γM Antibodies Research Articles

Antistreptococcal group a antibodies: Production after in vitro activation and hybridization of mouse spleen cells

Streptococcal group A polysaccharide-specific antibodies were raised by the method of somatic cell hybridization. Spleen cells of experimentally unprimed BALB/c and C57BL/6 mice were activated in vitro by the streptococcal vaccine and fused with the Sp2/0-Ag14 line at times 0, 35, 70, and 105 h thereafter. Hybridomas were obtained at all times independent of the addition of thymocyte-conditioned medium. Occurrence of specific hybridomas for the T cell-dependent A-CHO, however, required activation for greater than 35 h. Low responder C57BL/6 splenocytes fused at considerably higher fusion efficiency to yield specific hybridomas than high responder spleen cells 105 h after activation by antigen. The isotypes of A-CHO-specific antibodies comprised predominantly μ and k polypeptides; however, γ3, α, and λ polypeptide chains were also identified. All specific antibodies were agglutinating the group A streptococcal cells; this agglutination was fully inhibited by the addition of 1 % N-acetyl-D-glucosamine, the immune determinant sugar of the A-CHO. Three hybridomas obtained by fusion of BALB/c splenocytes 105 h after activation were cloned and grown as tumours in the peritoneal cavity of BALB/c mice. The monoclonal antibodies in the ascites did not precipitate the A-CHO but continued to agglutinate group A streptococcal cells in a hapten inhibitable fashion with different specificity profiles. Antibody from clone 21S36.1 was coprecipitable upon addition of A-CHO with a γG3 monoclonal hybridoma-derived antibody in a ratio of sol1 7 while the other two monoclonal γM antibodies and the S117 myeloma protein were not. This result suggests that antibody 21S36.1 recognizes one chain terminal determinant of the A-CHO.

γM Alloantibody Elicited by First-Set Renal Allografts: in Vivo and in Vitro Studies

Abstract γM alloantibody (allo γM) was produced in Lewis (Le) rats after a 5- to 7-day residence of Brown Norway (BN) renal grafts. The isolated, purified and isotopically labeled allo γM fixed to spleen, lung, liver and erythrocytes after intravenous injection into BN target rats, but did not fix to kidney unless the renal vein was clamped for 3 min. Allo γM fixed promptly in BN kidneys grafted into Le recipients. In such kidneys allo γM specifically bound to endothelia of glomerular and intertubular capillaries and of medium-sized arteries. It did not bind in vivo to renal tubular cells but did so in vitro. At least two γM antibodies were detected. One reacted with kidney, spleen cells and erythrocytes, the second only with spleen cells. Intravenous infusion of allo γM into target BN rats produced extensive pulmonary hemorrhages but the production of these lesions lacked specificity. Allo γM elicited by renal graft was strain but not organ specific, i.e., it reacted with transplantation antigens shared by kidney and by other organs and tissues of the immunizing strain. Its role in the rejection process could not be established.

The influence of polyvalency on the binding properties of antibodies

We present a general formalism for predicting the relationship between the binding affinities in several kinds of antigen-antibody interactions. The reference binding constant is that of monovalent antibody for monovalent antigen. The equations then allow one to predict the ratio of this constant to that for attaching divalent antibody to divalent antigen. We further develop the theory to consider the binding of multivalent antibodies to a multideterminant antigen particle, in the restricted case of small fractional occupancy of the determinant sites by antibodies. There is good agreement between the calculated results and certain data from the literature.In situations where multisite adherence to a single particle and cross-linking of discrete particles are both possible, the former is predicted to predominate strongly. That the opposite appears sometimes to be experimentally the case means that special features favoring agglutination reactions must be present in these instances.Finally, those factors that potentially make polyvalent γM antibodies more effective agglutinators than bivalent γG antibodies are delineated briefly.

Passive protection of mice against intraperitoneally injected Vibrio cholerae by G and M antibody.

Fractions of rabbit anti-Vibrio cholerae serum containing gammaG antibodies were compared with fractions containing gammaM antibodies for their ability to protect mice against lethal infection resulting from the intraperitoneal injection of organisms suspended in mucin. About twice as much gammaG as gammaM (estimated by quantitative precipitation) was required to protect against approximately 1,000 50% lethal doses when the antibody was given intraperitoneally 4 hr before challenge. When protective serum fractions were given subcutaneously, however, the amount of gammaM required to protect was increased about 40-fold, whereas gammaG was about equally effective subcutaneously and intraperitoneally.

