Fm. Fusi et al., PRODUCTION OF MOUSE MONOCLONAL-ANTIBODIES DIRECTED AGAINST THE OOLEMMA OF HUMAN AND HAMSTER OOCYTES BY INTRA-SPLENIC INJECTION OF OOCYTES, American journal of reproductive immunology [1989], 33(1), 1995, pp. 122-130
PROBLEM: To develop an additional approach for the study of oolemmal s
urface moieties involved in gamete interactions, we decided to obtain
monoclonal antibodies by intrasplenic injection of human and hamster o
ocytes in Balb/c mice. METHOD: Two Balb/c males were injected three ti
mes intrasplenically at 15-day intervalS with approximately 40 zona-fr
ee hamster and 3-5 zona-free human oocytes. After the third injection,
spleen cells were fused and hybridomas developed. We used a novel scr
eening system based upon the use of sections of frozen human and hamst
er eggs, tested by means of indirect immunofluorescence. The antibodie
s that we produced were evaluated for their ability to interfere with
the zona-free hamster eggs penetration by human spermatozoa. The B2B5
antibody was also developed as ascitic fluid and further characterized
. RESULTS: Seven antibodies reactive with hamster oocytes were produce
d. Six of them also reacted with human oolemmas. The binding was confi
ned to the oolemma, and no staining of the zona nor the cytoplasm was
present. One of these antibodies reduced the penetration of zona-free
hamster eggs by human spermatozoa. This antibody, B2B5, an IgM kappa,
was confirmed to interact with the oolemma by means of indirect immuno
fluorescence of fresh eggs and Covasphere binding. B2B5 did not react
with other human or hamster tissues except capacitated human spermatoz
oa. The reactivity with the oolemma of hamster oocytes was not lost af
ter egg penetration by human sperm. CONCLUSIONS: Intrasplenic immuniza
tion using zona-free human and hamster oocytes allows the production o
f anti-oolemma antibodies. A system of screening based upon the use of
sections of frozen eggs also allows an easy and quick scoring of many
supernatants. B2B5 monoclonal anti-oolemma antibody deserves further
studies in that is able to interfere with fertilization and its antige
n appears to be confined to the gametes surface.