Immunochemical studies on blood groups. LIV. Classification of anti-I and anti-i sera into groups based on reactivity patterns with various antigens related to the blood group A,B,H, Le a, Le b and precursor substances.

Quantitative precipitin assays were carried out with eleven anti-I and five anti-i cold agglutinin sera. Their reaction patterns with precursor substances and with certain antigens related to the A, B, H, Le(a), and Le(b) substances revealed at least six types of I and four types of i specificity. All of the I and i determinants appear to be represented in varying amounts on that fraction of the ovarian cyst precursor substance OG which was precipitated by 20% ethanol (OG 20% 2X). Only some of these determinants can be detected in the first periodate oxidation stages of A, B, and H substances, in cow substances, and in hydatid cyst fluid.

Studies on human antibodies. 8. Properties and association constants of human antibodies to blood group A substance purified with insoluble specific adsorbents and fractionally fluted with mono- and oligosaccharide.

Human antibodies to blood group A substance were purified by absorption on columns of insoluble polyleucyl hog blood group A + H substance and eluted first with N-acetylgalactosamine and then with an A active reduced pentasaccharide AR(L)0.52. The gammaM and gammaG antibodies in these eluates were separated by density gradient centrifugation. The antibodies were studied for their relative capacities to be inhibited by various blood group A active oligosaccharides. Antibodies eluted by the N-acetylgalactosamine could be inhibited by N-acetylgalactosamine, as well as by lower concentrations of A active tri- and pentasaccharides, while those eluted by the pentasaccharide AR(L)0.52 could only be inhibited by the two oligosaccharides, but not by N-acetylgalactosamine, indicating that the N-acetylgalactosamine eluate had more antibodies with smaller size combining sites than the AR(L)0.52 eluate. Measurements by equilibrium dialysis gave values ranging from 2 x 10(3) to 1 x 10(5)M(-1) and the values obtained with the AR(L)0.52 eluate were somewhat higher than those with the GalNAc eluate. Only one of three anti-A sera had gammaM anti-A in the AR(L)0.52 eluate, while all three had gammaM in the N-acetylgalactosamine eluate. Data on the precipitating, hemagglutinating, complement fixing, hemolytic properties of the eluted antibodies, and of their content of kappa and lambda light chains are given.

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid. I. Antigenicity of the glycolipids and the production of specific antibodies in rabbits.

Isomaltose oligosaccharides varying in size from two sugars, isomaltose (IM2), to seven sugars, isomaltohepatose (IM7), were coupled to stearylamine by reductive amination with sodium cyanoborohydride. Each compound was purified by column chromatography to yield a series of glycolipids containing oligosaccharides differing in length. Stearyl-isomaltotriose to stearyl-IM7 could be incorporated into liposomes and could render them agglutinable by specific antibodies to alpha 1 leads to 6 dextran and could be lysed if complement was also added, whereas those containing stearyl-IM2 were not agglutinated or lysed, indicating that stearyl-IM2 may not be protruding from the liposome surface sufficiently to react with the antibody. Stearyl-isomaltosyl oligosaccharides by themselves or incorporated into liposomes were equally antigenic when emulsified in complete Freund's adjuvant. They elicited pauciclonal responses, and the antibodies were alpha 1 leads to 6 specific, cross-reacted with dextran, and gave semirestricted isoelectric focusing patterns.

Immunochemical studies on blood groups LXII. Fractionation of hog and human A, H, and AH blood group active substance on insoluble immunoadsorbents of Dolichos and Lotus lectins.

The purified lectins from Lotus tetragonolobus and Dolichos biflorus were coupled to Sepharose 2B to make insoluble adsorbents for purification and fractionation of blood group A and H active glycoproteins. With both adsorbents, hog gastric mucin A + H blood substance (HGM), purified by phenol-ethanol precipitation, yielded fractions showing only A, only H, or AH activities. The AH fraction was obtained when the adsorbent column was overloaded with HGM and its A and H specificities seem to be carried on the same molecules since they were not separable by chromatography on either column. However A and H specificities of blood group substance from the stomach of a presumably heterozygous individual hog were both on the same molecules as they too could not be fractionated on either column. Analytical properties of the isolated fractions were generally similar to those of the unfractionated material, the purfied A substances had a higher galactosamine/fucose ratio than did the H substances. Although the original A + H showed very little specific optical rotation, the separated A and H substances rotated positively and negatively, respectively. The lectin-Sepharose adsorbents have also proven useful in isolating A or H substances directly from the crude commercial hog gastric mucin. Blood group A2 substance from a human ovarian cyst yielded two fractions on the Lotus-Sepharose column; the effluent did not interact with the Lotus lectin but precipitated the Ulex and Dolichos lectins and anti-A, and appears to contain type 1 H determinants. The other fraction reacted with Lotus and Ulex lectin as well as with Dolichos and anti-A.

Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

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.

Competitive binding assays using 3H-labeled blood group A substance and insolubilized Dolichos biflorus lectin or human anti-A were carried out, measuring competition by blood group A1 and A2 glycoproteins, and by unabsorbed anti-A sera, and with these sera absorbed with the A1 and A2 glycoproteins. With Dolichos lectin specific for (formula: see text) A1 substances had about 11 times as many determinants as did A2 substances, but the slopes of the lines in the competitive binding assays were the same. With insolubilized anti-A, A2 substances gave lines of lower slopes. Although individual A1 populations varied in the amounts giving 50% inhibition in the assays, as did A2 substances, the slopes of the lines for the A1 substances were the same and always higher than the slopes of the lines for the A2 substances. Competitive binding assays with unabsorbed anti-A sera and with these sera absorbed with insoluble polyleucyl A1 and A2 substances showed that partial absorption of polyleucyl A1 substances left antibodies of lower slope in the supernate, whereas absorption with polyleucyl A2 substance left antibodies (anti-A1) having the same or an even higher slope than the unabsorbed sera. The findings indicate that human A1 and A2 glycoproteins differ in their determinants, and that A2 specificity is determined by the type 2 chain in which the A trisaccharide (formula: see text) is linked beta 1 leads to 4 to DGlcNAc, whereas the A1 specificity is determined by the type 1 chain in which this trisaccharide is linked beta 1 leads to 3 to DGlcNAc; most of the determinants in the glycoproteins have a second LFuc linked alpha 1 leads to 3 and alpha 1 leads to 4 to the DGlcNAc of the type 2 and type 1 chains, respectively.

An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity.

In an attempt to account for antibody specificity and complementarity in terms of structure, human kappa-, human lambda-, and mouse kappa-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24-34, 50-56, and 89-97; variations in length have been found in the first and third of these. It is hypothesized that positions 24-34 and 89-97 contain the complementarity-determining residues of the light chain-those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.

Evidence indicating independent assortment of framework and complementarity-determining segments of the variable regions of rabbit light chains. Delineation of a possible J minigene.

Amino acid sequences of rabbit light chains show considerable evidence of independent assortment of framework (FR) and complementarity-determining (CDR) segments. This suggests that they are coded for by independent genetic units (minigaenes) and that individual light chains are assembled somatically by recombining these units. Identical FR sets with multiple members generally comprise chains with different specificities, whereas identical CDR sets tend to have chains of a single specificity. A J segment, which, by analogy with mouse light chains, is made up of the last two residues of CDR3 plus all of FR4, contained 18 different sets and could contribute to diversity generated by CDR3. The longest segment, FR3, had a very large number of sets. Evidence is presented showing that the number of sets could be substantially reduced by permitting FR3 to be formed by two independently assorting segments comprising residues 57-68 and 69-88.

Studies on human antibodies. IV. Purification and properties of anti-A and anti-B obtained by absorption and elution from insoluble blood group substances.

