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Biomedical subjects

A Vlug

Publications and source records attributed to A Vlug.

At least 19 recordsLinked to original sources
PubMed1997

Human B cells secreting immunoglobulin G to glutamic acid decarboxylase-65 from a nondiabetic patient with multiple autoantibodies and Graves' disease: a comparison with those present in type 1 diabetes.

Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) diabetes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmune disease. Antibodies to GAD in IDDM patients usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the second isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, we have generated three human IgG autoantibodies, termed b35, b78, and b96, to GAD65 from one patient with multiple autoantibodies to endocrine organs and Graves' disease. All three autoantibodies are of the IgG1 isotype, with islet cell activity, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation with a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD proteins, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino acids 240-435) whereas both b78 and b96 target the IDDM-E2 region of GAD65 (amino acids 451-570). Furthermore, examination of binding to recombinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD65. However, b35, b78, and b96 autoantibodies had different footprinting patterns after trypsin treatment of immune complexes with GAD65, again indicating different epitope recognition. Our results indicate that antibodies to GAD65 present in nondiabetic patients with multiple autoantibodies to endocrine organs show similarities to those in IDDM (by targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle differences in epitope recognition (such as binding to denatured and reduced GAD65 and by protein footprinting). Thus, the GAD65 epitopes recognized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.

Adult↗
PubMed1994

Nephelometric measurements of human IgG subclasses and their reference ranges.

IgG subclass measurements are generally performed with the radial immunodiffusion (RID) technique. With this method, results are obtained after an incubation period of 48-72 hours. We developed nephelometric assays on the Behring Nephelometer Analyzer (BNA) that allow a quantification of IgG subclass concentrations in a large number of samples quickly (less than 15 minutes for a complete IgG subclass profile) and reproducibly (intra-assay variation 2.5-5.5%, interassay variation 3.4-6.0% and inter-lab variation 5.4-10.3%). The nephelometric method was compared with the RID technique by analyzing the IgG subclass levels in sixty selected samples. For all IgG subclasses identical results and high correlation coefficients (r > 0.93) were found. In addition, the detection limits of the nephelometric method for all four IgG subclasses were identical or lower than those of the RID technique. Furthermore, the interlab variations of the nephelometric IgG subclass assays are lower than those of the RID method. However, the major advantages of the nephelometric assay are the speed, the minimal workload (automated IgG subclass determinations) and the possibility for automated bidirectional data transmission. Recently we have established new reference ranges for the human IgG subclasses in sera of adults and children. In order to validate these reference values we have measured the IgG subclasses in sera from 112 healthy children with the nephelometric method. In 1992, more than 2000 patient sera were tested by the nephelometric assay. A predominance of IgG2 abnormality was observed. In 9.8% of these sera the IgG2 concentration was decreased. Elevated IgG2 concentrations were found in 1.9% of the sera. Furthermore, the sum of the quantitated four IgG subclasses was similar to that of total IgG (less than 20% difference).

Adolescent↗
PubMed1992

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

Antibodies, Anti-Idiotypic↗
PubMed1992

On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

Antibodies, Monoclonal↗
PubMed1991

A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding.

The low-affinity human Fc gamma RIIa is encoded by a single gene with allelic variation, defined by low-responder and high-responder alleles (LR and HR). The HR Fc gamma RIIa transcript interacts strongly with murine (m) IgG1 complexes, in contrast to the LR Fc gamma RIIa. Furthermore, the transcripts can be discriminated by mAb 41H16, which recognizes an epitope expressed on the HR Fc gamma RIIa molecule. We report that this receptor is also polymorphic in its reactivity with human (h) IgG2. Binding studies using well-defined hIgG dimers revealed that LR Fc gamma RIIa molecules can efficiently bind hIgG2, in contrast to HR Fc gamma RIIa. Previous work of others showed one amino acid difference between the allelic forms of Fc gamma RII. We, however, found a second amino acid difference between both allelic forms. In this study, hybrid Fc gamma RIIa molecules were constructed to determine the epitope for mAb 41H16 and the binding domain for mIgG1 and hIgG2 complexes. Our data point to the importance of the amino acid at position 131, located in the second Ig-like domain of Fc gamma RIIa. When an arginine residue is present at amino acid position 131, the receptor is recognized by mAb 41H16. Furthermore, the receptor can bind mIgG1-sensitized indicator E, but binds hIgG2 dimers only weakly. When a histidine residue is present at this amino acid position, hIgG2 dimers do bind efficiently to Fc gamma RII, whereas mIgG1-sensitized E and mAb 41H16 exhibit a strongly diminished binding.

