Five hybridoma cell lines secreting monoclonal antibodies (MAbs) to a 65- kDa tumor-associated phosphoprotein (p65) were established. Purified to homogeneity, p65 was used as an immunogen to induce immune response in C57BL/6N mice. Splenocytes were fused with mouse myeloma cells and hybridoma lines were selectively subcloned. A rapid and sensitive sandwich type ELISA, using purified MAbs was established to measure markedly elevated amounts of p65 in sera obtained from both tumor-bearing rats and from cancer patients. The p65 from rat and human sources was added quantitatively to normal sera to construct standard curves. The average level of p65 in normal rat sera was 38 ng/ml ± 13 ng/ml (mean ± SD), and in sera from rats bearing mammary adenocarcinomas, the average value was 1005 ± 140 ng/ml. In normal human sera the mean level of p65 was 34 ± 35 ng/ml (mean ± SD) and sera of patients with variety of cancers had an average p65 value of 344 ± 57 ng/ml. More than 80 % of tested sera from adenocarcinoma-bearing rats (20/24) as well as from cancer patients (82/98) had p65 levels elevated two standard deviations above the mean. Overall the assay had a sensitivity of 80.9% and specificity of 85%. The purified IgG1 MAbs, with high titers and strong anti p65 specificities were also used to develop an immunohistochemical method to visualize the expression of p65 in rat tumor tissue sections. The HB2, HF11 and RE6 cell lines have proved to be quite stable in the ability to secrete anti-p65 MAbs.
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