Serological Properties of γG and γM Antibodies to the Somatic Antigen of Vibrio cholerae During the Course of Immunization of Rabbits

Direct- and passive-agglutinating, complement-fixing, and bactericidal properties of gammaG and gammaM antibodies produced in rabbits inoculated with live Vibrio cholerae were determined at intervals over a period of 345 days. Although gammaM antibody titers increased more rapidly than gammaG during the initial stages of antibody production, the titers of gammaG and gammaM declined proportionally during a 3-month rest period and increased proportionally after a booster injection. The relative titers of gammaM as determined in the four serological procedures remained fairly constant throughout the period of observation. In contrast, early gammaG was less effective than late gammaG in vibriocidal, complement-fixing, and passive-hemagglutinating activity. At no stage of immunization was the agglutinating ability of gammaG affected by 2-mercaptoethanol, but its complement-dependent activity was markedly reduced, more so in early serum than in late. The heat lability of early gammaG approached that of gammaM, but gammaG became more resistant to heat in later stages of immunization.

Nature of the fluid in serous otitis media.

Middle ear fluids from 123 patients with serous otitis media were analysed by comparing elements of the fluid with the patients' sera. Results of paper electrophoresis, immunoelectrophoresis, and disc electrophoresis showed no qualitative difference between serum and middle ear fluid. Most fluids further contained antibodies to rabbit erythrocytes, and the agglutination titers were remarkably similar to those in the corresponding sera. Even γM antibodies were present in serum concentrations. An antiserum produced by immunization of a rabbit with a pool of ear fluid behaved as an antiserum to whole human serum. No additional lines appeared in tests with ear fluid. The fluid is probably a transudate of plasma, produced as a result of a negative pressure in the middle ear due to a dysfunction of the eustachian tube.

Immunoglobulins and Antibody-Production in Amphibians

SYNOPSIS. Recent evidence suggests Ihat amphibians represent a key transition stage in the evolution of immunological competence. In particular, anuran amphibians exhibit a significant advance relative to more primitive vertebrates in their capacity to synthesize two distinct classes of immunoglobulins in response to antigenic stimulation.These two classes of antibody molecule resemble the γM and γG antibodies of man in size and polypeptide chain structure. Furthermore, cellular aspects of antibodyproduction in anura resemble those of mammals in the method of retention of antigen and the range of antibody-forming cells (lymphocytes to plasma cells). In addition to these parameters of antibody-formation, a state of specific nonresponsiveness (high zone tolerance) can be induced in adult anura by pretreatment with large doses of protein antigen. Studies of larval anurans establish that these forms become immunologically competent early in development, but possess only γM immunoglobulins. These larval stages provide feasible model systems for the study of the activation of genes which specify immunoglobulins. Antibody-formation in urodeles is discussed in relation to the emergence of immunological competence in amphibians.

Regulation of Homocytotropic Antibody Formation in the Rat

Abstract Adult rats about 3 months old were splenectomized or thymectomized and then immunized with dinitrophenylated Ascaris suum extracts (DNP-As) plus Bordetella pertussis 3 to 10 days after the operation. Homocytotropic antibody (HTA) formation against DNP-As was examined by passive cutaneous anaphylaxis (PCA) over a period of 45 days. HTA formation was found to be greatly enhanced and prolonged by splenectomy and thymectomy. Virtually no termination of HTA formation occurred. Combined extirpation of the spleen and thymus in adult rats had the same enhancing effect. Passive hemagglutination tests with sera treated or untreated with 2-mercaptoethanol showed that the production of γG and γM antibodies was unchanged. In contrast to adult thymectomy, neonatal thymectomy caused a loss, or at least a marked reduction, of the ability to produce HTA. The results suggest that adult splenectomy and thymectomy cause regulatory disturbances of HTA formation in the rat.

Foot-and-mouth disease virus in cattle and pigs: use of polyethylene glycol or dextran for purifying 19S gamma-M immunoglobulin from sera.

Polyethylene glycol (PEG) and dextran (DEX) were shown to be good selective precipitants of high molecular weight proteins and foot-and-mouth disease antibodies in bovine and porcine sera. Although PEG was less selective than DEX for 19S γM antibodies, the DEX precipitated at most, 60 to 90% of this protein. After two DEX precipitations and two density gradient purification steps, analysis of the product by immunoelectrophoresis, “disc” electrophoresis and ultracentrifugation indicated only a single component.

The proportion and structure of cells forming antibody, γG and γM immunoglobulins, and γG and γM antibodies

A double-label method was developed that utilized horseradish peroxidase (POX) as antigen and its reaction in substrate to detect antibody in cells and simultaneously used 125I-labeled monospecific antisera to detect Ig class. Three categories of cells may be detected, antibody forming elements, immunoglobulin producing cells, and cells synthesizing antibody of a known immunoglobulin class. In the light microscope the percentage of γG antibody producing cells was five times greater than the percentage of γM antibody producing cells. In the electron microscope about 1 6 of the γG antibody forming cells were lymphocytes and 5 6 were plasma cells. By contrast, of the γM antibody forming cells, 9 10 were lymphocytes and 1 10 plasma cells. By calculation roughly 70% of all antibody forming cells were synthesizing γG antibody and 12% were synthesizing γM antibody. Approximately 35% of all γG producing cells were making γG antibody, and 23% of all γM producing cells were making γM antibody. The double label method was found to be sensitive and specific, but inherent difficulties of the enzymatic reaction and its activation by antibody impaired its accuracy.

