SH-009

We first established a cell strain, SHIN-3, from human ovarian serous cystadenocarcinoma, and performed antigen analysis for CA125 which appeared to be massively secreted by the SHIN-3 cells. Protein digestion analysis revealed that a low molecular weight peptide of 49 kDa showed antigen activity. In the present study, we describe a mouse monoclonal antibody against this low molecular peptide, presumed to be part of the CA125-bearing antigenic molecule. Purified antigen prepared from culture supernatant was adsorbed to nitrocellulose membranes and injected intrasplenically in mice. Of the obtained 398 clones, 10 clones were selected by screening in an ELISA test. Of the 10, two were further selected, i.e. SH-9. This hybridoma produces IgG1 monoclonal antibodies. Immunoblotting analysis revealed that SH-9 recognizes the low molecular peptide used as the immunogen. Immunohistological examination with the SH-9 MAb revealed that the antigen reacted with the bronchial epithelium, cervical glands of the uterus and other various normal tissues. Of tumorous tissues, the antibody mainly reacted with ovarian tumours, but positive reactions were also observed with pulmonary adenocarcinomas or squamous cell carcinomas. Surprisingly the positive rate was high in mucinous tumour of the ovary, while no positive reaction was observed in serous tumours. Dot-blot assay using SH-9 revealed that 17/19 (90%) sera of lung cancer patients were positive for the titre suggesting that SH-9 may be useful to set up a serum test for lung cancers.

The SHIN-3 cell line producing CA125 was established from an ovarian cancer patient. Using the SHIN-3 cell line, we found that the low-molecular-mass antigen (about 50 KDa) might be the main antigenic determinant in CA125-immunoreactive species. A new monoclonal antibody to this low-molecular-mass was raised to examine a new cancer associated antigen by a hybridoma technique. Using enzyme linked immuno sorbent assay, ten clones were selected from among 398 clones. Two clones were IgG1 and eight were IgM. By immunostaining (ABC assay), a new antibody (named SH-9) reacted with normal pulmonary bronchus and uterine cervical glands. No positivity, however, was observed in endometriosis (adenomyosis). In tumorous lesions of ovary, SH-9 antibody reacted specifically with mucinous cystadenoma-benign, borderline or malignant. However, no positivity was found in serous cystadenocarcinoma. In any other carcinomas, only lung cancer (adenocarcinoma, squamous cell carcinoma) showed a clear positivity. Immuno blotting analysis showed that SH-9 antibody recognized a low molecular mass. Therefore, SH-9 is seen to be an extremely unique antibody when compared with OC125 biochemically and histochemically.