ASSESSMENT OF A POSITIVE SELECTION TECHNIQUE USING AN AVIDIN COLUMN TO ISOLATE HUMAN PERIPHERAL-BLOOD T-CELL SUBSETS

The current studies were designed to assess a new technique for positi vely selecting human T cells from whole peripheral blood mononuclear c ells using the minimal amount of monoclonal antibody required to bind the T cell to an avidin column indirectly via a biotin-conjugated seco ndary antibody. Positive selection of T cells has previously been avoi ded because the saturating amounts of antibodies required for other is olation procedures can lead to aberrant results in assays of T cell ac tivation and function. The avidin column technique for obtaining purif ied T cell subsets was compared to a multi-step procedure that include d negative selection panning. The positive selection technique was eas ily performed within 4 h whereas the negative selection technique requ ired a minimum of 12 h to complete. The avidin column technique proved to be a rapid and simple method for isolating T cell subsets of high purity and normal functional capabilities. Since minimal amounts of mo noclonal antibodies were used for the purification protocol, no consis tent inhibitory or stimulatory effect of the residual antibody was not ed in assays of activation and proliferation of positively selected T cells compared to T cells isolated by negative selection panning.