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A quantitative indirect immunofluorescent assay for zona pellucida antibodies
A dilution series of a standard antiserum, raised in rabbit, against intact porcine oocytes was tested by an indirect immunofluorescent assay under a variety of conditions. Maximal antibody binding was obtained in phosphate-buffered saline with sucrose and sodium azide as a serum diluent and wash buffer, with relatively long incubation and wash periods, and in some cases heat-inactivation of sera. Factors found to influence the antibody binding were: alteration of the zonae by fixation; the presence of sucrose, galactose or glycerol; addition of protein to the buffer; heat inactivation of serum; substitution of phosphate by borate buffer; and replacement of the labelled second antibody by labelled Protein A. Antisera produced against acid- and heat-solubilized zonae differed from the standard antiserum in their binding capacity.l Control sera (anti-porcine spleen, adjuvant injected, and normal) were all completely negative.
A dilution series of a standard antiserum, raised in rabbit, against intact porcine oocytes was tested by an indirect immunofluorescent assay under a variety of conditions. Maximal antibody binding was obtained in phosphate-buffered saline with sucrose and sodium azide as a serum diluent and wash buffer, with relatively long incubation and wash periods, and in some cases heat-inactivation of sera. Factors found to influence the antibody binding were: alteration of the zonae by fixation; the presence of sucrose, galactose or glycerol; addition of protein to the buffer; heat inactivation of serum; substitution of phosphate by borate buffer; and replacement of the labelled second antibody by labelled Protein A. Antisera produced against acid- and heat-solubilized zonae differed from the standard antiserum in their binding capacity.l Control sera (anti-porcine spleen, adjuvant injected, and normal) were all completely negative.