Reduction of interchain disulfide bonds converted some IgG incomplete antibodies to direct hemagglutinins. This conversion occurred whether antibody was free in solution or bound to the red-cell surface. Reduced antibody permitted to reoxidize in air no longer behaved as a direct agglutinin; reversion to an incomplete antibody did not occur when reoxidation was prevented by S-alkylation. These antibodies in low titer. Four sera containing an incomplete antibody of high titer were selected for the isolation and chemical modification of IgG; three (Gue, Les, and Tur) contained anti-D and one (Dai) contained anti-K. Other sera containing incomplete antibodies (anti-D, anti-C, anti-c, anti-E, anti-K, anti-S, anti-Fya, and anti-Jka) were tested without isolation of the IgG. The sera had not been heat-inactivated. Isolation and Characterization of IgG. Two methods were used for the isolation of IgG; in both, partial delipidation by centrifugation (20,000 X g for 30 min) was performed before isolation. In the first, serum Gue and serum Dai were fractionated by ammonium sulfate precipitation followed by anion-exchange chromatography on Whatman DE-52 cellulose equilibrated in 10 mM Tris-HCI/20 mM NaCl, pH 7.0. Monomeric IgG was obtained from the unadsorbed fraction by gel chromatography on LKB Ultrogel AcA 34 equilibrated in Tris-buffered saline (10 mM Tris-HCI/0.15 M NaCI/0.02% NaN3, pH 7.8). The purity of the isolated IgG fractions was established by immunoelectrophoresis in 1.3% agarose (Kallestad) in barbital buffer (pH 8.6, , = 0.05, containing 0.85 mM calcium lactate) against a sheep antiserum to whole human serum and class-specific antisera, and by 0.1% sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 7.0% gels (10).Since in serum Gue anti-D activity was restricted to the fast 'y/f3 fractions isolated from DE-52 cellulose in the gradient fractions, as observed with another example of anti-D (11), the other two sera (Les and Tur) were fractionated on QAE-Sephadex A-50 (12) followed by gel filtration on Ultrogel AcA 34 in Tris-buffered saline. The IgG fractions were assessed for purity as described above and were found to contain a trace contaminant of Al3 mobility, identified as transferrin by Ouchterlony analysis. Before testing, all samples were concentrated by dialysis under reduced pressure against phosphate-buffered saline (40 mM in phosphate, pH 7.2, A = 0.15) containing 0.02% NaN3. The concentration of IgG in whole.sera and in the IgG fractions was determined by single radial immunodiffusion and spectrophotometrically from the absorbance at 280 nm using Alm = 14.0, respectively.Reduction and S-Alkylation of IgG. Each of the IgG samples (about 10 mg/ml) was adjusted to pH 8.6 with 2 M Tris base and mildly reduced with 10 mM dithioerythritol (Sigma Chemical Co.) at room temperature. After 45 min, iodoacetamide (Sigma, twice recrystallized) was added to a final concentration of 25 mM; the samples were left for 1 hr in the dark and then dialyzed into phosphate-buffered saline for testing. R...
