Studies on Affinity Constants of Hapten‐Specific Monoclonal Antibodies Using an Antibody‐Immobilized ELISA

Abstract

An enzyme-linked immunosorbent assay to measure affinity constants of hapten-specific monoclonal antibodies has been developed. In this method an antibody-immobilizing format is used. Antibody affinity toward hapten-enzyme tracer is first calculated by using Scatchard's method. Using this affinity, antibody-hapten affinity is then calculated by following the principles of chemical thermodynamics and mass conservation. The affinity from this method agrees with the IC50 values in ELISA. This method is useful in early determination of affinity during monoclonal antibody development. Compared with the antigen-coated format, this method avoids the problem of distinguishing binding of unliganded antibody and singly liganded antibody to antigens on solid phase.

Characterization of the asialofetuin microtitre plate-binding assay for evaluating inhibitors of ricin lectin activity

“…The interpretation of the galactose-ricin dissociation constant is therefore uncertain, as is the magnitude of the dissociation constant, because the latter is a function of the ricin-asialofetuin dissociation constant, which, as explained above, is only an approximation. This may explain why the value of the dissociation constant (K i ) that was determined in the present work (83 M) is lower than that reported using other methods (149-590 M; Refs 7,[8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27].…”

“…where A 0 is the rate of absorbance change in the absence of inhibitor, A is the rate of change of absorbance in the presence of inhibitor of concentration I, K i is the ricin-inhibitor dissociation constant, R is the concentration of ricin and K r is the ricin-asialofetuin dissociation constant. 22 Equation (3) Fig. 5 (open circles), from which the linearity of the relationship is evident.…”

Characterization of the binding of cholera toxin to ganglioside GM1 immobilized onto microtitre plates

“…where A 0 is the rate of absorbance change in the absence of inhibitor, A is the rate of change of absorbance in the presence of inhibitor of concentration I, K i is the toxininhibitor dissociation constant, T is the concentration of cholera toxin and K t is the toxin-G M1 dissociation constant (Chu et al, 1995). The equation predicts that a plot of A 0 /A vs I will be linear with an intercept of 1 and a slope of [K i (1 + T/K t )] −1 ; K i therefore can be determined by knowing T and K t .…”

Detection of Antigen−Antibody Binding Events with the Atomic Force Microscope

“…The relative strength of the antibody-antigen interaction is estimated using their affinity constants (Harlow & Lane, 1988a) which can be determined experimentally by immunoassay (Chu et al, 1995), fluorescence methods (Bruggeman et al, 1995), and surface plasmon resonance (Pellequer & Vanregenmortel, 1993). However, none of these methods can be used to directly measure the strength of the antigenantibody bond.…”