Separation of antigen specific lymphocytes. I. Enrichment of antigen binding cells

Abstract

Normal mouse spleen cells were fractionated in dishes coated with thin layers of DNP gelatin or NIP gelatin, which were insoluble at 4°C. Highly viable cells were recovered from the dishes by melting the gel at 37°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 album (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 125I 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 125I 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.