Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports.
Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')2 and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combining sites. The affinity constant for IgG as well as F(ab')2 and Fab', 6 X 10(9) M-1, was identical when tested using fluid phase antigen (125I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5 X 10(9) M-1 at high antigen concentrations (1.3 X 10(-7) M) to 9 X 10(9) M-1 at low antigen concentration (1.3 X 10(-10) M). Binding constants for F(ab')2 and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis*; D001666:Binding Sites, Antibody; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D051379:Mice; D008819:Mice, Nude; D051381:Rats; D011916:Rats, Inbred F344
D000911:Antibodies, Monoclonal; D000941:Antigens; D005338:Fibrin Fibrinogen Degradation Products; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; C015214:fibrinogen D fragment