Specificity of oxytocin and vasopressin immunofluorescence

A total inhibition of immunofluorescence could not be obtained using the technique of preincubating either anti-vasopressin or anti-oxytocin plasmas with their homologous antigens. An alternative test of specificity was therefore developed. Lysine-vasopressin (LVP), arginine-vasopressin (AVP) or oxytocin were covalently bound to CNB-activated agarose beads. These hormone-coupled beads were then either placed immediately on glass slides and treated in the same way as tissue sections for immunofluorescence, or pre-incubated in test-tubes with the antibodies and then prepared for immunofluorescence. Fluorescence was measured quantitatively using a Leitz orthoplan microscope with epi-illumination and a photometer attachment. Without pre-incubation the anti-oxytocin plasmas produced intensive fluorescence on those beads containing their homologous antigen (oxytocin) but only slight fluorescence with the heterologous antigens (AVP or LVP). Anti-vasopressin plasmas produced intensive fluorescence of AVP-, LVP- and oxytocin-containing beads. Because the neurohypophysial hormones were bound to agarose beads, antibodies binding to these hormones could be removed by simple centrifugation. Anti-oxytocin and anti-vasopressin lost their fluorescence capacity after pre-incubation with oxytocin- or vasopressin-containing beads respectively, showing that all the antibodies important for fluorescence were bound to homologous antigens. Pre-incubation of anti-oxytocin or anti-vasopressin with beads coupled to their heterologous hormones completely removed the components binding to the heterologous hormone, leaving antibodies that showed fluorescence only with oxytocin or vasopressin respectively. This purification showed that in contrast to the findings with homologous antigens, not all of the antibody population bound to its heterologous antigen. Using non-purified anti-vasopressin, immunofluorescence was observed in neurohypophyses of homozygous Brattleboro rats. This was evidently due to those antibodies which bind to oxytocin, since the fluorescence was abolished after pre-incubating the antibody-containing plasmas with oxytocin-coupled beads. Immunofluorescence was still observed, however, in the suprachiasmatic nucleus of both Wistar and heterozygous Brattleboro rats, if purified anti-vasopressin was used. This was probably due to either vasopressin storage or production in these hypothalamic cells.