Kinetics of the autologous red cell agglutination test

Abstract

The kinetics of the autologous red cell agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-red blood cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin α on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab′ fragments were determined ( n = 4.80 × 10 5 sites/erythrocyte, K a = 0.43 × 10 7 M −1, K d = 23 × 10 −8 M for 1C3/86, n = 4.66 × 10 5 sites/erythrocyte, K a = 1.05 × 10 7 M −1, K d = 9.5 × 10 −8 M for 10F7MN. The binding studies were performed under the same conditions as the autologous red blood cell agglutination test. When 0.9 μg of anti-glycophorin Fab was added to 10 μl of blood 0.25 μg of 1C3/86 Fab was bound whereas 0.29 μg for 10F7MN Fab′ was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 μg of anti-peptide antibody gave half a maximal agglutination score. This is equivalent to 10 μg/ml circulating antibody. Under the agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal agglutination occurred when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum agglutination score of four occurred in the presence of 1 μg of anti-peptide antibody equivalent to 100 μg/ml circulating antibody whereas an agglutination score of 1+ was elicited by only 0.032 μg anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin per red cell.