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.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity; D005260:Female; D005455:Fluorescent Antibody Technique; D008722:Methods; D010063:Ovum / Q000276:immunology*; D011817:Rabbits; D013395:Sucrose / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D015044:Zona Pellucida / Q000276:immunology*
D000906:Antibodies; D013395:Sucrose