IgGl Antibodies Research Articles

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.

Induction of in vivo helper activity for murine antibody responses by macrophages pulsed with ovalbumin-monomethoxypolyethylene glycol (OA-mPEG) conjugates

Conjugates of protein antigens with an optimal number of monomethoxypolyethylene glycol (mPEG) chains of an appropriate molecular weight had been shown to suppress murine IgE responses to the unmodified antigen. To investigate the possibility that the tolerogenic capacity of these mPEG conjugates is attributable to a defect in macrophage (Mφ) presentation of their antigenic determinants, the activity of ovalbumin (OA)-mPEG conjugates when pulsed onto mouse peritoneal adherent cells (Mφ) was compared in this study with their activity in solution. Surprisingly, in contrast to the suppressogenic capacity of mPEG conjugates in solution, the OA-mPEG pulsed Mφ appeared to exert a helper effect when injected intraperitoneally (ip), i.e., after subsequent immunization with dinitrophenylated OA (DNP 3-OA) in Al(OH) 3, the mice showed accelerated IgE and IgGl antibody responses to OA and DNP. However, when Mφ were exposed to limiting concentrations of OA or OA-mPEG, markedly higher concentrations of OA-mPEG were required to yield pulsed Mφ, exerting a significant helper effect. It was concluded that although Mφ were capable of presenting the OA determinants of OA-mPEG conjugates to helper T (Th) cells, the preparations of modified antigen were presented less effectively than native OA.

A monoclonal antibody CIBr17 recognizes a myoepithelium-specific antigen in human mammary gland.

A murine monoclonal antibody (MAb) CIBr17, raised against the human breast carcinoma cell line PMC42, reacts specifically with myoepithelial cells in normal human breast. This IgGl antibody recognizes a approximately 110kDa glycoprotein that is expressed on the cell surface and junctional membranes of PMC42 monolayer cultures. The CIBr17 antigen is present in two major glycosylated forms with approximate pls of 5.2 and 5.5 respectively in PMC42 cells. The tissue specificity of CIBr17 was assessed on frozen sections of PLP-fixed tissues by means of a 4-layer immunoperoxidase technique. CIBr17 has reacted with a variety of epithelium-derived tissues and some smooth muscle cells. Within many epithelial tissues, CIBr17 has demonstrated specific staining of particular epithelial cell types. Within normal breast and most benign breast lesions, antibody CIBr17 stained only myoepithelial cells. No staining of luminal epithelium, basement membranes or stromal elements was observed. In sclerosing adenosis, CIBr17 stained areas of pronounced myoepithelial differentiation, while in duct epitheliosis variable staining of proliferating cells was observed. In breast carcinomas, CIBr17 demonstrated variable antigen expression. In most tumors, CIBr17 either did not stain any tumor cells or stained only a small number of tumor cells spread randomly throughout the tumor. In several ductal carcinomas, however, CIBr17 stained the majority of tumor cells.

1103 IgG1 ANTIBODY RESPONSES OF CHILDREN AND ADULTS IMMUNIZED WITH HAEMOPHILUS INFLUENZAE TYPE b (Hib) POLYSACCHARIDE VACCINE (PRP)

IgG was purified by gel filtration of sera from 3 adults immunized with PRP. The ratio of IgGl to IgG2 anti-PRP was determined in affinity purified anti-PRP. We also determined IgG1 anti-PRP by measuring anti-PRP radioantigen binding activity before and after column immunoabsorption of IgGl employing a highly specific monoclonal prepared locally (HG-11). 38–46% of the post-vaccine serum IgG anti-PRP was of the IgGl subclass. An ELISA was then used to measure IgG and IgGl anti-PRP antibody in sera from 16 normal adults; and from 97 children 12 to 83 mos of age and 8 adults immunized with PRP. Only 2 of 16 unimmunized adults had IgGl liters >1:49. The IgGl anti-PRP antibody 2 mo after immunization was age-dependent.12 of 35 children had high IgG antiPRP responses (post vaccine titers >1:400) but no detectable IgGl antibody (IgGl titer <1:49). Thus, IgGl is an important component of the IgG antiPRP response of some but not all immunized subjects. In addition, although serum IgGl reaches adult concentrations in the first year of life, most children of this age cannot respond to immunization with IgGl anti-PRP whereas many older children can.

