Separation of lymphocytes by specific adherence to cellular monolayers containing protein A of Staphylococcus aureus

Abstract

A technique was developed for the isolation of lymphocytes according to their surface antigenic markers. The method was based on the general reaction of protein A (SpA from S. aureus) with the Fc-part of an IgG antibody. Monolayers of S. aureus or Spa-coated sheep red blood cells (SpA-SRBC) fix antibody-charged cells specifically; non-fixed cells are easily removed by washing. Alternatively, the monolayers can be treated with a cell surface specific antibody prior to addition of non-charged cells. Monolayer-adhered cells were detached by the addition of lysostaphin (bacterial monolayer) or ammonium chloride (SpA-SRBC monolayer). As an example, Ig-bearing cells were isolated from mouse spleen lymphocytes using a specific rabbit anti-mouse Ig serum for charging either the cells or the monolayers. The recovery of Ig-bearing cells was approximately 84%. The purity of the cells was approximately 83% and the viability 89%. Antibody-SpA complexes on the surface of isolated cells were removed either by trypsin treatment or by cultivation. Ig on the cell surface is restored after 16 hr of cultivation.