Precipitin Curves Research Articles

Activation/Inhibition of mast cells by supra-optimal antigen concentrations

Mast cells (MCs) are tissue resident cells of hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activation of the FcεRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The FcεRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high (so-called supra-optimal) Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the FcεRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound FcεRI formed a monovalent complex with “its own Ag”, thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of FcεRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive FcεRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5′-phosphatase SHIP1) and they actively down-regulate MC effector responses. Thus, the analysis of MC signaling triggered by supra-optimal crosslinking holds great potential for identifying novel targets for pharmacologic therapeutic intervention to benefit patients with acute and chronic allergic diseases.

Design and Synthesis of High-Avidity Tetravalent Glycoclusters as Probes for Sambucus sieboldiana Agglutinin and Characterization of their Binding Properties

We designed and synthesized three tetravalent sialo-glycoclusters that had different separations between the terminal sialic acids and the linking carboxy groups of the ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate scaffold to serve as ligands for the sialic acid-binding lectin Sambucus sieboldiana agglutinin (SSA). The interaction between each glycocluster and SSA was characterized by hemagglutination inhibition, quantitative precipitation, and double-diffusion assays. For the precipitation assays, the precipitin curves indicated that the ligands and SSA bound in either a 1:1 or a 1:2 ratio, i.e., stoichiometrically. The strong interactions of these sialo-glycoclusters with SSA could be ascribed to a combination of multivalency and spacer effects. We also assessed the nature of the ligand-SSA complexes by isothermal titration calorimetry and dynamic light scattering. The results of those experiments indicated that formation of intermolecular complexes occurred at less than stoichiometric ratios of ligand to SSA concentrations and that, as the concentrations of the ligands increased, larger cross-linked aggregates formed. Large aggregates that were present concurrently with visible precipitation and with a particle size centered at ~600 to 800 nm were identified by dynamic light scattering.

A general theory for the precipitin reaction--based on Fc-mediated precipitation.

Fc-mediated immune precipitation is studied theoretically by computer simulation of precipitin curves and of zonal profiles of soluble immune complexes. Comparison with experiments indicates that Fc-mediated immune precipitation is caused by Fc-Fc interactions with association constants ranging from 10(5) to 10(7) liters/mole. The Langmuir adsorption isotherm is used for evaluation of these interactions. A general theory for the precipitin reaction is formulated, assuming it to be a three-stage process: 1st stage Formation of immune complexes, 2nd stage Precipitation due to coalescence of antibody-rich immune complexes, and 3rd stage Fc-mediated immune precipitation.

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-gamma. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcgammaRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.

Lectin structure-activity: the story is never over.

Advances in plant lectin biochemistry have made great strides during the past decade. Technical advances in biophysical techniques and molecular biology, the availability of synthetic oligosaccharides, and characterization of lectins with unique carbohydrate-binding properties are responsible for these advances. Studies in this laboratory support the view that interesting new discoveries are yet to be made. A new lectin was recently isolated from a fungus (Polyporous squamosus) that recognizes the Neu5Ac alpha2,6 Gal beta1,4 GlcNAc/Glc trisaccharide epitope with high affinity. The lectin does not interact with alpha2,3-linked Neu5Ac or Neu5Ac alpha2,6 GalNAc groups as occur in ovine submaxillary mucin. An unusual lectin with two distinctly different carbohydrate-binding sites is present in tubers of Xanthosoma sagittifolium (L). One species of sites recognizes clusters of oligomannosyl residues. The other type of binding site best accommodates a nonsialylated, triantennary oligosaccharide having LacNAc or Lacto-N-biose (Gal beta1,3GlcNAc) groups at its three nonreducing termini. The banana lectin has also been studied. It recognizes both alpha and beta1,3-linked glucosyl oligosaccharides, generates a precipitin curve with the branched trisaccharide Man alpha1,6[Man alpha1,3]Man, and binds to beta-glucans containing beta1,6-glucosyl end groups.

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

Steep Gradients of Inert Substances as Supports for Precipitin Reactions

Precipitin reactions were conducted in the wells of micro titer plates and the light "absorbance" at 405nm plotted as a function of the neg log2 dilutions of the antigens using the Titertek Multiscan apparatus. The procedure followed was to incorporate antibody in 20% sucrose and to diffuse the mixture into serial two-fold dilutions of antigen overlaid on the sugar-antibody columns in the wells. Where the antigen met its antibody in equivalence precipitin bands formed. The bands of precipitates remained suspended. The precipitates formed by mixing the antibody sugar mixture with the serial two-fold dilutions of the antigen were also recorded in the Titertek Multiscan apparatus. Tobacco mosaic virus and its IgY type antibody produced simoidal precipitin curves with the layering as well as the mixing techniques. The haemocyanin of the whelk and its IgY antibody yielded a precipitin diagram with the layering technique which gave evidence to three antigens. The three antigens in the haemocyanin was confirmed by two dimentional Laurell-electrophoresis. Semi-purified human colostral dimeric IgA and its IgY antibody produced a precipitin diagram which showed evidence of four of five antigens. With the mixing technique a single precipitin maximum was obtained preceded by a prozone. Rabbit Ig and its IgY antibody gave a precipitin curve with two maxima. The precipitin curve obtained when the reagents were mixed had one peak preceded by a prozone.

