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Production and characterization of monoclonal antibodies against bovine oviduct cell surface antigens
Abstract
A panel of monoclonal antibodies (MAbs) was generated against bovine oviduct cell surface antigens. Male mice were immunized with intact oviduct cells which had been collected by collagenase dissociation. Mice were boosted twice at three week intervals and then fused with Sp2/0-Ag14 hybridoma cells according to conventional procedures. The resulting antibody-secreting hybrid clones were identified by an indirect enzyme-linked immunoadsorbant assay (ELISA) of cell supernatants on intact oviduct cells. Sixty-seven clones were found to be positive. Of these, thirty-three stable clones were established. Indirect immunofluorescence on live cells was used to visualize the staining patterns of each antibody. This panel of MAbs was tested by ELISA for cross-reactivity to cells from other bovine tissues, namely ovary, uterus, trachea, spleen, liver and lymph node. The majority of these MAbs (20) were specific for antigens on the oviduct cell surface. One MAb (#27) cross-reacted only with uterine cells. Eight MAbs cross-reacted with tracheal cells as well as uterine cells. The remaining four MAbs cross-reacted with a number of different cell types. In addition, this panel cross-reacted with sheep oviduct cells (14/33) but not those of rabbit. Also, eleven MAbs cross-reacted with cow oviduct fluid (ODF) while fourteen cross-reacted with sheep ODF. To determine whether any of the MAbs recognized epitopes which were cycle stage-specific, ODF from both the luteal and estrus phases of the reproductive cycle was examined. ELISA data of ODF from cows did not indicate any obvious cycle stage variations. In contrast, ELISA data of sheep ODF did indicate some variations in antigen appearance between the estrus and luteal phases. Antigens that reacted with the MAbs were characterized by immunoblotting analysis of detergent-solubilized membrane antigens after SDS-PAGE fractionation under reducing conditions. Soluble antigens in cow and sheep ODF were also characterized by similar methods. Several MAbs detected broad diffuse bands which suggests that these antigens may be carbohydrate in nature. In an in vitro cell adhesion assay, MAb #27 prevented apical sperm head attachment to oviduct cells at concentrations as low as 5 ug/ml of purified antibody. None of the other MAbs appeared to have an effect on sperm binding. This effect is not due to nonspecific toxicity since inhibition of attachment is reversible. The oviduct can now be examined in greater detail using these MAbs as probes for the molecular components of the system. Such studies should bring to light details of cell surface composition, which will provide further insights into possible oviduct cell surface functions such as sperm storage.
Subject Area
Cellular biology|Physiology
Recommended Citation
Gerena, Robyn Lynn, "Production and characterization of monoclonal antibodies against bovine oviduct cell surface antigens" (1993). Doctoral Dissertations Available from Proquest. AAI9408277.
https://scholarworks.umass.edu/dissertations/AAI9408277