γG Antibodies Research Articles

Studies on Antigen-Antibody Complexes

Abstract The quantitative tissue uptake of soluble immune complexes made with rabbit γG antibodies, was studied in normal and complement-depleted rabbits. The immune complexes that were cleared quickly from the circulation were primarily localized in the liver; less than 1% of the injected complexes was found within the lungs, kidneys or spleen. The tissue distribution of complexes made with intact antibodies was unchanged in complement-depleted rabbits. Soluble complexes made with reduced and alkylated antibodies fixed complement ineffectively. There was no hepatic uptake of HSA-anti-HSA and decreased uptake of γG-anti-γG and γM-anti-γM complexes, made with reduced and alkylated antibodies. In the latter two instances residual hepatic uptake was due to human γG or γM antigens in the complexes. It was concluded that the in vivo interaction of the fixed tissue macrophages and γG class of antibodies in soluble immune complexes was not mediated by circulating complement components. However, studies with a Waldenström's macroglobulin suggested that the in vivo interaction of macrophages and γM depended upon the third component of complement. It appeared that the complexes had to contain more than two γG antibody molecules for rapid uptake by the RES to occur. The RES uptake of complexes did not depend on their physical size. The rapidly removed complexes fixed complement strongly in vitro, although their in vivo uptake by the RES did not depend on an intact complement system in the circulation.

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 Homocytotropic antibody (HTA) formation, as measured by the passive cutaneous anaphylaxis (PCA) test, was enhanced, although after a delay, and prolonged by 400 R whole body x-irradiation given shortly before through 8 days after immunization of rats with dinitrophenylated Ascaris extracts (DNP-As) plus killed Bordetella pertussis. Delayed enhancement and prolonged production were most striking when x-irradiation was given 1 day before to 2 days after initial immunization. An adjuvant effect of 400 R was produced in rats immunized with DNP-As alone omitting B. pertussis. As evidenced by passive hemagglutinin titers after 2-ME treatment, the production of γG antibody was greatly suppressed by 400 R x-irradiation. Two possible pathways to account for the irradiation effects on HTA formation were postulated, i.e., the enhancement of γM antibody and the suppression of 7S γG antibody resulted in a defective humoral feed-back regulation, and the depletion of certain populations of lymphoid cells, probably of thymic origin, caused cellular regulatory disturbances.

Studies of Opsonic Activity in Univalent 6.5S Rabbit Antibacterial Antibody

Abstract Further definition of the structural γG requirements for effective opsonic function was sought using rabbit univalent antibacterial hybrid γG antibody to several strains of Escherichia coli. Hybrids of immune and normal γG made at ratios of 1:4 and 1:10 showed complete loss of opsonic function while retaining their capacity to combine with bacteria as shown by Coombs'-type facilitation of bacterial agglutination reactions. Self-recombinants of immune γG retained some degree of opsonic activity. When hyperimmune rabbit antibody was reduced and alkylated alone with 0.02 M concentrations of mercaptoethanol, there was considerable loss of opsonic activity. These experiments further emphasize the structural integrity necessary in the Fc portion of γG for complete expression of opsonic function.

A Quantitative Assay for Cell-Bound Antibody Using a Radiolabeled Coombs-Type Antiserum to a γG Myeloma Protein

Abstract Harder and McKhann have introduced an isotopic antiglobulin technique for measuring antibodies to cell-surface antigens (1). Sparks et al. have refined the technique to detect alloantibodies formed after first-set rejection of skin grafts (2). Further, Ting and Herberman have used it to study the humoral antibody response to a tumor-specific cell surface antigen (3). Two additional refinements are reported in this paper. The first is the preparation of a Coombs antiserum by immunizing rabbits with a highly purified mouse myeloma protein of γG type, thereby producing highly specific antibodies which cross-react with normal mouse γG globulin (4). The second is the purification of these antibodies by use of an insoluble antigen complex. The resulting purified Coombs antibody is radiolabeled and binds avidly to cell surfaces coated with mouse γG antibody. A very desirable end result, namely direct proportionality between the quantity of mouse antibody incubated with the cells and the amount of radiolabeled Coombs serum bound to the cell surface, has been obtained.

Effect of tonsillectomy and adenoidectomy on nasopharyngeal antibody response to poliovirus.

