title: 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 alpha 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 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 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 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram 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 microgram of anti-peptide antibody gave half a maximal agglutination score. This is equivalent to 10 micrograms/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 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per red cell.
authors.affiliation: St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Catimel
authors.forename: K M
authors.initials: KM
authors.lastname: Wilson
authors.forename: B E
authors.initials: BE
authors.lastname: Kemp
mesh_terms: D000906:Antibodies / Q000032:analysis; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis*; D001666:Binding Sites, Antibody; D004912:Erythrocytes / Q000276:immunology* / Q000378:metabolism; D015488:HIV Antigens / Q000032:analysis; D015497:HIV-1 / Q000276:immunology; D006386:Hemagglutination Tests; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D018272:Porins / Q000032:analysis / Q000276:immunology; D011233:Precipitin Tests; D011485:Protein Binding; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D015488:HIV Antigens; D007128:Immunoglobulin Fragments; D010455:Peptides; D018272:Porins