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

Binding experiments with tobacco mosaic virus and specific Fab fragments were analysed by means of two types of Scatchard plot. The first type, which makes no assumption regarding antigen valence, yielded a series of curves which, when extrapolated, revealed that the effective antigenic valence of the virus was 800. The various plots obtained with different Fab concentrations showed that the affinity constants of TMV antibodies depended on antibody concentration, and they did not substantiate earlier claims that TMV antibodies are homogeneous. The second type of Scatchard plot, which is commonly used for monovalent haptens, yielded a series of lines, the slope of which varied with Fab concentration. The linearity of these plots appears to be an artefact produced by the large antigen valence of tobacco mosaic virus, and does not indicate homogeneity of association constants. The forward binding reaction between Fab and TMV is practically irreversible and is largely dependent on an increase in entropy. It appears that previous workers who investigated this system and reported that anti-TMV antibodies are homogeneous were misled because they assumed an incorrect antigenic valence and used equations that are unsuitable for antigens with a very large valence.