Immunoglobulins of the Hamster

Abstract Hamster γ1, γ2, γA and γM globulins have been identified by immunoelectrophoresis developed with antisera from rabbits injected with antigen-hamster antibody complexes. Binding of labeled antigen to hamster γ1 and γ2 antibodies was observed in sera obtained 1 to 8 weeks following injection of hapten-protein conjugates emulsified in complete Freund's adjuvant. Binding to γM antibodies was limited to weeks 1 and 2 and binding to γA antibodies was demonstrable only at week 3. Indirect evidence for the existence of a reagin-like homocytotropic antibody was presented.

The Search for Antibodies with Molecular Uniformity

Publisher Summary This chapter discusses the physical, chemical, and biological aspects of γM antibodies in depth. The chapter describes the physical characteristics, chemical composition, and subunit structure of the typical circular γM pentomers and the relationship of these molecules to low molecular weight γM-like proteins. The characteristics of the interaction of γM antibodies with antigens—for example, the nature and number of antigen combining sites—and the interaction of γM antibodies with the complement system are compared to the corresponding properties of γG antibodies. The biosynthesis and metabolism of γM antibodies and their peculiar role in the immune response are also discussed in the chapter. The chapter presents a brief summary related to the hallmarks of immunoglobulin heterogeneity and the criteria that must be used to judge the restricted heterogeneity or molecular uniformity of an antibody preparation. The chapter discusses human antibodies to certain selected antigens that appear to possess less heterogeneity than normal γ-globulin.

Complement-Fixing Activity of Antibodies Associated with Different Classes of Immunoglobulins

Abstract Antibodies belonging to different immunoglobulin classes and subclasses exhibit characteristic immunochemical properties. Among these properties, which serve to differentiate between these immunoglobulin classes, is the ability to fix C′. Using a C′1a-fixing system comprised of human anti-γA isoantibodies and type A red cells, both γG and γM antibodies have been shown to fix C′1a, whereas γA antibody in both monomeric and polymeric forms lacks this ability. It was also found that monospecific aggregates of γG and γM fixed C′, whereas aggregated γA failed to do so. Among the subclasses of γG globulins, evidence has been obtained that γG1, γG2, and γG3 globulins fix C′ and C′1a on aggregation but γG4 globulin does not. Although antibodies of both the γG and γM type have been shown to fix C′, whether the same mechanisms of fixation exist for both classes has not yet been resolved.

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.

Studies on Antigen-binding Activity of Macroglobulin Antibody Subunits and Their Enzymatic Fragments

Abstract To gain insight into reasons for the presence of only five combining sites in γM antibodies, studies were undertaken to determine the number of antigen-binding sites on γM subunits and on Fabµ fragments of a γM rheumatoid factor anti-γ-globulin from a patient with mixed cryoglobulins. The rheumatoid factor contained both κ and λ light chains and reacted with human and rabbit γG. Combining ratios were determined by quantitatively determining the amount of complex formation by analytical ultracentrifugation. Each monomeric subunit had only one effective antigen-binding site, and there were no inactive subunits. All of the 3.7 S Fabµ fragments, prepared by trypsin digestion of the subunits, were active and each had at least one effective binding site per fragment. These results indicate that there are 10 potential combining sites in the intact molecule although only five are available for antigen binding. Studies were performed also on the nature of the cross-reaction of this antibody with rabbit γG. These experiments showed that the amount of complex formed with rheumatoid factor subunits and their antigen was the same whether the antigen consisted of rabbit γG alone, human γG alone, or a mixture of the two. Since there is no evidence for antibody subunits which combine with only one species of γG, the binding sites appear to resemble those in other cross-reacting systems in which each combining site may be occupied by either antigen. In contrast to the excellent binding activity of γM antibody subunits for rabbit γG, Fabµ fragments showed little or no binding activity for rabbit γG.

Serological characterization of gamma-M(19 S) and gamma-G(7 S) rabbit antibiodies in response to Entamoeba histolytica antigens.