Induction of Fc receptors and immunoglobulin-binding factors in T-cell clones

The interaction of immunoglobulins (I& with their receptors [Fc receptors (FcR ii)] on the cell membrane leads to different events which depend on the cell type and the physico-chemical form of the Ig. For instance, interaction of mast cells with IgE and antigen induces their degranulation and the release of inflammatory mediators (Ishizaka and Ishizaka, 1975). The same phenomenon can also be achieved in the mouse by IgGl (Nussenszweig et al., 1964) and IgG2a antibodies (Da&on et al., 1982). Interaction of macrophages with antibody-coated particles induces phagocytosis (Silverstein et al., 1977). Binding of K-cells to IgG-sensitized target cells results in cytolysis of the targets (Perlman et al., 1972). Besides their capacity to induce effector activities in cells of the immune system, and possibly in relation to this capacity, Ig isotypes have been shown to regulate the membrane expression of their respective FcR. Incubation of lymphocytes with antigen-complexed IgG in the solid phase results in the disappearance of FQ R from the cell surface ~‘modulation”) (Cordier et ai., 1977). This phenomenon is followed by a loss of the proliferative response of the modulated cells to mitogens (Ryan and Henkart, 1976) and, in case of T-cells, of the capacity of modulated cells to act as

STABILITY OF RADIOIODIMATED MONOCLONAL ANTIBODIES (MoAb) AND THEIR F(ab')2 AND Fab FRAGMENTS IN VITRO

Knowledge of the stability of radioiodinated MoAb and their fragments in vitro is necessary for their practical application and commercialization. Using molecular-sieving (TSK-3000) high performance liquid chromatography (HPLC), cellulose acetate electrophoresis (CAE) at 11 and 45 minutes and solid phase immunoreactivity, we have observed the in vitro stability of a B cell lymphoma antibody (Lym-1, IgG2a) and its Fab fragment, and a mammary cancer antibody (B6.01, IgG1) and its F(ab‘)2 fragment. Each species was iodinated (I-125) with chloramine-T (C-T) at one microgram C-T per microgram of MoAb and one atom of iodine per molecule of MoAb, and were stored at 4°C. All four species showed mild degradation by 3 days, but this was only slowly progressive for the whole antibodies up to the final observation time of 21 days. F(ab’)2 more rapidly degraded between 6 and 14 days, and Fab between 3 and 6 days, as well as subsequently. The results by HPLC and CAE were comparable and were confirmed by tests of immunoreactivity. In summary, there was no significant difference in degradation in vitro between an IgG2a and an IgGl antibody iodinated in an identical manner, and these molecules were reasonably stable for 14–21 days at 4°C. Fragments of these MoAb iodinated in an identical manner were less stable, and this seemed to be size-related. This could be a reflection of the relatively greater impact of the addition of an atom of iodine to a smaller species, but more likely reflects an inherently reduced stability of the fragments.

IgG subclass distribution of myasthenia gravis thymoma-associated antiskeletal muscle antibodies.

Myasthenia gravis patients with thymoma often have serum antibodies directed against human skeletal muscle. We have developed an enzyme-linked immunosorbent assay to investigate the subclass distribution of these antibodies. Five thymoma patients all had IgG4 antibodies, whereas a patient who developed myasthenia gravis after bone-marrow transplantation had only IgG1 antibodies. The data suggest that IgG1 is the first subclass to appear in the autoantibody response against skeletal muscle.

Immunoglobulin gene expression in transformed lymphoid cells.

Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.

Acute effects of ethanol on biosynthesis and glycosylation of IgGl(kappa) antibody molecules in cultured P3/X63-Ag8 myeloma cells.

Protein synthesis in cultured P3/X63-Ag8 mouse myeloma cells was inhibited by acute exposure to ethanol. However, the synthesis of IgGl antibody, as a percentage of total protein synthesis, increased slightly. Experiments using actinomycin D suggest that the overall inhibition of protein synthesis by ethanol occurs at the translational level. Following an L-[35S]methionine pulse, cultured P3/X63-Ag8 cells contained one light antibody polypeptide and two heavy antibody polypeptides. One of these heavy chains was shown to be the unglycosylated precursor of the other, mature molecule. Only the glycosylated polypeptide is a normal constituent of secreted IgGl antibody. The glycosylation of the immature heavy chains occurred more rapidly during a 1-hr isotopic pulse in cells exposed to ethanol (0.1 v/v % and above), than in unexposed control cells. The observed effects of ethanol on antibody glycosylation may be related to the increased susceptibility of alcoholic patients to infections. Ethanol may also affect the synthesis of other glycoproteins in myeloma cells and other tissues.

