Detection and separation of lymphocytes with specific surface receptors, by using microparticles

Abstract

Horse anti-(human lymphocyte) globulin was immobilized together with fluorescein labelled dextran in spherical microparticles of polyacrylamide (AHLG-particles). The particles had a diameter of 1-5 micrometer and a density of 1.12g/cm3, with globulin exposed on the surface. Human lymphocytes bearing the antigen (thymus-derived lymphocytes) bound the particles, which were easily detected by fluorescence microscopy. In this way, about 58% of circulating human lymphocytes were able to bind AHLG-particles at 23 degrees C. Non-specific binding was low, only 3% when human serum albumin was present in the buffer, and only 4% when non-specific horse globulins were incorporated in the microparticles. The cell-particle complexes could be separated from cells that had not reacted by density-gradient centrifugation in Ficoll/metrizoate. The viability was not changed after the separation procedure. The number of cells binding AHLG-particles corresponded well the the relative amount of T-cells. When the cells binding AHLG-particles were separated from the lymphocytes, the number of T-cells decreased remarkably, indicating that the antibodies bind preferably to the T-cell population. Concanavalin A immobilized in microparticles was sufficiently exposed to initiate the agglutination of the lymphocytes. The agglutination was completely inhibited by preincubating the microparticles with alpha-methyl mannoside.