Evaluation of equilibrium constants from precipitin curves: Interaction of α-crystallin with an elicited monoclonal antibody

A simple procedure, based on the precipitin curve and the antibody:antigen ratios of the precipitates, is described for evaluation of the intrinsic association constant ( k) governing the interaction between a multivalent antigen and a bivalent antibody. Its application is illustrated with a study of the interaction between α-crystallin and an elicited monoclonal antibody, which is shown to exhibit essentially identical affinities ( k = 9 × 10 4 M −1) for the α m and α c forms of the antigen.

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose, bisected hybrid, and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.

Characterization of high titer antithyroglobulin antibodies

Few autoantibodies directed against thyroglobulin (TgAbs) have been fully characterized in man. The present study was designed to characterize TgAbs from patients with unusually high titers (greater than 1:5(12] using tanned red cell hemagglutination technique (TRC). IgG was isolated from the sera of subjects with Hashimoto's thyroiditis (n = 4), subacute thyroiditis (n = 1) and Graves' disease (n = 1) using DEAE-Sephacel chromatography. Isolated TgAbs were substituted as first antibody in a double antibody thyroglobulin (Tg) RIA and the Ka's were determined by Scatchard analysis. Molecular ratios of antibody to antigen, TgAb: Tg, were calculated from quantitative precipitin curves. The clonality of each antibody was determined using agarose isoelectric focusing and 131I labeled Tg as an autoradiographic probe. All six TgAbs were polyclonal. The Ka's were on the order of 10(9)-10(10). In two sera TgAb:Tg ratios of 20:1 and 8:1 were obtained. These results are significant when compared to previously characterized Tg autoantibodies which have been of low titer, low Ka (10(5], and have been directed towards a restricted portion of the Tg molecule (TgAb: Tg ratios of 2:1 to 6:1). In view of their high affinity constants and recognition of a less restricted portion of the Tg molecule, some of the TgAb's with unusually high titers behave more like Tg heteroantibodies than autoantibodies.

Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus.

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361-369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS (approximately f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H2O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of high mannose and bisected hybrid type glycopeptides.

We have previously reported that concanavalin A (ConA) is precipitated by a high mannose type glycopeptide (Brewer, C. F. (1979) Biochem. Biophys. Res. Commun. 90, 117-122; Bhattacharyya, L., and Brewer, C. F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). In the present study, we have investigated the ability of a series of high mannose and bisected hybrid type glycopeptides to bind and precipitate the lectin. The modes of binding of the glycopeptides were studied by nuclear magnetic relaxation dispersion (NMRD) techniques, and their affinities were determined by hemagglutination inhibition measurements. The stoichiometries of the precipitation reactions were investigated by quantitative precipitation analysis. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that certain high mannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, we have identified two protein binding sites on each glycopeptide: one site on the alpha(1-6) arm of the core beta-mannose residue involving a trimannosyl moiety which binds with high affinity (primary site); and the other site on the alpha(1-3) arm of the core beta-mannose residue involving an alpha-mannose residue(s), which binds with lower affinity (secondary site). These two types of sites bind to ConA by different mechanisms. Certain bisected hybrid type glycopeptides were found to possess only the primary ConA binding sites, but not the secondary sites, and hence were able to bind but not precipitate the lectin. Other related glycopeptides have only the secondary type sites and thus exhibit low affinity and are unable to precipitate the protein. The results are related to the possible structure-function properties of cell-surface glycopeptides.

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides.

In the preceding paper (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glycopeptides are bivalent for concanavalin A (ConA) binding. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbisected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relaxation dispersion techniques, and their affinities were determined by hemagglutination inhibition measurements. We find that certain bisected complex type oligosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were investigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected complex type oligosaccharides are bivalent for lectin binding. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic relaxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 alpha-mannose residues on the alpha(1-6) and alpha(1-3) arms of the core beta-mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose residue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glycopeptides on the surface of cells.

