Preparation and specificity of antisera for radioimmunoassay of 4-methoxyestrogen

Abstract

4-Hydroxyestrogens, which play physiologically important roles, are metabolized to 4-methoxyestrogens by catechol O-methyltransferase. For radioimmunoassay of 4-methoxyestrone (4-MeOE₁), antisera were prepared in rabbits by immunization with two types of hapten-bovine serum albumin (BSA) conjugates. The antiserum A was elicited with the C-2 conjugate in which 4-MeOE₁ was coupled to BSA by a Mannich reaction. Antiserum B was raised against 4-methoxyestradiol 17-hemisuccinate-BSA conjugate. The antisera A and B bound to approximately 50% of tritium-labeled 4-MeOE₁ at final dilutions of 1:47600 and 1:420000, respectively. The association constants of antisera were determined as 6.1×10⁹ and 4.6×10¹⁰ M^-1 using Scatchard plots. The linearity of standard curve (log-logit plots) ranged from 10 to 1000 pg for antiserum A, while the plots were linear over the range of 2 to 1000 pg for antiserum B. The antiserum A showed cross-reactions to a considerable extent with estrone derivatives substituted at the A-ring. In contrast, the antiserum B underwent no cross-reactions with any compound except 4-methoxyestradiol(107%).