Preparative separation of human T cells reactive with the OKT4 monoclonal antibody

Abstract

A cell separation technique is described which can provide large numbers (i.e. greater than 5 x 10(7)) of human lymphoid cells which are positively and negatively selected for their ability to react with the OKT4 monoclonal antibody. Peripheral blood lymphocytes are depleted of plastic-adherent cells and then exposed to OKT4 antibody. Unbound OKT4 antibody is removed by washing and then this OKT4-exposed cell population is placed on plastic Petri dishes that are coated with affinity-purified goat anti-mouse immunoglobulin. Cells which do not bind the OKT4 antibody (OKT4- cells) can be obtained by gently washing the nonadherent cells from the plates, while the adherent (OKT4+) cells can be recovered by vigorous pipetting. This procedure yields functional OKT4+ and OKT4- cell populations with a purity which approaches that obtained by separation using the fluorescence-activated cell sorter. The principal advantages of this plate separation technique are that: (1) large numbers of functional cell populations can be obtained; (2) very small quantities of monoclonal antibody (e.g. 1:10(4) dilution of ascites) are required; and (3) sterility of the cell preparations can be maintained quite easily.