Precipitation of the D-galactose specific lectin from Erythrina indica by a triantennary complex type oligosaccharide

We have previously demonstrated that a high mannose type glycopeptide is bivalent for binding Concanavalin A (Con A) and can precipitate the lectin (Bhattacharyya L. and Brewer, C.F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). The present results show that a triantennary complex type oligosaccharide containing nonreducing terminal galactose residues can precipitate the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina indica (EIL). The interactions of the oligosaccharide with EIL was investigated by quantitative precipitin analysis. The equivalence point of the precipitin curve indicated that the glycopeptide is trivalent for EIL binding. These results indicate that each arm of the oligosaccharide can independently bind separate lectin molecules leading to precipitation of the complex. These findings are discussed in terms of the possible biological structure-function properties of complex type oligosaccharides.

Precipitation of Concanavalin A by a high mannose type glycopeptide

The interactions of a high mannose type glycopeptide with Concanavalin A has been investigated by quantitative precipitation analysis. The equivalence points of the precipitin curves indicate that the glycopeptide is bivalent for lectin binding. These results and others demonstrate that there are two lectin binding sites per molecule of the glycopeptide: one site on the α(1–6) arm of the core β-mannose residue involving a trimannosyl moiety, and another site on the α(1–3) arm of the core β-mannose residue involving an α(1–2) mannobiosyl group. The two sites are unequal in their affinities, and bind by different mechanisms. These results are related to the possible structure-function properties of high mannose type of glycopeptides on the surface of cells.

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512.

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.

Effects of polyethylene glycol and dextrans on immunoprecipitations: A two-cross immunodiffusion study

Precipitating titers and immunochemical titers obtained in a wide range of antigen-to-antibody concentration ratios by the two-cross immunodiffusion technique are compared with the corresponding laser light scatter precipitin curves. The two-cross immunodiffusion technique has also been applied to investigate whether polyethylene glycol of molecular mass 6000 and dextrans of molecular masses from 10,000 to 2,000,000 enhance the immunoprecipitation processes of the system human serum IgG-rabbit immune serum at pH 5.5 and 8.1 at 20°C. It was found that the significant increase of precipitating titers of both precipitating components in the presence of polyethylene glycol is a consequence of a strong decrease of solubility of the primary antigen-antibody complex. The decrease of solubility does not affect the immunochemical titer of the immune serum, indicating stoichiometrical invariancy of the precipitate at the equivalence. The apparent strong decrease of diffusion coefficients of both antigen and antibody in 20- and 40-g/liter polyethylene glycol solution is attributed to increase of viscosity of the solutions and to a partial self-association of protein molecules due to steric exclusion. In 40-g/liter polyethylene glycol solutions at pH 5.5 every fourth molecular entity of antigen and every third molecular entity of antibody are present in the form of a two-molecular self-associate, whereas in 20-g/liter polyethylene glycol solutions only 1% of antigen molecules and 8% of antibody molecules are associated. With the increase of pH to 8.1 the self-association of protein molecules is strongly further enhanced. Dextrans in 20-g/liter solutions, without regard to their relative molecular masses, do not influence precipitating titers and solubility of the antigen-antibody system at equivalence and do not enhance self-association of protein molecules. The strong decrease of diffusion coefficients of immunoglobulin G antigen and antibodies in dextran solutions is solely attributed to the increase of viscosity of the dextran solutions; hence there was no evidence of interaction of dextrans with serum IgG proteins.

Spot immunoprecipitate assay in gel compared with immune reactions in solution and applied to antibody titration

Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable ( r = 0.9979) working range for quantification between 0.1 and 0.83 μg protein in 3 μl samples ( ≥ 0.03 g/l ). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8–9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 μl sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.

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.

Effect of pH on the precipitin reaction of human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides containing 3,4-pyruvylated d-galactose and 4,6-pyruvylated d-galactose

Precipitin reactions of two human monoclonal macroglobulins specific for Klebsiella capsular (K) polysaccharides containing 3,4-pyruvylated d-galactose (K30) and 4,6-pyruvylated d-galactose (K21) have been studied over a pH range of 4.0–7.0. One macroglobulin, IgM WEA, that does not precipitate at pH 7.0 with Klebsiella K21 polysaccharide containing 4, 6-pyruvylated d-galactose, reacts at pH 4.5 and at 4.0 gives a precipitin curve that is identical with those found over the entire pH range with Klebsiella K30 and K33 containing 3, 4-pyruvylated d-galactose as the non-reducing end group. This is in contrast to a second monoclonal protein IgM MAY which reacts identically with K21, K30 and K33 over the entire pH range. As the pH decreases to 4.0, other polysaccharides having 4, 6-pyruvylated d-glucose and d-mannose also precipitate to some extent with IgM WEA and to a considerable extent with IgM MAY. The decreased reactivity of both IgM MAY and IgM WEA with modified polysaccharides in which the 3, 4- and 4, 6-pyruvylated d-galactose was esterified or reduced provided additional information on the role of the carboxyl group in the specificities of their combining sites.