Separation of lymphocyte sub-populations using antibodies attached to staphylococcal protein A-coated surfaces

Abstract

Plastic surfaces which had been coated sucessively with IgG, Staphylococal protein A (SpA) and diluted whole antiserum specific for cell membrane antigens, were used successfully to separate rabbit lymphocyte sub-populations. This method does not require the purification of SpA or antibody, and uses only small quantities of antiserum. The specificity of the cell separation method was demonstrated using various rabbit "b" locus anti-allotype sera, and the number of cells removed by the SpA-antiallotype system correlated well with the number of Ig+ve or "B" lymphocytes detected by other methods. Optimal conditions for cell attachment could readily be determined using multi-well plastic trays, and the adherent cells rapidly quantitated. Using 125I-labelled rabbit IgG (Rb IgG) or sheep IgG (Sh IgG) it was possible to quantitate the amount of IgG coupled to plastic surfaces, or binding to SpA-coated surfaces. By using 125I-labelled (As4) Rb IgG it was also possible to estimate the amount of antibody bound to SpA. The wide application of the SpA-antibody method relative to other affinity-antibody methods is discussed.