CLINICAL ASPECTS OF AUTOIMMUNE HEMOLYTIC ANEMIA IN CHILDREN (AIHA)

Fourteen patients with AIHA between the ages of 10 weeks to 18 years were investigated. Of these 10 had warm reactive autoantibodies (6 anti-dl, 1 anti-nl, 1 anti-Rho, 1 non-specific, 1 Coombs negative IgGl antibody) and 4 had cold reactive autoantibodies (3 anti-l and 1 non-specific antibody). At the time of initial diagnosis the hemoglobin levels varied from 2.8 to 8.7 gm/dl. Four patients had reticulocytopenia at the onset. In 2 patients the haptoglobin levels were normal. One patient received no treatment, one only required a blood transfusion and 12 were treated with corticosteroids of which 11 responded successfully. The patient who failed to respond to corttcosteroids also did not respond to exchange transfusion and immunosuppresslve drugs but responded following splenectomy. Eleven were idiopathic and 3 had the following associated conditions: mycoplasma pneumonitis, systemic lupus erythematosus and a “panhematoimmunopathy” (anti-neutrophii autoantibody, anti-platelet autoantibody and anti-dl). In most of these patients the course of AIHA was transient, due to warm autoantibodies and responsive to steroids. Coombs negative AIHA is an uncommon entity requiring detailed immunohematologic investigations including the use of the autoanalyzer and elution studies for its identification.

Control of IgE and IgGl antibody production in mice

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.

Acceleration of intradermal catabolism of guinea pig IgG1 and IgG2 antibodies by challenge with antigen.

The intradermal catabolism of antibodies injected in guinea pigs to provoke skin reactions was studied using 125I-labeled guinea pig IgG1 and IgG2 anti-ovalbumin antibodies. Disappearance of both the IgG1 and IgG2 antibodies from injected sites was accelerated by intravenous injection of the antigen. The antigen-antibody complexes produced in vitro were also catabolized more rapidly than free antibodies, when estimated using 125I-labeled antibodies. On the other hand, the catabolism of normal IgG2 was not influenced by local anaphylactic reaction elicited by IgG1 antiovalbumin antibody coexisting at the sites. Therefore, the enhanced catabolism of antibodies on challenge was not caused by increased vascular permeability due to anaphylactic reactions, but by more rapid elimination of immune complexes formed at the sites. The Fc parts of IgG1 and IgG2 antibodies played an essential role in the enhancement of catabolism since the catabolism of the F(ab')2 fragments was not accelerated by complex formation with ovalbumin, but rather reduced.

Suppressor T cells in mice made unresponsive to skin allografts.

WE have established1 that long-lasting, strain-specific unresponsiveness to H-2 incompatible skin allografts can be induced in 30–50% of adult CBA mice by pretreatment with a single injection of a crude extract of liver from the graft donor strain, combined with a dose of Bordetella pertussis vaccine and a short course of antilymphocyte serum (ALS). Lymphoid cells removed from mice bearing healthy well-established allografts were reactive against graft donor strain antigens when tested in mixed lymphocyte culture (MLC), cell-mediated lympholysis tests (CML) and in graft versus host assays (GvH)2. Haemagglutinating and cytotoxic antibodies specific for the donor strain were invariably absent from the serum of unresponsive mice although small quantities of IgGl antibodies have been detected by a more sensitive technique3. We have so far been unable to detect any strain-specific suppressive activity in sera using MLC, CML and GvH tests2 or when passively transferring sera to skin-grafted mice.

Production of antibodies of identical idiotype but diverse immunoglobulin classes by cells derived from a single stimulated B cell.

The availability of anti-phosphocholine antibody of the TEPC 15 idiotype from the clonal progeny of a single precursor cell, stimulated in vitro, permitted the demonstration of monoclonal antibodies with as many as three immunoglobulin classes with identical variable regions. This demonstration was dependent on sensitive radioimmunoassays which showed a one to one relationship between the total anti-phosphocholine antibody produced by a clone and the sum of anti-phosphocholine antibody of the different classes as well as the amount of antibody of the TEPC 15 idiotype. The class distribution was confirmed by isoelectric focusing identification of IgM, Igta, and IgGl antibodies of the TEPC 15 idiotype produced by single clones which showed characteristic pI values for each immunoglobulin class. Thus, within the generative phase of a single antibody-producing cell clone, various heavy chain constant regions can be linked to the same variable region, and single precursor cells have the capacity to generate progeny expressing at least three distinct immunoglobulin classes.

Effect of varying the immunizing dose of ovalbumin on the production of non-precipitating IgG1 and IgG2 antibodies in guinea pigs.

Effect of varying the immunizing dose of ovalbumin on the production of non-precipitating IgG1 and IgG2 antibodies in guinea pigs.