Poliovirus antibody levels in nasopharynx and serum were studied before and after tonsillectomy and adenoidectomy in 40 children previously immunized with live poliovaccine. Before operation, γA poliovirus antibody was present in the nasopharynx of all children, but no γG or γM antibody was detectable. Shortly after operation low levels of γG antibody appeared in the nasopharynx of 50 per cent of subjects. The response was short lived, and disappeared in two months. Pre-existing γA antibody in the nasopharynx declined sharply after operation in all children studied. Mean antibody titers decreased three to four times, and individual titers in several children four to eight times. Four children who had had antibody levels in the nasopharynx failed to demonstrate any such antibody activity after operation. Reduced or absent antibody activity persisted for as long as seven months. In nine children with intact tonsils the antibody response in the nasopharynx after immunization with live poliovaccine was two to four times higher than in eight children whose tonsils had previously been removed.

Receptors for human gamma G globulin on human neutrophils.

Cell surface receptors for human gammaG antibodies directed against bacterial antigens were demonstrated on human neutrophils using an in vitro bacteriocidal-phagocytic assay. These results were confirmed by adherence of sensitized erythrocytes to monolayers of neutrophils or monocytes. Erythrocytes sensitized indirectly with antibacterial gammaG antibodies after passive sensitization with bacterial antigens adhered to both neutrophils and monocytes. Erythrocytes sensitized directly with conventional anti-D gammaG antibodies adhered only to monocytes, while those sensitized with the hyperimmune anti-CD gammaG antibody Ripley adhered to both monocytes and neutrophils. Adherence of anti-Rh or antibacterial gammaG antibodies to monocytes and neutrophils could be inhibited by whole gammaG, myeloma globulins of the gamma(1) or gamma(3) subclasses, or Fc fragments, but not by Fab fragment. These results indicate that receptors for the Fc portion of human gammaG antibodies exist on both neutrophils and monocytes, and that gammaG antibodies differ in their ability to attach to these two cell types. Differences in the behavior of the gammaG antibodies studied may be related to differences in the density of antibodies on the erythrocyte surface and receptors on the phagocytic cells.

Distribution of Echovirus Antibody in Serum, Nasopharynx, Rectum, and Spinal Fluid After Natural Infection with Echovirus Type 6

By employing the techniques of radioimmunodiffusion and autoradiography and by using (32)P-labeled echovirus 6 as the antigen, the distribution of echovirus 6 antibody in serum, nasopharynx, rectum, and spinal fluid was studied in a group of children with naturally acquired echovirus type 6 infection, with or without meningeal involvement. The response in serum was characterized by sequential appearance of gammaM, gammaG, and rarely of gammaA antibody. The secretory response in the nasopharynx and rectum was limited to secretory gammaA immunoglobulin. The response in spinal fluid was limited to gammaG immunoglobulin. gammaG echovirus antibody appeared in the spinal fluid with or without demonstrable replication of virus in the spinal fluid. It is suggested that, after short-term acute infection with enteroviruses, the appearance of gammaG antibody in spinal fluid may largely be a reflection of increased transport of serum antibody, particularly of gammaG immunoglobulin class.

γG Erythrocyte Autoantibodies: Comparison of in Vivo Complement Coating and in Vitro “Rh” Specificity

Abstract Panreacting γG antibodies were eluted from the red cells of 35 patients with positive direct Coombs' tests. Twelve patients had γG coated cells and 23 had γG + C coated cells. Six of 12 γG and 6 of 23 γG + C eluates showed “Rh” specificity by their failure to react with the Rhnull blood. After absorption with Rhnull cells, an additional γG eluate and seven γG + C eluates showed “Rh” specificity. In these eluates specificity was obscured by the presence of additional antibodies of other specificities and was not apparent until antibodies lacking “Rh” specificity were removed by absorption with Rhnull cells. These findings suggest that the γG antibodies from γG and γG + C coated cells are similar and provide additional support for the concept that surface configuration of red cells as well as the characteristics of antibodies involved may be important in determining whether γG autoantibodies coat erythrocytes with C.

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.

