Anti-5-hydroxytryptamine antibodies: studies on their cross-reactivity in vitro and their immunohistochemical specificity

Abstract

Antibodies to 5-hydroxytryptamine (5-HT) were obtained from 4 rabbits after injections of 5-HT coupled to bovine serum albumin by means of paraformaldehyde (PF). Two methods were used to monitor the development of antibodies (AB): the one based on the "in vitro" competitive binding properties of the antibodies with 3(H)5-HT, the other, on their "in situ" binding properties to endogenous 5-HT, using the peroxidase-anti-peroxidase immunohistochemical technique, applied to paraffin embedded sections of cat brainstem. No pharmacological processing, detergents or proteolytic enzymes were used. The specificity of the antiserum was tested by competitive procedures with 20 analogs using the "in vitro" and "in situ" techniques. "In vitro" studies were performed with 5-HT free analogs and with analogs previously coupled with PF to lysine. Radioimmunological tests showed that the antibodies recognize mainly the ethylamine (CH2-CH2-NH2)-chain of the free analogs and that the best specificity was obtained with the 5-HT conjugate (5-HT-lysine-PF). The results suggest that the hapten is coupled through the phenolic positions C4 or C5. The "in situ" immunohistochemical extinction assays also revealed a distinct specificity for 5-HT. Possible optical and ultrastructural applications are illustrated in the raphé nuclei of the cat. These results confirm the reliability of radioimmunological tests for studying the specificity of AB directed against haptens, provided that haptens and analogs tested were first chemically transformed to resemble the immunogen (herewith lysine-PF coupling) with regard to its antigenic structure.