Murine IgA Research Articles

The isolation of an allergen from extracts of Dermatophagoides pteronyssinus using a murine IgA myeloma with a specificity for phosphorylcholine.

The presence of a high molecular weight glycoprotein component bearing the hapten phosphorylcholine (Pc) has been demonstrated in some but not all extracts of house dust mite using a phosphorylcholine specific IgA myeloma protein (S107). The Pc bearing component was isolated from house dust mite extracts by precipitation at equivalence using whole myeloma serum and the isolated fraction was found to be allergenic as judged by a direct RAST assay using a highly house dust mite allergic human serum pool. RAST inhibition studies using free hapten and direct RAST studies using pneumococcal polysaccharide C-substance indicated that the Pe moiety did not contribute significantly to the immunodominant portion of the isolated allergen. Further studies indicated that the S107 isolated material was immunochemically different from the tridacnin reactive material and the con A reactive material previously isolated fom extracts of house dust mite.

Physicochemical and immunochemical characterization of the high molecular weight allergens from dermatophagoides pteronysisinus with particular reference to the tridacnin, Con-A and S107 reactive components.

High molecular weight allergens which react with the S107 murine IgA myeloma and with the lectins Concanavalin A (Con-A) and tridacnin have been identified and characterized in extracts of the mite Dermatophagoides pteronyssinus. Mite extract from two different manufacturers were fractionated by Con-A affinity chromatography. The major antigen in both the Con-A bound and unbound fractions, which was antigenically indistinguishable, reacted with tridacnin and the S107 myeloma. This high molecular weight, multi-reactive antigen corresponded to allergen Dpt 4, suggesting that Dpt 4 exists as isoallergens, each differing in the degree of glycosylation or in the type of carbohydrate moiety comprising the glycan part of the allergen. Mite media extracts also reacted with Con-A, tridacnin and the S107 myeloma but were not allergenic per se.

Effect of interchain disulfide bond on hapten binding properties of light chain dimer of protein 315.

The hapten binding characteristics of the covalent light chain dimer, derived from the murine IgA secreted by plasmacytoma MOPC-315, to two nitroaromatic compounds, epsilon-N-(2,4-dinitrophenyl)-L-lysine and 4-(alpha-N-alanine)-m-nitrobenz-2-oxa-1,3-diazole, were investigated by differential spectroscopic titrations. The binding curves for both haptens were found to display sigmoidity similar to that reported earlier for the reduced and alkylated dimer held together by noncovalent bonds only. However, the presence of the interchain disulfide bond in the covalent dimer was found to cause marked changes in its binding properties. The data, like those obtained for the noncovalent dimer, fit the allosteric model of Monod, Wyman, and Changeux in which binding of the first hapten to the dimer causes a conversion of both sites of the protein molecule from a lower to a higher affinity conformation. However, the binding parameters show that both the affinity and the positive cooperativity in the interaction between haptens and the covalent dimer are significantly enhanced. The differences in the parameters of the binding and of the allosteric transition caused by the presence of the interchain disulfide bond demonstrate the existence of longitudinal interactions in immunoglobulin derivatives. These properties of the light chain dimer make it a potential model for the receptors present on thymus-derived lymphocytes.

M467: A Murine IgA Myeloma Protein that Binds A Bacterial Protein

Abstract We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner than could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.

Contributions by ionic and steric features of ligands to their binding with phosphorylcholine-specific immunoglobulin IgA H-8 as determined by fluorescence spectroscopy.

The murine myeloma IgA H-8 Fab' fragment which exhibits a binding specificity for phosphorylcholine was assayed for its ability to bind with a number of charged ligands. Monitoring of the ligand-induced changes of protein fluorescence provided a fast and accurate method of determining the equilibrium binding constants. The binding data along with fluorescence spectral properties of the protein permitted an assessment of the relative importance of some binding parameters as well as an evaluation of certain ionic and steric contributions made by ligands exhibiting significant binding affinity for the antibody fragment. Among the conclusions reached is that the dielectric of the binding site microenvironment is important in determining the strength of binding and that hydrophobic groups surrounding a quaternary cationic ligand are important in creating an appropriate binding site of low dielectric value.

Idiotypic cross-reactivity of human and murine phosphorylcholine-binding immunoglobulins.

The idiotypic cross-reactivity of mouse and human monoclonal immunoglobulins with binding activity for phosphorylcholine (PC) was investigated, using an idiotypic antibody elicited against the PC-binding human IgMFR. The isolated FR heavy chain proved to be a better inhibitor for the reaction of IgMFR with anti-FR than the FR light chain, but the intact protein was necessary for full idiotypic expression. PC was an inhibitor only at concentrations greater than 10(-3) M indicating that the idiotypic antibody was not combining site-directed. Among the murine PC-binding IgA myeloma proteins, MOPC 167 was found to be the best inhibitor, but its inhibitory capacity was about 4 orders of magnitude lower than that of the homologous IgMFR. McPC 603 was an even weaker inhibitor, while TEPC 15 effected no better inhibition than human monoclonal immunoglobulins without PC-binding activity. MOPC 167 has a most similar binding specificity to IgMFR as indicated by the high affinity for choline of these two proteins. TEPC 15 and McPC 603, on the other hand, exhibit a much lower affinity for choline. In addition to their similarity in specificity, proteins FR and MOPC 167 show important structural similarities within parts of their heavy and light chain variable domains. The data provide some evidence for the existence of idiotypic cross-reactivity between the two species man and mouse.