Passive Anaphylaxis in Mice with γG Antibodies

Abstract Mast cells collected from certain mouse strains (e.g., A, C57BL) were shown to be significantly less susceptible than those collected from other strains (e.g., C3H, DBA/2) to in vitro passive anaphylactic sensitization by purified anti-hapten antibodies. Antibodies of isologous, homologous and heterologous (rat) origin were used with identical results. No relation between the presence or absence of a complement component (MuB1 Ag, C5) and susceptibility to sensitization was found, and no clear pattern of inheritance of this characteristic could be demonstrated.

Appendix and γM-Antibody Formation

Abstract Intravenous injection of 200 µg of alum-precipitated bovine γ globulin into irradiated (900 r) rabbits, whose appendix and bone marrow had been shielded during irradiation, gave rise to an early hemagglutinin response predominantly of the γM immunoglobulin type. This effect was exaggerated by direct injection of antigen into the appendiceal artery or into the lumen of the appendix. Infection by either route of antigen freed of aggregated material by centrifugation gave little immune response and appeared to inhibit formation of γM antibody on subsequent challenge with alum-precipitated antigen. Late formation of γG antibody and the development of memory (ability to give an early γG response after secondary challenge) were not affected by irradiation of the appendix, but were inhibited in animals whose bone marrow was not shielded during irradiation.

Chemical Modification of Human and Rabbit Antistaphylococcal γG Antibodies: Effect of Acetylation, Carbamylation and Amidination on Opsonic Activity

Abstract Opsonically active γG-globulins isolated from five patients with staphylococcal subacute bacterial endocarditis and two hyperimmunized rabbits were subjected to acetylation, carbamylation and amidination to determine if substitution of chemical groups in the γG molecule would result in loss of opsonic activity. The degree of chemical substitution was monitored by the ninhydrin reaction. In vitro opsonic capacity of all γG preparations tested decreased with progressive acetylation or carbamylation. No preparations were opsonically active after substitution of 50% of the free amino groups. The ability of the antibodies to react with staphylococcal antigens was not significantly diminished by these treatments. Both acetylation and carbamylation produced a marked increase in the net negative charge of γG globulin and significantly altered antigenic determinants of both light and heavy chains. Limited alteration of γG opsonins was obtained with ethylacetimidate which substitutes only at α and ε amino groups. Amidination of up to 87% of the free amino groups did not alter the charge of the γG preparations and resulted in only minor changes in antigenic determinants of the light and heavy chains. The ability of the antibody to combine with staphylococcal antigens was retained. The opsonic capacity of human γG was abolished by substitution of 65% of the free amino groups. A comparable degree of amidination of rabbit γG globulin had no significant effect on its opsonic activity.

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.

The Effect of Carbamylation, Amidination and Benzylation on the Ability of Rabbit γG Antibody to Produce Passive Cutaneous Anaphlaxis in Guinea Pig and Rabbit Skin

Abstract The effect of chemical modification of amino groups of rabbit γ G antibody on its PCA activity in guinea pig and rabbit skin was studied, with particular attention to high degrees of carbamylation which have profound effects on the antibody's ability to fix complement and to aggregate with antigen. It was concluded that there is no evidence at present for the selective inactivation of specific PCA sites on the Fc portion of the antibody molecule by these reagents. Rather, the observed loss of PCA reactivity in rabbit skin could be attributed to the impaired ability of the antibody to fix complement. Loss of PCA activity in guinea pig skin required greater than 90% carbamylation of the antibody molecule and could be explained in terms of the nonspecific effects on the molecule produced by such treatment. It was further observed that the loss in PCA activity under these conditions could be potentiated in a system in which cross-reacting antigen was used.

The Latency Requirements and Rate of Evolution of Passive Cutaneous Anaphylactic Reactions Produced by Untreated and Carbamylated Rabbit γG Antibody