Alleles↗
PubMed1990

Multiple levels of analysis of an IGHG4 gene deletion.

Human immunoglobulin heavy chain constant region (IGHC) genes constitute a typical multigene family, usually comprising eleven genes on the telomere of chromosome 14 (14q32). In this region, deleted and duplicated haplotypes have been reported to exist with considerable frequency. Their origin is the result of either unequal crossing-over or looping out excision. In this paper, we report the characterization of a new type of deletion, involving the IGHG4 gene, in a subject who also carries a larger deletion of a previously described type on the second chromosome. Employment of several methods (polymerase chain reaction, standard Southern blot, pulsed field gel electrophoresis, serological techniques) to analyze these deleted haplotypes has resulted in a level of accuracy in their characterization that has not been achieved in previous cases. The site of recombination responsible for the IGHG4 deletion was restricted to a 2.5-kb region 3' of the G4 gene; this rules out any possible involvement of the S regions in the recombination process. The usefulness of the various techniques in the characterization of the deletions is also discussed, together with possible future applications in the field.

Base Sequence↗
PubMed1989

Binding characteristics of dimeric IgG subclass complexes to human neutrophils.

Immune complexes were prepared by incubation of human IgG paraproteins with F(ab')2 fragments of the mAb K35 against the kappa-L chain of human IgG. The composition of these complexes was analyzed by centrifugation over sucrose gradients, by gel filtration, by RIA with either IgG Sepharose or K35 Sepharose and by double-labeling studies. The results indicated that the complexes consist of saturated tetramers composed of two IgG molecules cross-linked by two F(ab')2 fragments of the mAb. These complexes were used to study the binding of the different IgG subclasses to human neutrophils at 4 degrees C. Human neutrophils bound IgG3 complexes approximately three times faster than IgG1 complexes. Binding of IgG2 or IgG4 dimers to the neutrophils was undetectable. The same number of IgG1 complexes and IgG3 complexes bound to the neutrophils, but considerable inter-donor variation was found (mean number of Fc gamma R per neutrophil: 190,000, range 120,000 to 400,000). The Ka for the binding of IgG1 complexes to neutrophils (median 11 x 10(7) M-1) was lower than the Ka for the binding of IgG3 complexes (median 47 x 10(7) M-1). Competition studies between labeled IgG1 complexes or IgG3 complexes and unlabeled complexes showed that the Fc gamma R of human neutrophils do not display an IgG subclass specificity. Incubation of neutrophils with a mAb against the FcRIII completely blocked the binding of IgG1 complexes and IgG3 complexes. Incubation with a mAb against the FcRII reduced the affinity of the complexes for the neutrophils but had no effect on the maximum number of complexes bound. This indicates that one complex may bind simultaneously to one FcRIII and to one FcRII.

Animals↗
PubMed1989

Activity of two types of Fc receptors, Fc gamma RI and Fc gamma RII, in human monocyte cytotoxicity to sensitized erythrocytes.

