Isolation of Ia positive human leukocytes by a direct rosette assay

Abstract

A direct rosette assay (DRA) using ox erythrocytes coated with monoclonal antibody (AF-10) to human Ia-like antigen (OE-anti-Ia) is a rapid and convenient method for detection and isolation of Ia-like antigen-bearing cells. Various cell lines and peripheral blood lymphocytes were tested by DRA. Cell lines expressing Ia-like antigen formed rosettes, while cell lines lacking Ia-like antigen did not. In a highly purified T cell fraction, a small number of T cells formed rosettes; but in a Con A-stimulated T cell fraction, about 40% of the cells formed rosettes. The majority of purified B cells and monocytes formed rosettes. Rosette-forming and non-rosette-forming mononuclear cells could be separated by centrifugation on Ficoll-Urografin. Rosette-forming cells reacted positively by indirect immunofluorescence (IIF), while non-rosette-forming cells reacted negatively. The results obtained by DRA were consistent with those obtained by IIF with OKIa ∗1 and AF-10. DRA is thus suitable not only for detection of Ia-like antigen-bearing cells, but also for separation of Ia-like antigen-positive (Ia +) and -negative (Ia −) cells.