Monoclonal antibodies to mitotic cells.

Abstract

Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, monoclonal antibodies to mitotic cells were raised. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa [human cervical carcinoma] cells were fused with SP2/0-Ag14 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species [human, mouse, hamster, chicken, frog, nematode, mosquito, rat] tested were isolated. Chromosomes and cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins for mitotic cells were separated by electrophoresis in NaDodSO4[sodium dodecyl sulfate]/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The 2 antibodies, designated MPM-1 and MPM-2, recognize a family of polytpeptides with apparent molecular masses of 0.40 to > 200 kilodaltons (kDA). Both antibodies reacted strongly with 3 polypeptide bands of 182 kDa, 118 kDA and 70kDA. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.