Inhibition of cryoprecipitation of murine IgG3 anti-dinitrophenyl (DNP) monoclonal antibodies by anionic DNP-amino acid conjugates

Previously we have demonstrated that eight out of nine IgG3 monoclonal antibodies (mAb) obtained from autoimmune MRL-lpr/lpr mice were able to self-associate and to precipitate in the cold (Gyotoku et al., J. Immunol. 1987. 138:3785). To determine whether the cryoprecipitation of IgG3 mAb is enhanced or inhibited in the presence of specific ligand, we have established eight IgG3 mAb reactive with 2,4-dinitrophenol (DNP) hapten: four mAb were obtained from fusion of spleen cells of C57BL/6 mice immunized with 2,4,6-trinitrophenylated keyhole limpet hemocyanin, three from 129/Sv and one from BALB/c immunized with DNP-lipopolysaccharide. Five of them induced cryoglobulins composed exclusively of the IgG3 mAb. The binding of negatively charged monomeric DNP-amino acid conjugates completely inhibited the cryoprecipitation of all the five cryoprecipitating anti-DNP IgG3 mAb, while the incubation with positively charged or neutral DNP-amino acid conjugates had variable effects: increase, inhibition or no change of the cryoprecipitation. In addition, positively charged DNP-amino acid conjugates were able to induce the cryoprecipitation of one of the non-cryoprecipitating anti-DNP IgG3 mAb. Our data showed that (a) IgG3 mAb derived from non-autoimmune strains of mice, similar to IgG3 mAb derived from an autoimmune MRL-lpr/lpr strain, possessed the unique property to self-associate and were able to form cryoglobulins in most cases; (b) although the Fc-Fc interactions of IgG3 mAb play a decisive role in IgG3 cold solubility, IgG3 cryoprecipitation was markedly influenced after interacting with their specific ligand, depending on the charge of the hapten-amino acid conjugate. This suggested that even minor interferences with the electrostatic equilibrium of the IgG3 by the binding of charged hapten molecules induced dramatic changes in the solubility of the IgG3 mAb at low temperature