Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.
1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled  anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays
D000327:Adsorption; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification*; D002417:Cattle; D005934:Glucagon / Q000097:blood; D013006:Growth Hormone / Q000097:blood; D006168:Guinea Pigs; D006728:Hormones / Q000097:blood*; D006801:Humans; D007074:Immunoglobulin G; D007328:Insulin / Q000097:blood; D007457:Iodine Radioisotopes; D010281:Parathyroid Hormone / Q000097:blood; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods
D000888:Antibodies, Anti-Idiotypic; D006728:Hormones; D007074:Immunoglobulin G; D007328:Insulin; D007457:Iodine Radioisotopes; D010281:Parathyroid Hormone; D013006:Growth Hormone; D005934:Glucagon