Entamoeba histolytica (DKB strain) antigen mixed with an equal volume of complete Freund's adjuvant was injected at multiple sites into three rabbits. Primary response sera were drawn 1, 2, and 3 weeks after the first injection. Two injections of antigen were given in the fourth week, at 3-day intervals. The secondary response sera were drawn 5, 6, and 12 weeks after the beginning of immunization. A steady hemagglutination (HA) titer of 1:128 was obtained with all the three primary response sera, while the secondary response sera showed HA titers of 1:2000 to 1:8000. The 0.1 M 2-mercaptoethanol treatment of these sera reduced, but did not completely obliterate, their HA activity. The HA activity was detected in both the pooled γM and γG classes of immunoglobulins of the primary and secondary response sera; the immunoprecipitating activity was confined to the γG class. 2-Mercaptoethanol treatment completely destroyed the antibody activity of γM fractions without affecting that of the γG fraction. Repeated immunoelectrophoresis runs of the whole sera and pooled γM and γG fractions showed variable numbers of precipitin bands at the γG position but none at the γM and γA positions.

The Heterogeneity of Combining Sites of Anti-Benzylpenicilloyl Antibodies Obtained from Individual Rabbits: Changes in Combining Sites of γG and γM Antibodies During the Immune Response

Summary Nine rabbits were immunized with benzylpenicilloyl-human γG conjugate (BPO-HGG). Radioiodinated γG and γM were prepared from the anti-BPO-HGG sera of individual rabbits collected 10 days and 10 weeks after the first immunization. These antibodies were charged on a specific immunoadsorbent and eluted step by step with three haptens corresponding to constituents of the BPO molecule. Three antibody fractions were separated. The antibody fractions were adapted to 6-acetylaminopenicilloic acid (nuclear portion), phenylacetylglycine (acyl side chain portion) and benzylpenicilloyl-ε-aminocaproate (whole BPO molecule), respectively. The binding properties of these antibodies for BPO antigens were examined by equilibrium dialysis. The binding affinity of antibodies directed to the whole BPO molecule was stronger than that of antibodies directed to smaller parts of the BPO molecule. By PCA reactions the responses of antibodies complementary to the smaller parts of the BPO molecule were shown to be weaker than those of antibodies complementary to the whole BPO molecule. The combining sites of γG and γM antibodies changed with time after the immune response. At a late stage in the immune response a significant increase was observed in antibodies adapted to the whole BPO molecule. These changes of combining sites were more characteristic in γG than in γM. However, at an early stage of immune response no significant differences were observed between γG and γM. It is concluded that changes in the binding affinity and heterogeneity of antibodies during the immune response are caused by changes in the combining sites of the antibodies.

A “monoclonal” type, low molecular weight protein related to γM-macroglobulins

A unique “monoclonal” gamma globulin was detected in the serum of a patient with malignant disease of the plasma cell-lymphatic system. This protein had antigenic determinants common to 19S γM-globulin (macroglobulin) but there were marked physicochemical differences. The clinical and pathologic findings in this patient differed from those usually found in patients with multiple myeloma or Waldenström's macroglobulinemia. Evidence obtained from density gradient and gel filtration studies of whole serum indicated that the γM antigenic determinants were located primarily on molecules smaller in size than 19S-globulin. The anomalous gamma globulin, isolated from serum, had a sedimentation coefficient ( s 20, w) of 6.5S, consisted of μ-type heavy chains and λ-type light chains and differed slightly from the monomeric subunits of a reduced alkylated 19S γM-globulin. This protein may be related to low molecular weight γM antibodies previously described in a normal serum and in several disease states.

Examination of virus-gamma-M antibody complexes by electron microscopy. I. Configuration of gamma-M antibodies and mode of attachment to antigen.

Abstract Poliovirus-γM antibody complexes were examined by the negative staining technique in the electron microscope. The virus was purified by equilibrium centrifugation, and rabbit γM antibodies by zone centrifugation or gel filtration. The specificity of the poliovirus-γM antibody reaction was established by the use of heterologous antigen and γM fractions from normal sera. N- and H-virus particles aggregated separately in the presence of γM antibodies against both these antigens. The size of specific poliovirus aggregates and the density of the cross-linking network of γM antibodies increased as the relative γM antibody concentration went up. With intermediate or high relative antibody concentrations single virions or virions in the periphery of aggregates were often surrounded by an aura of loops. The maximal length of the turn of the loops was 220–250 A and their width 35–40 A, suggesting that they represented γG-like 7 S subunits of the γM molecule. The maximal number of subunits contained in one γM molecule appeared to be 5 or 6. The distribution of the lengths of γM antibodies bridging virions had a mode and median of about 330 A and maximal value of 360–370 A.

Comparison of the Opsonic Activity of γ-G- and γ-M-Anti-Proteus Globulins

Summary The opsonic activities of γG and γM antibodies to Proteus mirabilis were studied in a leukocyte-bacterial phagocytosis system. γG antibody was superior to γM antibody in enhancing phagocytosis. This superiority was a reflection of the influence of antibody on uptake of bacteria rather than an effect on intracellular fate of the organisms. This difference in the function of antibodies in the two classes of immunoglobulins may explain certain clinical observations which indicate γG antibodies are more protective against infection than γM antibodies.