Separation of antigen-specific lymphocytes. I. Enrichment of antigen-binding cells.
Normal mouse spleen cells were fractionated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin, which were insoluble at 4 degrees C. Highly viable cells were recovered from the dishes by melting the gel at 37 degrees C. NIP3- gelatin layers bound approximately 0.1% and DNP4-gelatin layers 0.5% of normal spleen cells. Increasing numbers of low affinity cells were bound with increasing DNP density of the adsorbent. The binding to insoluble DNP-gelatin was hapten-specific since it was inhibited by DNP-lysine, soluble DNP-gelatin or DNP-BSA but not by soluble gelatin or bovine serum albumin (BSA). It was also inhibited by a polyvalent rabbit antimouse Ig. DNP-gelatin was detected on the surface of cells recovered from DNP-gelatin-coated dishes by 125-I-labeled anti-DNP Ig. The cell surface bound DNP-gelatin could be removed by treatment with collagenase. Collagenase treatment did not detectably affect cell viability or surface receptors. More than 90% of DNP-gelatin binding cells were labeled with a polyvalent 125-I-labeled antimouse Ig before or after collagenase treatment under conditions known to label B lymphocytes. Furthermore, the specific antigen-binding capacity of the purified cell populations could be demonstrated after treatment with collagenase. Purified DNP4-gelatin binding cells contained more than 100 times as many DNP-RFC than unfractionated cells. The enrichment of NIP-RFC in the cell population recovered from NIP3 gelatin-coated dishes was more than 200-fold.
D000327:Adsorption; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002384:Catalysis; D002469:Cell Separation; D004140:Dinitrophenols; D000939:Epitopes; D006241:Haptens; D007104:Immune Adherence Reaction; D007136:Immunoglobulins / Q000032:analysis; D007457:Iodine Radioisotopes; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D008808:Mice, Inbred CBA; D011817:Rabbits / Q000276:immunology; D012710:Serum Albumin, Bovine; D012756:Sheep / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology
D004140:Dinitrophenols; D000939:Epitopes; D006241:Haptens; D007136:Immunoglobulins; D007457:Iodine Radioisotopes; D012710:Serum Albumin, Bovine