New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes

Abstract

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.