Studies on Human Antibodies

Abstract

Antibodies to the blood group A and B substances in human sera are extraordinarily heterogeneous. Sera generally contain vG, ~,M, and ~A anfi-A or anti-B (1-6). Differences in properties of various sera by precipitation (4, 5), hemagglutination (4, 5, 7-9), hemolysis (8, 10, 11), complement fixation, (10-12) and fixation to skin (4, 13) have been found. To understand the extent of this heterogeneity it is essential to prepare and study blood group antibodies of the various immunoglobulin classes from single donors. In an earlier study from this laboratory (5), insoluble blood group A and B substances were prepared by attaching polyleucyl side chains to them, using the N-carboxyanhydride method (14). Large volumes of anti-A could be specifically absorbed on these products and, after washing to remove nonspecific protein, a considerable proportion of the antibody was eluted with N-acetylgalactosarnine (GalNAc). This eluate could be fractionated into ~,M anti-A and ~,G anti-A; these two anti-A fractions showed striking differences with respect to the relative capacities of mono-and oligosaccharides to inhibit precipitation by antigen. With the ~,G anti-A, a reduced pentasaccharide was a much better inhibitor on a molar basis than a trisaccharide which in turn was better than N-acetylgalactosamine, while with the ~,M anti-A, all three compounds were equally potent (5).The present study extends these observations, adding the characterization of a second population of antibodies obtained by eluting with a specific blood group A pentasaccharide (ARs0.52) the anti-A antibodies which had remained absorbed on the insoluble antigen after elution with N-acetylgalactosamine.

Immunochemical studies on blood groups LXVI. Competitive binding assays of A1 and A2 blood group substances with insolubilized anti-A serum and insolubilized A agglutinin from Dolichos biflorus.

“…Ethyl chloroformate insolubilized (32,33) human anti-A serum Chris D2 (34) and D. biflorus (23) inselubilized on cyanogen bromide-activated Sepharose 2B (30,(35)(36)(37) were prepared as described earlier (26). Whole anti-A serum was acidified by adding 1.0 M sodium acetate-acetic acid buffer, pH 4.6, to a final concentration of 0.20 M (pH 4.9-5.0).…”

“…Such antibodies must have sites large enough to accommodate the whole determinant. The antibodies from Chris anti-A have been purified (34) according to size of the combining site by elution from PL-Hog A with DGalNAc followed by the ARL 0.52 pentasaccharide with the structure (5, 7) LFucal 2 DGaINAcal--*3DGaI~I--*4DGIcNAc~I--*6R.…”

Purification and characterization of a blood-group A reactive hemagglutinin from the snail Helix pomatia and a study of its combining site

“…minant have been shown to be heterogeneous both with respect to size of combining sites (Kaplan and Kabat, 1966;Moreno and Kabat, 1969), heavy and light chains, and genetic markers (Yount et al, 1968;Dorner et al, 1969). Partial fractionation of antibodies with respect to size of combining site can be accomplished by specific elution with haptens representing the entire antigenic determinant or smaller parts of it (Schlossman Gelzer and Kabat, 1964a,b;Moreno and Kabat, 1969). Antibody heterogeneity seriously limits the study of sequence and the three-dimensional arrangements of amino acids in the combining region.…”

The Reactions of IgG and IgM Anti-A and Anti-B Blood Group Antibodies with 125I-Labelled Blood Group Glycoproteins

“…Reduction of IgM anti-B to subunits was accompanied by a marked fall in equilibrium constant, suggesting that IgM anti-B is attached to red cells by at least two combining sites [3]. Moreno and Kabat [12] were unable to demonstrate binding of A hapten to IgM anti-A by equi librium dialysis, suggesting that the equilibrium constant of each binding site is extremely low. Moreno and Kabat [12], however, also experienced difficulty in obtaining IgM anti-A in eluates from columns of A glycoprotein and they suggested that this might be related to their high valency and hence to a higher binding constant than IgG anti-A for a multivalent glycoprotein antigen.…”