Insoluble blood group substances prepared by copolymerization of soluble blood group substances with N-carboxy-L-leucine anhydride were used to absorb blood group antibodies from two human, high-titered anti-A sera. After the absorbants were washed free of nonspecific serum proteins, blood group antibodies were eluted either with pH 3.62 acetate buffer, or at neutral pH with monosaccharide haptens of the A or B antigenic determinants (N-acetyl-D-galactosamine or D-galactose respectively). The purified anti-A antibodies were characterized, immunoelectrophoretically, as gammaM-, gammaA-, and gammaG-immunoglobulins. These were further separated into gammaM- and gammaG-fractions by gel filtration or density gradient centrifugation. Both gammaM- and one of the two gammaG-antibody fractions contained K and L light chain determinants; the remaining gammaG-fraction was comprised, almost totally, of type K molecules. Precipitability of the purified anti-A immunoglobulins by blood group A substance varied from 43 to 89%. The agglutinating activity per unit N of the isolated gammaG-anti-A was found to equal, in one case, and to exceed, in the second, that of the gammaM-antibodies from the same individuals. The marked differences between gammaM- and gammaG-antibody fractions in quantitative hapten inhibition studies were interpreted to mean that the antibody-combining site of the isolated eluted gammaG-anti-A was significantly larger than that of the eluted gammaM-anti-A. Whether these data connote differences in combining site size between entire immunoglobulin classes in an individual serum or simply reflect the properties of highly selected antibody populations cannot be stated at present.

Glycosylation of a VH residue of a monoclonal antibody against alpha (1----6) dextran increases its affinity for antigen.

We have observed that antidextran hybridomas with potential N-linked glycosylation sites in VH have higher affinity for polymeric dextran and for isomaltoheptaose than those lacking potential glycosylation sites. In these studies we have used gene transfection and expression techniques to verify that the carbohydrate addition sites in VH were used. The carbohydrate of the VH region was accessible for binding by the lectin Con A. By ELISA analysis it was demonstrated that the aKa of the antibody for dextran was influenced by the presence of carbohydrate in VH, with the aglycosylated antibody having an aKa 15-fold lower than its untreated counterpart. The aKa for antigen of antibodies that contain carbohydrate only in their constant region was unaffected by lack of carbohydrate. Thus, not only the amino acid sequence of the variable region but also its carbohydrate moieties can determine the magnitude of the antigen-antibody interaction.

Immunochemical studies on mouse myeloma proteins reactive with dextrans or with fructosans and on human antilevans.

Four BALB/c IgA mouse myeloma proteins (W3129, W3434, QUPC 52, and UPC 102) reactive with dextran, four myeloma proteins reactive with fructosans, three IgA (W3082, UPC 61, and Y5476), and one IgG2a (UPC 10), and two human antilevans were studied immunochemically. Quantitative precipitin and inhibition assays showed that W3129, W3434, and QUPC 52 had specificities for isomaltose oligosaccharides similar to those previously found with alpha(1 --> 6)-specific human antidextrans. W3129 and W3434 were most complementary to IM5 but W3129 reacted equally with IM4 and IM3 while W3434 had a greater affinity for IM4 than IM3. QUPC 52 had a larger combining region and was most complementary to IM6. Protein UPC 102 (IgA), like MOPC 104E (IgM) (27), was most complementary to the alpha(1 --> 3)-linked trisaccharide, nigerotriose, and thus differed from J558 (29), which was inhibited best by nigeropentaose. UPC 102 was similar to J558 but they differed from MOPC 104E in their reactions with non-alpha(1 --> 3)-linked disaccharides. The fructosan-specific myeloma proteins fell into two groups with different specificities. The first group, W3082 (IgA), UPC 61 (IgA), and the previously studied J606 (IgG3) (28, 29), reacted with inulin and W3082 and UPC 61 appeared to have identical specificities for beta(2 --> 1)-linked fructofuranosyl residues with maximum complementarity for the tetrasaccharide betaDfructofuranosyl (2 --> 1)betaDfructofuranosyl(2 --> 1)betaDfructofuranosyl(2 --> 6)Dglucose while protein J606 was inhibited best by the trisaccharide betaDfructofuranosyl(2 --> 1)betaDfructofuranosyl(2 --> 6)Dglucose. W3082 and UPC 61 also differed from J606 in their behavior toward sucrose and betaDfructofuranosyl(2 --> 6)Dglucose as compared with alphaDglucosyl(1 --> 3)Dfructose (turanose). The second group containing myeloma proteins UPC 10 (IgG2a) and Y5476 (IgA) behaved similarly to human antilevans in that neither reacted with inulin nor were they inhibited by the beta(2 --> 1)-linked fructose oligosaccharides. Unlike the beta(2 --> 1)-specific proteins, they reacted with perennial rye grass levan that contained over 90% beta(2 --> 6)links. The differences in specificity and site size among homogeneous mouse myeloma proteins reactive with the same antigenic determinant are completely consistent with the concept that they represent products of homogeneous clones selected from the known heterogeneous population of antibody-forming cells.

Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran.

Binding constants of the dextran-reactive BALB/c mouse IgA myeloma proteins W3129 and QUPC 52 have been determined for each member of the isomaltose series of oligosaccharides and for methyl alphaDglucoside. Protein W3129 has maximum complementarity for isomaltopentaose (IM5) deltaf degrees = 7,180 cal/mol) with 55-60% of the total binding energy directed against methylalphaDglucoside. Protein QUPC 52 gives maximum binding with isomaltohexaose (IM6) (deltaF degrees = -5,340 cal/mol) and has about 70% of its total binding energy for isomaltotriose (IM3), but at most only 5% for isomaltose (IM2) or methyl alphaDglucoside. Protein W3129 precipitates with branched dextrans high in alpha (1 yields 6) linkages and reacts with but does not precipitate a synthetic alpha (1 yields 6)-linked linear dextran. Protein QUPC 52 precipitates both branched and linear dextrans. Thus, the immunodominant group for protein W3129 is mimicked by methyl alphaDglucoside and this protein reacts exclusively at the terminal nonreducing ends of alpha (1 yields 6)-linked dextran chains. Protein QUPC 52 has an immunodominant group which is expressed by IM3 but not smaller oligosaccharides and this protein can react at nonterminal locations along alpha (1 yields 6)-linked dextran chains. Precipitation of linear dextran seems to be a valid although not quantitative assay for antidextrans with nonterminal specificity. Quantitative precipitin reactions with branched and linear dextrans suggest that alpha (1 yields 6)-specific human antidextrans are mixtures of molecules having terminal and nonterminal specificities and that the fraction of each type can vary among individuals. Rabbit antisera against IM3 or IM6 coupled to bovine serum albumin also appear to contain antibodies with nonterminal specificity for dextran chains although a large fraction has terminal specificity. Low molecular weight clinical dextran N-150N (congruent to 60,000) reacted more like linear dextran than like its parent native-branched dextran B512. This is thought to result from an abundance of nonterminal determinants in clinical dextran N-150N but a very small number of functional terminal determinants per molecule. An appreciation of terminal and nonterminal specificities and of the different immunodominant structures in isomaltosyl chains has proven to be of a great value in understanding the immunochemical reactions of dextrans. Moreover, certain previous findings with fructosan-reactive mouse myeloma proteins and human antilevans (55, 84) also suggest terminal and nonterminal specificities for levan chains.

Immunochemical specificity of the combining site of murine myeloma protein CAL20 TEPC1035 reactive with dextrans.

The immunochemical specificity of the combining sites of murine myeloma protein CAL20 TEPC1035 was studied by quantitative precipitin and precipitin inhibition assays. Myeloma protein CAL20 TEPC1035 precipitated with only three dextrans, B1355S4, B1498S, and B1501S, with high proportions of alpha(1 leads to 3) linkages, but not with any other dextrans, glycogen, and pullulan. Inhibition tests with various sugars show that the combining site of myeloma protein CAL20 TEPC1035 is most complementary to panose, a trisaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc. Panose was 3.3 times more potent than a tetrasaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc alpha(1 leads to 4)DGlc and 8, 23, 42, > 42 times more active than maltose, nigerose, isomaltose, and kojibiose, respectively. These findings were paralleled by their binding properties as determined by affinity electrophoresis. The association constants (Ka) of these three dextrans to myeloma protein CAL20 TEPC1035 ranged from 3.8 X 10(3) ml/g to 5.02 X 10(3) ml/g. The association constant of inhibitor (Kia) of panose was 8.19 X 10(3) M-1. Myeloma protein CAL20 TEPC1035 is an antidextran with specificity different from those of other murine myeloma antidextrans and from human antidextrans reported previously and its combining site size is at least as large as a trisaccharide. The binding constant of methyl alpha-D-glucoside (7.2 X 10(2)) was 73% of that of panose and comparable to that of myeloma protein W3129 (9.4 X 10(2)) with a cavity-type site and 600 times lower (1.6 X 10(0)) for QUPC52 with a groove type site, indicating that the terminal nonreducing residue is held in a cavity. Inhibition data with various alpha(1 leads to 4)-linked oligosaccharides also indicate that the internal portions of these inhibitors may react directly with a portion of the combining site. These findings suggest that myeloma antidextran CAL20 TEPC1035 has a partial cavity-type combining site in which the terminal nonreducing dGlc alpha(1 leads to 6) moiety is held in a cavity with the other two sugars forming a groove. However, oligosaccharides with one or more alternating [leads to 3DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 3)DGlcl leads to] units with and without terminal nonreducing DGlc alpha(1 leads to 6) or DGLc alpha(1 leads to 3) side chains remain to be tested to determine whether structures known to be present in the three dextrans which precipitate CAL20 TEPC1035 may not prove to be more active than panose.

