Factors affecting the properties of insolubilized anitbodies.
IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site. The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions. The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1-0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites. On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed.
D001665:Binding Sites; D003488:Cyanogen Bromide; D004958:Estradiol; D004964:Estriol; D004970:Estrone; D007074:Immunoglobulin G; D007700:Kinetics; D011485:Protein Binding; D012995:Solubility
D007074:Immunoglobulin G; D004970:Estrone; D004958:Estradiol; D004964:Estriol; D003488:Cyanogen Bromide