Conversion of incomplete antibodies to direct agglutinins by mild reduction: evidence for segmental flexibility within the Fc fragment of immunoglobulin G.

Abstract

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...

Changes in quaternary structure of IgG upon reduction of the interheavy-chain disulfide bond.

“…DISCUSSION Reduction of the interheavy-chain hinge disulfide of the IgG molecule interferes with several of the effector functions of the antibody. The complement-fixing ability of the antibody is destroyed (7,8), incomplete antibodies are converted to direct hemagglutinins (25), and the binding of whole antibody to membrane-bound Fc receptors is abolished by mild reduction (26,27). However, studies of the structure of monomeric antibodies have provided little understanding of the effects of cleavage of this disulfide bond.…”

Distinct functions of monoclonal IgG antibody depend on antigen-site specificities.

“…These data as well as those obtained with nonprecipitating equine antibodies of high affinity to the p-azophenyl-fl-lactoside (Lac) haptenic group coupled to protein carrier molecules rule out univalence and low affinity as explanations for functional differences (51). Because mild reduction by 10 mM DTT did not convert the low affinity anti-Av-CHO antibodies into lytic ones, these antibodies do not correspond structurally to the incomplete anti-Rh antibodies (28). Depending on the functional properties of anti-Av-CHO antibodies residing in the determinant specificity for this linear poly-rhamnosyl moiety, the associative model of complement activation and not the allosterie model appears to account for the phenomenon (52).…”

“…Lysis was expressed as a percent against water-lysed Av-CHO-SRBC and SRBC controls in supernates. These experiments were also performed in the presence of 10 mM dithio-threitol (DTT), Clelands reagent, Calbiochem for two low affinity antibody fractions of single-band purity (28).…”

Use of Fab Anti-IgG in Phenotyping IgG-Sensitised Red Cells

“…Heat can be used to elute the sensitising antibody from red cells [ 1 ], as can chloroquine diphosphate (CDP) [2] or ZZAP [3]. These methods have disadvantages in that they cannot always dissociate the red cell-bound immunoglobulin without causing damage to either cell integrity or antigen reactivity [1,3,4], Another method for phenotyping, but not requiring the antiglobulin technique, is to use chem ically modified antisera [5].…”