The Preparation and Specificity of Antibody to Thyrotropin Releasing Hormone1

Abstract

Synthetic thyrotropin releasing hormone (L-pyroglutamyl-L-histidyl-L-prolineamide) (TRH) was coupled to bovine serum albumin (BSA) with bis-diazotized benzidine (BDB). Recovery after dialysis of 14C-TRH included in the reaction mixture indicated that the product contained 37.5 μg TRH/mg albumin. The TRH-BDBBSA conjugate in doses calculated to contain 200 and 1000 ng TRH was biologically inactive whereas 20 ng TRH significantly increased rat serum TSH in vivo. Immunization of rabbits with the conjugate yielded significant quantities of anti- TRH antibody which was detected by reaction of diluted antiserum with 125I-TRH. Using 125I-TRH a double antibody radioimmunoassay for TRH has been developed in which as little as 0.1 ng unlabeled TRH can be detected. A variety of TRH analogues were tested and all had little reactivity in this immunoassay. The most potent of these was L-pyroglutamyl-D-histidyl-L-prolineamide which had 1.08% of the activity of TRH. Addition of the amino acid constituents of TRH, various other peptides, and bovine albumin was without effect. These studies indicate that the anti-TRH serum produced by this technique is highly specific and sensitive. (Endocrinology90: 722, 1972)