Immunochemical studies of tobacco mosaic virus—I: Refutation of the alleged homogeneous binding of purified antibody fragments

Antibody affinity measurements

“…and This transformation allows the construction offid vs nf plots from which the value of K and of the antigen valence s can be derived (Rappaport, 1959;Day, 1972;Hardie and Van Regenmortel, 1975). In such a plot ( Fig.…”

“…At any ratio of the reactants, the antigenic valency corresponds to the maximum number of antibody molecules that can be bound simultaneously (Van Regenmortel, 1988). For instance, in the case of tobacco mosaic virus which contains 2130 identical protein subunits, the antigenic valency is 800 and not 2130 as has sometimes been assumed (Anderer et al, 1971;Hardie and Van Regenmortel, 1975). This is true also under conditions of antigen excess when only a few antibody molecules will bind to each virus particle and each antibody molecule has the choice to bind to any of the available 21 30 antigenic sites.…”

“…The antigenic valence must first be calculated from the binding data according to Eqn (4), but since r=ny/sB, it is then possible to construct rjc vs sr plots. One oddity of this representation is that linear plots are always obtained with large multivalent antigens even when the antibody preparation that is used is highly heterogeneous as is normally the case with antiserum (Hardie and Van Regenmortel, 1975). The linear appearance of such plots has led some authors to the mistaken conclusion that their antibody preparations were homogeneous with respect to affinity constant (Mamet-Bratley, 1966;Anderer et al, 1971).…”

“…When sufficient quantities of the reagents are available and the antigen is much larger than the antibody, free antibody separated from the complex by ultracentrifugation can be measured spectrophotometrically (Hardie and Van Regenmortel, 1975). Smaller quantities of labelled reagents can be separated in an air-driven, high speed centrifuge such as the Beckman Airfuge or in the Beckman T L 100 ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA, USA).…”

Operational aspects of antibody affinity constants measured by liquid‐phase and solid‐phase assays

“…An important parameter for understanding the activity and biological significance of antibodies is the strength of their binding to the antigen expressed as the equilibrium constant K (Steward and Steensgaard, 1983). Several experimental techniques have been used for determining the K value of viral antibodies, such as ultracentrifugation (Hardie and Van Regenmortel, 1975) or equilibrium filtration (Fazekas de St Groth and Webster, 1961). These methods allow the equilibrium to be reached in solution and then use a physical principle to separate free from bound antibodies.…”