Biology of Reproduction, Vol 43, 260-270, Copyright © 1990 by Society for the Study of Reproduction

ARTICLES

Mouse antibodies to human zona pellucida: evidence that human ZP3 is strongly immunogenic and contains two distinct isomer chains

RB Shabanowitz
Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill 27599.

Polyclonal antibodies were raised in mice by immunization with solubilized human zonae pellucidae (ZP). Dot-blot analysis and ELISAs showed very strong immunoreactivity against human and pig ZP, and less immunoreactivity against rabbit and mouse ZP. One- and two-dimensional (1D and 2D) Western blot analysis detected immunoreactivity to three human ZP-specific bands. Under 1D nonreducing conditions, bands were detected at Mr 97,000, 61,000, and 51,000; the lower molecular weight (MW) bands showed the strongest reaction. Three acidic bands were detected under nonreducing 2D Western blot analysis, one at Mr 85,000- 107,000, and two closely apposed, parallel, and more acidic bands at Mr 47,000-55,000 and Mr 55,000-62,000. The lower MW bands displayed considerable microheterogeneity, with 8-13 charge isomers detected; the lower MW form (ZP3L) appeared to be more strongly reactive than the higher MW form (ZP3H), and displayed a more intense labeling towards the basic end of the isomer charge train. The human ZP antiserum detected electrophoretically separated heterologous ZP from pigs, rabbits, and mice, and was also shown to inhibit homologous sperm-zona binding in in vitro assays. This is the first study to report that human ZP3 consists of two distinct isomer chains.

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