ABSTRACT
Monoclonal antibodies against human thyroid stimulating hormone (hTSH) are among the key reagents required in the development of an immunoradiometric assay (IRMA) for hTSH in serum. In this study we have produced and characterized twelve hTSH monoclonal antibodies. Hybridomas were generated by fusion of B-lymphocytes from mice immunized with hTSH and myeloma cells. Clones producing antibodies against hTSH were selected using ELISA (Enzyme Linked Immunosorbent Assay). Antibodies from the selected and cloned hybridoma cells were purified by affinity chromatography and their reactivities were tested by ELISA against a panel of antigens, i.e., hTSH, bovine serum albumin (BSA), and milk protein, and also by studying the binding of these monoclonal antibodies with the radioiodinated antigens (hTSH, BSA, and milk protein). It was observed that some of the hTSH monoclonal antibodies produced were polyreactive, reacting with hTSH as well as with unrelated antigen BSA, while others were monoreactive, reacting only to hTSH.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Thyrotropin/immunology , Animals , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas , Iodine Radioisotopes/chemistry , Mice , Milk Proteins/chemistry , Milk Proteins/immunology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Thyrotropin/bloodABSTRACT
We report on the superiority of a radioimmunoassay (RIA) system wherein the second antibody (Ab2) is incorporated as a pre-incubated complex with the primary antibody (Ab1) for the assay of haptens like triiodothyronine (T3) and thyroxine (T4). Separation of the antibody bound and free antigen (Ag) was accomplished by 8% polyethylene glycol (PEG) (final concentration) following a single incubation of less than 1 h. The other advantages of this system are a 15-fold reduction in the quantity of Ab2 (without any need for increasing the concentration of PEG) and the consequent savings in cost.
Subject(s)
Radioimmunoassay/methods , Thyroid Hormones/analysis , Costs and Cost Analysis , Humans , Reagent Kits, Diagnostic/economicsABSTRACT
125I labelled monoclonal antibody (MoAb) to thyroid stimulating hormone (TSH) was used as a tracer for assessing the suitability of polyethylene glycol (PEG) for precipitation of MoAb and MoAb-antigen (AG) complexes. It was found that PEG can be advantageously used under certain conditions favouring insolubility and could thus be a non-specific separation reagent for radioimmunoassays based on MoAbs.
