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Title: Characterization of antigens on the surface of human spermatozoa using monoclonal antibodies
Author: Kerr, Lorraine Elizabeth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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A panel of murine monoclonal antibodies was raised against human sperm plasma membrane in order to identify and characterize molecules important in fertilization. Twelve monoclonal antibodies were produced and of these nine belong to the IgM subclass while the remaining three are of the IgG2a subclass. The antibodies were purified from bulk culture supernatants using affinity chromatography - the IgG antibodies on a protein A column and the IgMs on a column made using a commercially available anti-mouse IgM antibody coupled to an inert support. These purified antibodies were then used to characterize their respective antigens biochemically and assessed for their biological activity using a variety of in vitro tests of sperm function. All twelve antibodies recognized a molecule which was present on the entire surface of human spermatozoa. Eight antibodies cross-reacted with both marmoset and boar spermatozoa as well as with human leucocyte subclasses. The other four immunoglobulins were highly specific for human sperm cells and did not cross-react with any of the other cells tested. In western blotting experiments, the monoclonals recognized epitopes present on a variety of molecules. Only one antibody, spm 4/4, failed to recognize any proteins when transferred to nitrocellulose. When tested for their ability to interfere with the acrosome reaction, four antibodies significantly inhibited the percentage of spermatozoa in a population undergoing a sponataneous acrosome reaction. Subsequently, two of these antibodies, spm 1/1 and spm 1/2, were shown to cause a significant decrease in the percentage of sperm which were induced to undergo the acrosome reaction by the calcium ionophore A23187. The interaction between spermatozoa and the zona pellucida was assessed using salt-stored human zonae and two immunoglobulins, spm 1/2 and spm 4/6, significantly reduced the mean number of sperm bound to the zona pellucida. These two antibodies also caused a marked inhibition of sperm-oolemmal fusion as assessed using the hamster egg penetration test. When this test was performed at various sperm concentrations, both of these antibodies exhibited a dose dependent decrease in the penetration rate. One further antibody, spm 4/7, was found to have biological activity in that it stimulated the interaction between sperm and the oolemma.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available