Abstract
The application of monoclonal antibody technology is a rapidly expanding area of research, encompassing most areas of the biological sciences. The potential of the technique to provide standard but uniquely specific analytical reagents, which can be used singly or in combination is overwhelming by its ever increasing application. In this work the main aims were to produce specific high affinity monoclonal antibodies to the human anterior pituitary hormone, Thyroid Stimulating Hormone (TSH); to characterise the monoclonal antibodies and demonstrate their application in relation to human TSH. Two indirect enzyme-linked immunosorbent assays (ELISA), and a solid phase separation radioimmunoassay (RIA) system were developed for screening of the hybridoma cultures. The immunogenicity of TSH in the spleen cell donor mice was monitored by standard TSH double antibody RIA. Eight fusion experiments were done before stable positive cultures were established from the fusion experiments. Eleven positive primary cultures and 27 positive monoclonal cultures were frozen down and stored at -196°C in liquid nitrogen. Some difficulty was encountered in recovery of some of these cultures from the frozen state. Six monoclonal antibody cultures have been characterized with regard to titre in culture supernatant and ascitic fluid, antibody content, immunoglobulin class, sub-class and light chain content. The antibody chain composition of the preparations was also investigated. Cross reactivity in relation to the other closely related anterior pituitary hormones: luteinizing hormone (LH), follicle stimulating hormone (FSH), and the placental hormone human chorionic gonadotrophin (HCG) whole molecule and the a and B subunits were investigated. The antigenic determinant specificity of these antibodies was also investigated by competitive binding between radiolabelled monoclonals and unlabelled monoclonals. The monoclonals all had high titres and high affinity. Only one of the monoclonal antibodies investigated displayed any recognisable cross reactivity to HCG and FSH but this was still minimal. All six monoclonal antibodies were used in an indirect immunocytochemical immunoperoxidase procedure and demonstrated to be highly suitable for the localization of TSH in tissue. Four preparations of human anterior pituitary were examined, in addition to human placenta, kidney, and gut. Possible cross reacting animal TSH's were also investigated using baboon, dog, rat and ferret pituitary sections. The use of the monoclonals together with the immunoperoxidase technique offers quite distinct advantages of improved specificity, a permanent source of specific pure antibody and improved sensitivity (low observed background), and permanent stained records. Finally one of the monoclonals was investigated for use in a two-site microtitre enzyme-linked immunosorbent assay (ELISA) with an anti-h-TSH polyclonal anti sera. An enzyme-linked immunosorbent assay was developed with a detection limit of 4 mU/1 based on the 95% confidence limit from zero. The assay showed good intra- and inter-assay precision over the whole range of the standard curve, and as such may be suitable for clinical application.