Chow S N, Chen K W, Su S L, Tung J, Lee C Y
Department of Obstetrics and Gynaecology, National Taiwan University Hospital, Taipei, Republic of China.
Biotechnol Appl Biochem. 1988 Apr;10(2):137-42.
By using an improved hybridoma technique with a semisolid medium of methylcellulose for initial cloning, numerous high affinity monoclonal antibodies against human thyroid stimulating hormone (TSH) were generated. These antibodies were characterized with respect to their subunit and epitope specificity. Epitope analysis of antibodies specific to the beta-subunit of TSH was performed by a sandwich pairing procedure. Based on the results of this analysis, it was concluded that there are four distinct TSH-specific epitopes on the beta-subunit of TSH; these are designated a, b, c, and ab. The five antibodies binding to epitopes a, b, and c are not mutually exclusive. However, the antibody binding to epitope ab prevents further binding of other antibodies to epitope a or b, but not to epitope c. This epitope analysis enabled us to combine three high affinity monoclonal antibodies, each of which reacts with epitopes a, b, and c, respectively, in a typical sandwich enzyme immunoassay. One was immobilized on polystyrene beads and the other two were conjugated with horseradish peroxidase and served as the second antibodies. This enzyme immunoassay can be performed within 90 min and with a minimum sensitivity of 0.2-0.3 microIU/ml.
通过使用改进的杂交瘤技术，以甲基纤维素半固体培养基进行初始克隆，产生了众多针对人促甲状腺激素（TSH）的高亲和力单克隆抗体。对这些抗体的亚基和表位特异性进行了表征。通过夹心配对程序对TSHβ亚基特异性抗体进行表位分析。基于该分析结果，得出结论：TSHβ亚基上存在四个不同的TSH特异性表位；分别命名为a、b、c和ab。与表位a、b和c结合的五种抗体并非相互排斥。然而，与表位ab结合的抗体可阻止其他抗体进一步结合表位a或b，但不影响与表位c的结合。这种表位分析使我们能够在典型的夹心酶免疫测定中组合三种高亲和力单克隆抗体，每种抗体分别与表位a、b和c反应。一种固定在聚苯乙烯珠上，另外两种与辣根过氧化物酶偶联并用作第二抗体。这种酶免疫测定可在90分钟内完成，最低灵敏度为0.2 - 0.3微国际单位/毫升。
