Preparation and characterization of chemically defined oligomers of rabbit immunoglobulin G molecules for the complement binding studies.
Pure dimers, trimers, tetramers and pentamers of rabbit non-immune IgG (immunoglobulin G) or antibody IgG were prepared by polymerization in the presence of the bifunctional cross-linking reagent dithiobis (succinimidylpropionate). Oligomerization was performed either in the presence of polysaccharide antigen and specific monomeric antibody (method A) or by random cross-linking of non-immune rabbit IgG in the absence of antigen (method B). By repeated gel-filtration chromatography, samples prepared by both methods exhibited a single band in analytical sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The electrophoretic mobilities of samples prepared by method A were slightly greater than those for the corresponding samples prepared by method B. This might suggest a role played by antigen in the orientation of IgG molecules within the clusters, which may be more compact than those formed by random cross-linking. The average numbers of cross-linker molecules per oligomer varied between 3 and 6 for clusters made by method A and between 1 and 3 for clusters made by method B. Ultracentrifugal analyses of the oligomers yielded sedimentation coefficients (S20,w) of 9.6S for the dimer, 11.2S for the trimer, 13.6S for the tetramer and 16.1S for the pentamer. Comparison of the observed sedimentation coefficients with those predicted by various hydrodynamic models suggested these oligomers possessed open and linear structures. Reduction of the cross-linking molecules converted oligomers into monomeric species of IgG. C.d. spectra of some oligomers studied in the range 200-250 nm were essentially the same as that of monomeric IgG molecules, thus strongly suggesting no major conformation changes in IgG molecules within clusters. These oligomers were found to be stable for up to 2 months when stored at -70 degrees C.
D000818:Animals; D002942:Circular Dichroism; D003165:Complement System Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013388:Succinimides
D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D013388:Succinimides; D003165:Complement System Proteins; C011240:dithiobis(succinimidylpropionate)