Expression of recombinant human thyrotropin receptor in myeloma cells.
Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.
Tokyo Research Center, Tosoh Corporation, Kanagawa.
T
T
Matsuba
M
M
Yamada
H
H
Suzuki
A
A
Kanai
O
O
Isozaki
T
T
Yoshida
T
T
Tsushima
K
K
Yasukawa
D000818:Animals; D001323:Autoantibodies / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane; D005285:Fermentation; D006111:Graves Disease / Q000097:blood; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics* / Q000473:pathology; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D013552:Swine; D014407:Tumor Cells, Cultured
D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D009838:Oligodeoxyribonucleotides; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; C094372:thyrotropin-binding inhibitory immunoglobulin