Bovine serum albumin-GABA-His-Pro-NH2: an immunogen for production of higher affinity antisera for TRH.

Dissatisfaction with current methods for the production of immunogens for raising antisera to TRH stimulated us to synthesize the hapten, GABA-His-Pro-NH2. Coupling of this hapten to bovine serum albumin at a molar ratio of 18:1 by means of a water-soluble carbodiimide produced an immunogen which stimulated the rapid production in New Zealand white rabbits of antisera with an affinity (2.42 +/- 0.3 X 10(9) l/mol) for TRH, some 8-fold higher than that of antisera (0.33 +/- 0.03 X 10(9) l/mol) raised by immunization with a conjugate produced by the currently accepted bis-diazotized-benzidine bridging technique. These higher affinity antibodies when used in a standard TRH radioimmunoassay permitted the detection of less than 1/pg of TRH per assay tube and showed an extremely low affinity for the two major metabolites of TRH, p-Glu-His-Pro-COOH and His-Pro diketopiperazine (Ka 4.84 X 10(4) and 4.0 X 10(4) l/mol, respectively). Application of this newer radioimmunoassay to the measurement of TRH in brain tissue yielded measurements of TRH content similar to those determined by current RIA methods. Chromatography of whole crude brain extracts revealed one major immunoreactive peak corresponding to authentic TRH. We conclude that immunization of rabbits with this hapten rapidly produces antisera with a high affinity for TRH suitable for the development of a very sensitive TRH radioimmunoassay.