The epitope associated with the binding of the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1 to a human monoclonal macroglobulin, IgMNOV.

The fine structure of the combining site of human mAb IgMNOV to poly-alpha(2----8)linked NeuNAc, the epitope of the group B meningococcal and E. coli K1 polysaccharides, has been probed using RIA and ELISA. Inhibition by oligomers ranging from 2 to 12 residues was used to assay binding to IgMNOV by group B meningococcal polysaccharide preparations (GBMP) or by poly(A). The inhibitory properties of the oligomers were almost identical in both assays of the binding of GBMP to horse IgM (H46). This evidence and the finding that both GBMP and poly(A) precipitated IgMNOV equally per unit weight indicated that the epitope of poly(A) must mimic an equivalent epitope on GBMP despite the absence of any apparent common structural features in the two molecules. Unlike most carbohydrate-anticarbohydrate systems in which the site is saturated by oligomers of up to six or seven sugars, all the anti-alpha(2----8)NeuNAc systems above required much larger oligomers. Because these oligomers are larger than the maximum size of an antibody site the epitope must be conformationally controlled, and this has been confirmed by nuclear magnetic resonance spectroscopy. However, despite the above similarities, GBMP and poly(A) were differentiated in that only GBMP bound to H46. Smaller linear molecules obtained by delipidating the GBMP, as well as periodate-oxidized GBMP with its nonreducing end oxidized or linked covalently to BSA, bound to and precipitated IgMNOV and H46. This showed that, despite their differences, terminal nonreducing ends were not involved and that both epitopes were located in the conformationally controlled inner residues of the GBMP. The difference thus must reside in the ability of IgMNOV and H46 to recognize different structural aspects of the same conformationally controlled inner residues. The ELISA data indicate that both IgMNOV and H46 have groove-type sites that bind exclusively to an epitope located on the acidic side of the inner residues. The differences determining the ability of IgMNOV and the failure of H46 to cross-react with poly(A), poly(I), and denatured DNA, may depend on differences in the degree of protonation required by each antibody, and this may be clarified by a study of the effects of pH on the precipitin behavior of IgMNOV and H46.

Immunochemical studies on blood groups. XLVII. The I antigen complex--precursors in the A, B, H, Lea, and leb blood group system--hemagglutination-inhibition studies.

A partially purified blood group-like substance obtained from milk showed I activity with 2 of 21 anti-I sera. With these antisera, certain human ovarian cyst substances considered to be precursors of the A, B, H, Le(a), and Le(b) substances also showed I activity comparable to the milk material. Strong I activity could be produced by one-stage periodate oxidation and Smith degradation of human ovarian cyst A and B substances, or of hog mucin A + H substance, or by mild acid hydrolysis of human saliva or ovarian cyst blood group B substance. The two sera indicate that I specificity appears at intermediate stages in the biosynthesis of the A, B, H, Le(a), and Le(a) substances. Anti-I sera differ strikingly in their specificities, indicating substantial heterogeneity of the I determinants.

Studies on human antibodies. V. Amino acid composition of antidextrans of the same and of differing specificities from several individuals.

Human dextran-antidextran-specific precipitates from individuals immunized with dextrans containing either one or two antigenic determinants, were analyzed for their content of various amino acids. Large differences in certain amino acids were found among alpha-(1 --> 6)-specific antidextrans. Wide variations were also observed in antidextran fractions from a given individual specific for alpha-(1 --> 6)- and alpha-(1 --> 2)-linked glucose residues.