T-cell regulation of murine IgA synthesis.

In studies reported here, the polyclonal activator lipopolysaccharide was used to stimulate the synthesis and secretion of IgM, IgA, and IgG in cultures of mouse lymphoid cells. The total immunoglobulin of each class which resulted was measured by specific double-antibody radioimmunoassays. The effect of Con A-activated T cells from various tissues on such immunoglobulin synthesis was then assessed. Variations in regulatory T-cell activity among the various lymphoid tissues for IgA but not for IgM or IgG was observed. In particular, Peyer's patches T cells were found to contain a high level of IgA T-cell helper activity compared to that of spleen or peripheral lymph node. The independent variation of T-cell regulatory activity for IgA as compared to that for IgM and IgG among the different tissues is most consistent with there being a separate subset of T cells specifically regulating IgA. The significance of these findings for the understanding of the secretory immune system is discussed.

Age-related changes in the T-cell regulation of murine IgA biosynthesis

Age-related changes in the T-cell regulation of murine IgA biosynthesis

The binding of analogs of phosphorylcholine by the murine myeloma proteins McPC 603, MOPC 167 and S107

The binding of three sets of compounds, analogs of phosphorylcholine, to the murine IgA myeloma proteins McPC 603, MOPC 167 and S107 has been studied by the method of hapten inhibition of precipitation. The three sites were of the general formulae (CH 3) 3N +(CH 2( n COOH, (CH 3) 3N +(CH 2) n CH 2OH and (CH 3) 3N +(CH 2) n Ph. All three proteins showed similar behavior with the carboxylate compounds, maximum activity occurring at n=3, the closest analog of phosphorylcholine. In contrast, the phenyl and alcohol series behaved in a different fashion with each protein. In the case of S107 the phenyl compounds were almost as effective as the carboxylate ones. Only MOPC 267 showed strong binding of the hydroxyl compounds. Thus, despite their structural similarities and common reactivity to the phosphorylcholine determinant the three proteins show significantly different reactivities towards related compounds.

The Formation of Active Hybrid Immunoglobulins from the Heavy and Light Chains of β(1, 6) D-Galactan Binding Murine Myeloma IgA's S10 and J539

Abstract Murine myeloma immunoglobulin (IgA, κ) J539, which shows enhanced tryptophanyl fluorescence on ligand binding, and S10, which shows reverse-sign changes in tryptophanyl fluorescence on ligand binding (RLIF, see below), have been reduced, alkylated, and dissociated into their light (L) and heavy (H) chains. Two hybrid recombinants, H10L539 and H539L10, have been prepared and the 7S material has been isolated by chromatography. The binding behavior of these recombinants was studied with a number of ligands. Both recombinants showed activity with β(1→6) linked galactose ligands comparable to the native immunoglobulins. The ligand-induced fluorescence changes of the recombinants paralleled those of the heavy chain donor. For the recombinant H10L539, two different galactose-ligands caused fluorescence changes in opposite directions. It was quantitatively shown that binding of these ligands, nevertheless, took place in the same combining region. The idiotype of each recombinant resembled that of the heavy chain donor.

Regulation of Immune Response by Autogenous Antibody against Receptor

BALB/c mice repeatedly immunized with Pneumococcus R36A vaccine produce antibodies to phosphorylcholine having the TEPC-15 myeloma idiotype (murine IgA myeloma protein that binds phosphorylcholine). The plaque-forming cell response to phosphorylcholine shows a decrease with repeated immunizations. In contrast, spleen cells from multiply immunized mice responded better in vitro than spleen cells from nonimmunized mice. The serum of animals immunized four or five times agglutinates TEPC-15-coated sheep erythrocytes. Inhibition of hemagglutination shows that the agglutinating activity is directed against the TEPC-15 idiotype. Sera from these mice, when added to cultures of normal spleen cells, specifically suppress the response to phosphorylcholine. The suppressive activity in the serum can be removed by solid absorption with TEPC-15. Evidently, repeated immunization with antigen induces two kinds of antibody responses: one directed against antigen and the other directed against the antibody to the antigen. It is proposed that this "auto" antibody against receptor is involved in the regulation of the immune response.

A method for demonstrating mono- and oligomeric units of IgA myeloma proteins in the mouse

A antigen-antibody crossed electrophoresis technique, described by Laurell, was modified, the first step-electrophoresis being performed in polyacrylamide gel. This allowed separation of molecules not only by their charge but also by their difference in molecular size. Various murine IgA myeloma proteins, when analyzed in this way with a monospecific rabbit antiserum against mouse IgA, showed different precipitin profiles, each one typical for a particular myeloma protein. A proportional distribution of the various molecular size units of IgA can be obtained directly from a serum sample without any prior purification by this procedure.