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Double Determinant Immunoassay to Measure a Human High-molecular-weight Melanoma-associated Antigen¹

Columbia University College of Physicians and Surgeons, New York, New York 10032 [P. G., A. K. N., R. R. S. K., S. F.], and Regina Elena Institute, Rome, Italy [P. G. N.]

Using monoclonal antibodies to distinct determinants of a human high-molecular weight melanoma-associated antigen (HMW-MAA), a double determinant immunoassay has been developed. The assay is specific and reproducible. Its sensitivity is influenced by the incubation time of antibodies with antigen sources and the combination of antibodies, as well as by the pH of the buffer and the incubation time used to coat plates with antibodies. Testing with the double determinant immunoassay of Nonidet P-40 extracts of human cell lines and of surgically removed normal and malignant tissues has confirmed the restricted tissue distribution of the HMW-MAA. In addition, significant differences have been found in the level of HMW-MAA in mealnoma cell lines, as well as in melanoma lesions removed from different patients and from different sites of a given patient. The amount of HMW-MAA shed by various melanoma cell lines does not correlate with their cell surface expression and with their level in Nonidet P-40 extracts. Interferon and hyperthermia increase the shedding of the HMW-MAA by melanoma cells.

¹ This work was supported by NIH Grants A1 19189, CA 32609, CA 32619, CA 32634, and CA 32635 and by a Biomedical Research Support Grant (BRSG CA 32609-01).

² Supported by a fellowship in Cancer Immunology of the Cancer Research Institute of New York.

³ Supported by a National Cancer Institute postdoctoral fellowship.

⁴ To whom requests for reprints should be addressed, at Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, N. Y. 10032.

Received 8/17/82. Accepted 3/18/83.

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