We investigated the cytotoxicity of human monocytes mediated by two types of receptors for the Fc portion of IgG, Fc gamma RI and Fc gamma RII. Erythrocytes sensitized with human IgG (EA-human IgG) were used to assay Fc gamma RI function, and erythrocytes sensitized with mouse IgG1 (EA-mouse IgG1) were used to assay Fc gamma RII. Both types of Fc gamma R were observed to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which was further characterized by using different monoclonal anti-Fc gamma R antibodies (MoAb) and monomeric IgG. Lysis of EA-human IgG was inhibited by both monomeric human IgG and mouse IgG2a in a dose-dependent way, and also by anti-Fc gamma RI MoAb 10.1. Cytolysis of EA-mouse IgG1 was inhibited by monomeric mouse IgG1 and by two anti-Fc gamma RII MoAb, IV.3 and CIKM5. Antibodies of the mouse IgG2b isotype affected neither type of ADCC. The effectiveness of cytotoxicity mediated by either of the Fc gamma R was studied by means of targets sensitized with a calibrated number of IgG molecules. At least 20 times more IgG molecules per target cell were necessary to obtain half-maximal cytotoxicity mediated by Fc gamma RII than for Fc gamma RI-mediated cytolysis. Furthermore, the previously described polymorphism of Fc gamma RII was also reflected in Fc gamma RII-dependent cytotoxicity. These studies demonstrate that Fc gamma RII can mediate ADCC, although a higher degree of target cell sensitization is required than for Fc gamma RI-mediated ADCC.

Animals↗
PubMed1989

Monoclonal antibodies against IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1): their usefulness in HAI and capture ELISA.

Monoclonal antibodies (McAbs) were produced against the IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1). Four out of the six McAbs described in this paper showed in the haemagglutination assay cross-reactivity with some or all IgG-coated cell samples. In the haemagglutination inhibition assay, all six McAbs are useful as typing reagent for the above allotypes. In this assay, two of the McAbs show two different specificities, which depend on the Ig-coated cell sample used. Five McAbs are useful for allotyping in a capture ELISA. The results with four of these are promising for the development of a quantitative determination of Gm allotypes.

Animals↗
PubMed1988

Dot-immunobinding assay as an accurate and versatile technique for the quantification of human IgG subclasses.

A dot-immunobinding assay on nitrocellulose membranes has been developed for the quantification of human IgG subclasses using subclass-specific monoclonal antibodies. The advantages of this technique can be summarized as follows: (1) possibility of rapid semi-quantitative evaluation and/or precise quantitation from the same dot-pattern; (2) simple procedure with very good reproducibility; (3) sensitivity for nanogram concentrations of individual subclasses, therefore applicable not only to serum but also to other body fluids with a low content of IgG; (4) very small amounts of test material needed; (5) very good correlation of results with other techniques (ELISA, radial immunodiffusion) but without some of the inherent problems of the latter methods.

Antibodies, Monoclonal↗
PubMed1987

Characterization of two Fc receptors for mouse immunoglobulins on human monocytes and cell lines.

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti-CD3-induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1.

Animals↗
PubMed1986

Monoclonal antibodies against different domains of human IgA: specificities determined by immunoblotting and haemagglutination-inhibition.

The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.

Antibodies, Monoclonal↗
PubMed1986

Characterization of the humoral immune response in Sudanese leishmaniasis: specific antibody detected by class- and subclass-specific reagents.

Sera from Sudanese patients with either visceral or mucosal manifestations of infection with Leishmania were investigated to determine the class of antibodies against the causative parasite in an enzyme-linked immunosorbent assay (ELISA) with intact promastigotes as antigen. All patient sera had significantly more IgG and more antiparasite IgG than did control Sudanese or control Dutch sera; little or no IgM or IgA antiparasite antibody activity was detected. When sera specific for the subclasses of IgG were used to detect the anti-promastigote antibodies, these were found in IgG1- and IgG3-specific ELISA but not in those for IgG2 or IgG4.

Adolescent↗
PubMed1985

The quantification of human IgG subclasses in reference preparations.

Three laboratories took part in an investigation about the possibilities of quantitative determination of IgG subclasses. Polyclonal monospecific antisera were used in combination with different kinds of calibrators to assess the IgG subclass levels in three W.H.O. reference preparations. IgG 1 could be determined with satisfactory precision; for the other subclasses the quantification was less precise. The precision was dependent on the antisera for IgG 1, IgG 2 and IgG 4 but mainly on the calibrators for IgG 3. Mass units are to be preferred to relative units. W.H.O. reference preparation 67/97 is proposed as a reference for determination of IgG subclasses in mass units. The target values are 5.0 g/l for IgG 1, 2.6 g/l for IgG 2, 0.4 g/l for IgG 3 and 0.5 g/l for IgG 4. These values add up to a total IgG value close to earlier W.H.O. estimates.

