimulated by pooled normal serum without bTSH) which is most likely due to the lower number of hTSHR expressed per JP26 cell compared to JP09 cells. Due to their low nonspecific stimulation by normal sera, 40,000 JP26 cells per well of a 96 well plate was found to be the optimal cell number. Moreover, a serum dilution of 1/10 yielded the highest stimulation results of bTSH and thyroid stimulating antibodies (TSAB). Applying these optimal conditions for the determination of TSH-receptor antibodies only 10 of 34 TSBAB positive serum samples detected with JP26 cells were detectable with the JP09 cell line. In 197 patients with Graves' disease treated with antithyroid drugs 111 (56%) were TSAB positive. 34 (40%) of 86 TSAB negative patients were positive for thyrotropin stimulation blocking antibodies (TSBAB). CONCLUSION: Improvement of CHO bioassay conditions for the determination of TSH receptor antibodies leads to increased bioassay sensitivity which is most pronounced for the detection of TSBAB. Whether this increased sensitivity for the determination of TSBAB is of prognostic significance for detecting possible sequential changes in the individual TSHR AB profile of patients with Graves' disease, remains to be determined by further follow up studies.
BACKGROUND: With the aim of assaying TSH-receptor antibodies (TSHR AB) and to distinguish different TSHR AB activities, several in vitro bioassays for the determination of TSHR AB have been developed. The bioassay using the CHO cell line expressing the hTSH-receptor (hTSHR) is the most sensitive and least cumbersome assay system. However, up to now, the CHO cell bioassay has been performed with cells expressing much higher TSHR numbers than normal thyroid epithelial cells and with variable assay conditions. DESIGN AND PATIENTS: To determine the optimal CHO cell bioassay conditions we therefore systematically investigated the influence of different assay parameters on the sensitivity of the CHO cell bioassay for the detection of TSHR antibodies in 197 patients with Graves' disease treated with antithyroid drugs and tried to define optimal assay conditions. RESULTS: Compared to JP09 cells (90,000 hTSHR/cell), JP26 cells (2000 hTSHR/cell) exhibited a higher stimulation index (maximal bTSH-stimulated cAMP production/cAMP production stimulated by pooled normal serum without bTSH) which is most likely due to the lower number of hTSHR expressed per JP26 cell compared to JP09 cells. Due to their low nonspecific stimulation by normal sera, 40,000 JP26 cells per well of a 96 well plate was found to be the optimal cell number. Moreover, a serum dilution of 1/10 yielded the highest stimulation results of bTSH and thyroid stimulating antibodies (TSAB). Applying these optimal conditions for the determination of TSH-receptor antibodies only 10 of 34 TSBAB positive serum samples detected with JP26 cells were detectable with the JP09 cell line. In 197 patients with Graves' disease treated with antithyroid drugs 111 (56%) were TSAB positive. 34 (40%) of 86 TSAB negative patients were positive for thyrotropin stimulation blocking antibodies (TSBAB). CONCLUSION: Improvement of CHO bioassay conditions for the determination of TSH receptor antibodies leads to increased bioassay sensitivity which is most pronounced for the detection of TSBAB. Whether this increased sensitivity for the determination of TSBAB is of prognostic significance for detecting possible sequential changes in the individual TSHR AB profile of patients with Graves' disease, remains to be determined by further follow up studies.
Authors: Filippo Galli; Isabella Manni; Giulia Piaggio; Lajos Balogh; Bruce D Weintraub; Mariusz W Szkudlinski; Valerie Fremont; Rudi A J O Dierckx; Alberto Signore Journal: Thyroid Date: 2014-06-20 Impact factor: 6.568