Sperm preparation affects reactivity of monoclonal antibodies.
The polyacrylamide gel electrophoresis (SDS-PAGE) patterns of extracts prepared from thrice-washed human sperm were compared with extracts of swim-up and non-rise sperm. For sperm extraction sodium deoxycholate (DOC), octylphenoxypolyethoxyethanol (NP-40), dithiothreitol (DTT), lithium 3,5-diiodosalicylate (LIS), or 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) were used. A pellet of pooled thrice-washed semen was divided and extracted with these detergents. Similar gel electrophoresis profiles, as revealed by silver staining, were observed with the LIS, DOC, NP-40 and CHAPS extracts, whereas DTT extracts lacked the 150 and 200 kDa bands. Different thrice-washed semen pools varied in their protein pattern, probably due to the presence of sloughed germinal cells, white blood cells and sloughed cells from the reproductive tract. Swim-up sperm extracts had a different protein profile from that of thrice-washed pools but pools of swim-up sperm had a more constant protein pattern than those of thrice-washed semen pellets. Monoclonal antisperm antibodies did not always recognize antigens of the same molecular weights in western blots when extracts of swim-up sperm versus thrice-washed sperm were compared. In immunoblot studies with monoclonals (produced against thrice-washed sperm, cloned five times, reactive by ELISA and sperm function tests), 4/12 reacted with 0.3% NP-40 extracts of thrice-washed sperm and 5/12 with 0.3% NP-40 extract of swim-up sperm. These findings suggest that definition of antigens by monoclonal antisperm antibodies will be influenced by the nature of sperm extract employed.
Gupta
S K
SK
Eastern Virginia Medical School, Department of Obstetrics and Gynecology, Jones Institute for Reproductive Medicine, Norfolk.
Fulgham
D L
DL
Alexander
N J
NJ
D000911:Antibodies, Monoclonal; D000941:Antigens / Q000302:isolation & purification; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification; D013081:Sperm Motility; D013094:Spermatozoa / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D011506:Proteins