Abstract The amounts of antibody necessary to produce passive cutaneous anaphylaxis (PCA) with and without a latent period were compared, and the rate of evolution of the reaction was studied in both cases as a function of antibody dose. Differences in the morphologic appearance of the reaction in these two cases were defined. It was demonstrated that there is no absolute requirement for a latent period in the production of PCA in guinea pig skin. Rather, latency in this system must be defined in terms of enhanced anaphylactic sensitization as a function of time between the administration of antibody and challenge with antigen. Quantitative comparisons between the amount of antibody needed to produce these two types of PCA reactions and the amount of antibody needed to produce anaphylaxis with soluble immune complexes suggest that the underlying mechanism of all three may be similar. Studies using carbamylated antibody with marked reduction in antigen-aggregating ability suggest that small immune complexes are involved in the production of PCA when antibody and antigen are injected separately. Therefore in analogy with the situation in which soluble complexes are used, where the active complex has been shown to contain two molecules of antibody, it is argued that PCA reactions involve the binding of antigen to at least two molecules of antibody present at adjacent sites on an effector cell. If this is so, then latency, as defined above, may be determined by a time-dependent reassociation of antibody and antigen on the cell surface until optimal configurations are attained.

Human IgG and IgA Diphtheria Antitoxins in Serum, Nasal Fluids and Saliva

Abstract The site of production of γA and γG in human nasal fluids was investigated by measuring antibody responses to diphtheria toxoid injected parenterally or given topically as nose drops. The amount of radioactive toxoid combined with γA or γG was measured separately by precipitating the immonoglobulin from serum or exocrine fluids with rabbit antisera specific for each immunoglobulin class. That this measurement reflected the amount of each class of antibody present was shown by its correlation with neutralization titers and by measurements of antibody avidities. The bulk of γA antitoxin in nasal fluids was demonstrated to be 11 S, exocrine γA by its elution characteristics from Sephadex G-200 and by its reaction with antibody to secretory piece. Comparisons of γA and γG antibody levels found in serum, nasal fluids, saliva and sputum after parenteral or topical administration of toxoid indicated that most nasal fluid γA and some of the nasal fluid γG were made locally in response to local antigenic stimulation.

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.

Chronic autoimmune hemolytic anemia in childhood with cold antibodies, aplastic crises, and familial occurrence.

SummaryA report is given of two sisters who developed chronic autoimmune hemolytic anemia in their fourth and twelfth years of age, respectively. The serological findings were different in the two cases. In patient 1, a typical cold agglutinin with a titer of 256 at 4oC was found in the serum, and only complement on the red cells by the direct Coombs' test. The serum hemolysed trypsinized red cells at 37oC. Her sister had warm type hemolytic anemia with a γG antibody on her red cells. In case 1, the clinical course has been severe, with several exacerbations of the hemolytic process accompanied by erythroid aplasia in the bone marrow.Whereas acute autoimmune hemolytic anemia with cold antibodies is not rare in children, and may be elicited by mycoplasma or viral infections, chronic autoimmune hemolytic anemia is rarely seen before adulthood. The typical, chronic cold hemagglutinin disease with hemoglobinuria and Raynaud's phenomena has not been described in childhood, and was not present in our case. It is suggested that the disease in the case presented may have started as the result of an acute infection, and that for one reason or another (genetic predisposition?) the process has not shown the usual self‐limited course.Although a familial–probably genetic– predisposition exists for several autoimmune diseases, familial occurrence of autoimmune hemolytic anemia has rarely been observed. The authors have found nine convincing reports in the literature, and in only one of the reported families did the hemolytic anemia develop in childhood. In families predisposed to autoimmune diseases, a variety of γ‐globulin disturbances and autoimmune disorders are often found. The presence of a more fundamental, genetic aberration of the immune apparatus, preventing a normal immune homeostasis, may therefore be postulated.A brief comment is made on the possible mechanism of the repeated periods of erythroid aplasia in the patient with cold antibodies. It is suggested that a second antibody, causing hemolysis of trypsinized red cells at 37oC may be of importance by damaging the red cell precursors. The presence of this antibody may also possibly explain why the severity of the clinical picture of the patient did not seem to be related to exposure to cold.

An Incomplete Cold-Reactive γG Antibody with i Specificity in Infectious Mononucleosis

A new cold reactive antibody directed against fetal (i) cells is described in 90 % of patients with infectious mononucleosis. It appears to be the 7 S counterpart of the previously described 19S anti-i antibody. The 7S anti-i antibody interacts with a 19S anti γ globulin and the combination is responsible for the majority of the cold agglutinin phenomena previously shown in this disorder. The highest titers were detected in patients with severe hemolytic anemia, leukopenia, thrombocytopenia or combinations thereof.