Studies on human antibodies. VI. Selective variations in subgroup composition and genetic markers.

The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types. Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major gammaG1-type. However, a high preponderance of molecules of the minor gammaG2-subgroup was found for antibodies to dextran, levan, and teichoic acid. These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens. Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others. The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals. Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals. Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios. The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda. The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of gammaG2-heavy chains and kappa light chains. By these criteria as well as others, it closely resembled myeloma proteins.

Combining site specificities of mouse hybridoma antibodies to dextran B1355S.

The combining sites of 12 mouse hybridoma antibodies to dextran B1355S have been characterized by quantitative precipitin assay. All antibodies preferentially bind the immunizing antigen B1355S and two other class I dextrans, B1498S and B1501S, but show substantial differences in the extents to which they cross react with class I dextrans, suggesting their clustering into five groups. Three myeloma proteins, CAL20 TEPC1035, J558, and MOPC104E, which bind dextran B1355S, each fall into a different group. There appears to be a substantial, but imperfect, correlation of DH region structure and individual idiotypic determinants with dextran binding patterns. Proteins with RY DH segments and IdI (J558) idiotypes are in groups 1 or 3, and proteins with YD DH segments and IdI (MOPC104E) idiotypes are exclusively in group 5. However, identical patterns of precipitin curves accompany very different sequences in CDR3. Antibodies of group 1, which react only with class II dextrans, differ the most in primary sequence, a finding suggesting that subsites responsible for cross reactivity with class I dextrans may be blocked and that this may be effected by side chains of different amino acids. This finding delineates a new aspect of the relationship of variability in amino acid sequence to antibody complementarity.

A human monoclonal macroglobulin with specificity for alpha(2----8)-linked poly-N-acetyl neuraminic acid, the capsular polysaccharide of group B meningococci and Escherichia coli K1, which crossreacts with polynucleotides and with denatured DNA.

We have described an IgM antibody from a patient with macroglobulinemia specifically reacting with poly-alpha(2----8)N-acetyl neuraminic acid (NeuNAc) the capsular polysaccharide of two important human pathogens, group B meningococcus and E. coli K1. This antibody has a narrowly defined specificity in its interactions with polysaccharides, being unable to bind poly-alpha(2----9)NeuNAc or alternating poly-alpha(2----8)alpha(2----9)NeuNAc. However, it shows interesting crossreactivity with seemingly unrelated polynucleotides and denatured DNA, supporting the hypothesis that charged groups with a given spacing may determine the specificity of antigen-antibody interactions on otherwise dissimilar molecular structures. Despite the crossreactivity with denatured DNA and polynucleotides, the antibody does not appear to have adverse effects in the patient. The antibody protects newborn rats against E. coli K1 infection, as well as the standard horse antiserum H46, and one would expect it to prove useful in humans as an adjunct to antibiotic therapy in infections with group B meningococcus and E. coli K1. We have attempted to clone the antibody-producing cells from peripheral blood, and have shown that the relevant cells are present and can be cultured.

Immunochemical similarities between monoclonal antibacterial Waldenstrom's macroglobulins and monoclonal anti-DNA lupus autoantibodies.

Six monoclonal IgM from patients with Waldenstrom's macroglobulinemia that react with Klebsiella polysaccharides were tested for their ability to bind to nucleic acid antigens. One of the macroglobulins bound to the polynucleotide poly(G), and one bound to poly(G), poly(I), and single-stranded DNA. The reaction with the polynucleotides was specifically inhibited by the Klebsiella polysaccharide K30. A monoclonal lupus anti-DNA antibody (16/6) was found to react weakly with the Klebsiella polysaccharides K30 and K21. Five of the Waldenstrom macroglobulins shared an idiotypic determinant with the 16/6 anti-DNA antibody. The reaction between the macroglobulins and the antiidiotype serum was specifically inhibited by Klebsiella polysaccharides, an indication that the idiotypic marker was in the antigen-binding site of the macroglobulins. These results indicate the existence of widely dispersed conserved variable region genes that encode idiotypically related immunoglobulins with the capacity to bind to both bacterial polysaccharides and nucleic acids. Such genes can be expressed by patients with either Waldenstrom's macroglobulinemia or systemic lupus erythematosus.