Chromatographic fractionation of rhesus monkey (Macaca mulatta) IgG subclasses using deae cellulose and protein A-sepharose.
Rhesus monkey serum was applied to a protein A-Sepharose column at pH 7.3. IgG was not detectable in the unbound effluent. Bound protein was eluted with a pH gradient of citrate-phosphate buffer. Two major elution peaks reproducibly resulted at pH 4.85-4.9 and pH 4.35-4.43, respectively, with only slight variability among individuals. Since the immunoelectrophoretic mobility of IgG in the peak at pH 4.85-4.9 was slower than that of the peak at pH 4.35-4.43, serum was first fractionated by ion exchange chromatography into 2 fractions: the first did not bind to DEAE cellulose in 0.01 M phosphate buffer pH 7.8, the second did bind and was eluted by an NaCl gradient. Each DEAE fraction was then further fractionated on protein A-Sepharose. IgG in the first DEAE fraction bound to protein A at pH 7.3 and eluted in a single peak at pH 4.72-5.0. IgG in the second DEAE fraction also bound to protein A, but eluted in 2 peaks at pH 4.65-5.1, and 4.25-4.6, respectively. All 3 IgG fractions eluted in the same position from an A5m column, had immunodiffusion reactions of identity with anti-human gamma chain and were composed of similar heavy and light chains judged by SDS polyacrylamide gel electrophoresis. However, IgG eluting from protein A at the low pH (4.25-4.6) had a reaction of partial identity with other IgG fractions when tested by immunodiffusion against rabbit anti-rhesus gamma chain, suggestive of an antigenically different subclass. Analysis of rabbit antisera prepared against the 3 IgG fractions confirmed the occurrence of at least 3 antigenically distinct rhesus monkey IgG subclasses. These 3 subclasses have been provisionally designated IgG I, IgG II and IgG III.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D014644:Genetic Variation; D007074:Immunoglobulin G / Q000235:genetics / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008253:Macaca mulatta / Q000097:blood; D008297:Male
D007074:Immunoglobulin G