Humans↗
PubMed1985

Serum immunoglobulins, including IgG subclasses, in Vietnamese leprosy patients.

Levels of serum immunoglobulins were measured in healthy Vietnamese and in leprosy patients. Healthy Vietnamese had higher levels of IgG, IgA, IgM, and IgE than did healthy Dutch controls, as well as higher levels of three of the four subclasses of IgG (IgG1, IgG2, IgG3). Lepromatous leprosy patients had significant increases in all classes and subclasses of immunoglobulins, except for IgG2, in comparison with local controls. Tuberculoid leprosy patients had more IgG1, IgG3, and IgG4 than did local controls and had higher total IgG levels. The patients had no increase in autoantibodies against tissue antigens compared to local or Dutch controls.

Autoantibodies↗
PubMed1983

H-2 control of the cytotoxic antibody response against a newly defined MuLV-related cell-surface antigen: G(B10.A).

H-2-congeneic C57BL mice with milk transmission of B-tropic murine leukemia virus (V+ mice) have a much higher lymphoma incidence than the same strains without milk-transmitted virus (V- mice). Gene(s) within the major histocompatibility complex (H-2) influence virus titers, lymphoma incidence, lymphoma type and the anti-MuLV envelope antibody response. In this paper, we report that the prevalence of cytotoxic antibodies to virus-induced lymphomas is also regulated by the H-2 complex. Milk transmission of MuLV resulted in the formation of cytotoxic antibodies against primary virus-induced C57BL lymphomas. These antibodies detect an antigen that is also present on the RADAI tumor-cell line, and on normal spleen cells of young adult B10.A (H-2a) mice of both V+ and V- sublines, but not on spleen cells of young adult B10 (H-2a) mice of either subline. These cytotoxic antibodies were detected in the sera of B10V+ and B10.A(5R)V+ animals, but not in the sera of B10.AV+ mice. This indicates that the prevalence of these antibodies is controlled by a gene in the K- and/or I-A region of the H-2 complex. The presence of these cytotoxic antibodies in serum is recessively inherited. The specificity of the cytotoxic antibodies was investigated with a standard panel of transplantable tumor-cell lines. Of these, only the RADAI cells expressed the target antigen in direct cytotoxicity tests and by absorption. The ability of B10V+ sera to lyse the B10.AV+ and RADAI tumor cells is ascribed to antibody activity against a new MuLV-related cell-surface protein: G(B10.A). Immunochemical analysis and absorption experiments with different types of purified MuLV and MuLV-infected cell lines indicate that the cytotoxic antibodies belong to low-avidity IgM antibodies that are directed to MuLV.

Animals↗
PubMed1981

Genes of the H-2 complex regulate the antibody response to murine leukemia virus.

The influence of the major histocompatibility complex of the mouse (H-2 complex) on the antibody response against murine leukemia virus (MuLV) was investigated after 3 different ways of virus presentation (milk transmission of virus, spontaneous virus activation, and immunization with inactivated virus). The antibody response against MuLV was measured in the sera of H-2 congenic C57BL V+ sublines (V+ denotes positive for milk-transmitted MuLV), (B10.AV+ X C57BL)F1 mice, (C57BL X AKR)F1 mice and of C57BL animals after immunization with inactivated AKR virus. The pattern of immune responsiveness of the different C57BL strains to MuLV was independent of virus replication and was similar for the 3 ways of virus presentation. Serum antibody levels were controlled by 2 genes within the H-2 complex. The first gene (Ir-MuLV-1) was located in the I-A region and was complemented by a second gene (Ir-MuLV-2), which was situated in the regions to the right of the I-B region. Presence of 2 responder alleles (Ir-MuLV-1+,2+) led to an optimal antibody response (H-2b haplotype). Animals that only expressed a responder allele in the I-A region (Ir-MuLV-1+,2-) were intermediate responders. Animals lacking a responder allele in the I-A region (Ir-MuLV-1-,2+ or Ir-MuLV-1-,2-) were low responders.

Animals↗