Detection of Thy-1.2 membrane complexes shed from thymocytes and lymphoma cells by an immunoradiometric assay

Abstract

A direct binding immunoradiometric assay (IRA) for Thy-1 antigen was developed to study the properties of membranous complexes shed from murine thymocytes and lymphoma cell lines. Monoclonal anti-Thy-1.2 antiserum was iodinated and used to study the shedding from AKR (Thy-1.1) and C3H(Thy-1.2) thymocytes, and S49.1(Thy-1.2), S49-Thy-1 − and BW5147(Thy-1.1) continuous lymphoma cell lines. Culture supernatant fluids or purified shed complexes were allowed to bind to microtiter plates followed by measurement of the binding of iodinated anti-Thy-1.2. The assay was found to be completely specific for the Thy-1.2 allotype, and in conjunction with antibody coated wells could detect Thy-1 solubilized from cells with N-P40 detergent. Shed complexes containing Thy-1 from thymocytes and lymphoma cell lines were analysed by isopycnic centrifugation with continuous potassium tartrate gradients (5–40%). Shed complexes had a buoyant density of 1.06–1.10 g/cm 3 as compared to 1.15–1.17 g/cm 3 expected for murine leukemia virus or 1.20–1.24 g/cm 3 expected for mycoplasma. We concluded that the shed membranous complexes had a buoyant density similar to plasma membrane and the complexes were similar from both thymocytes and lymphoma cell lines. Thy-1 was not associated with virus, mycoplasma or other particles found in the gradients and Thy-1 was not found in the unsedimented fraction. The release of Thy-1 from thymocytes and cultured cell lines results in only one defined density of particles which may participate in cellular communication or in the survival of malignant cells.