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Sample records for allergen-specific igg antibodies

  1. Passive immunization with allergen-specific IgG antibodies for treatment and prevention of allergy

    PubMed Central

    Flicker, Sabine; Linhart, Birgit; Wild, Carmen; Wiedermann, Ursula; Valenta, Rudolf

    2013-01-01

    IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans. PMID:23182706

  2. Allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens: an in vitro study.

    PubMed

    Siman, Isabella Lima; de Aquino, Lais Martins; Ynoue, Leandro Hideki; Miranda, Juliana Silva; Pajuaba, Ana Claudia Arantes Marquez; Cunha-Júnior, Jair Pereira; Silva, Deise Aparecida Oliveira; Taketomi, Ernesto Akio

    2013-01-01

    One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%-51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.

  3. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    PubMed

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  4. Allergen-specific IgG as a mediator of allergy inhibition: Lessons from mother to child

    PubMed Central

    2017-01-01

    ABSTRACT Allergen-specific IgG produced by immune mothers is associated with less predisposition to allergy development in their children. This finding has been described by several groups over the last few decades, but the mechanisms by which maternal IgG can inhibit allergy development are still not fully understood. With the purpose of summarizing past investigations, we review the literature on murine models of maternal immunization with allergens and on immune regulation in humans after passive therapy with purified IgG. Based on our review, a new hypothesis about these mechanisms is presented, which may provide a foundation for the future development of therapies to inhibit allergy development. PMID:27808600

  5. Measurement of allergen-specific IgG in serum is of limited value for the management of dogs diagnosed with cutaneous adverse food reactions.

    PubMed

    Hagen-Plantinga, E A; Leistra, M H G; Sinke, J D; Vroom, M W; Savelkoul, H F J; Hendriks, W H

    2017-02-01

    Conflicting results have been reported in the literature in terms of the usefulness of serological testing for IgG against food allergens in dogs with cutaneous adverse food reaction (CAFR). The aim of the present study was to evaluate the suitability of a commercially available IgG ELISA for identifying food allergens in dogs, by challenging dogs with specific food ingredients, selected on the basis of IgG reactivity in serum samples. A total of 24 adult dogs with CAFR were enrolled into the study and 16 healthy dogs were included as a control group. Blood samples were obtained for measurement of specific IgG antibodies against 39 commonly used pet food ingredients by ELISA. Participating owners were surveyed to obtain information on their pet's dietary history. Eleven healthy control dogs and 12 dogs with CAFR were subsequently challenged in a blinded cross-over design experiment with both positive and negative food ingredients, selected on the basis of the ELISA test results. There was substantial individual variation in ELISA test results to the various food allergens, but no significant difference in IgG reactivity comparing the CAFR and control groups. None of the control dogs developed any clinical signs of an allergic reaction during the dietary challenge study. In the CAFR group, six of 12 dogs developed clinical signs after the negative challenge, and two of nine dogs developed clinical signs after the positive challenge. It was concluded that the ELISA test for dietary allergen-specific IgG is of limited value in the management of dogs with CAFR.

  6. Update in the Mechanisms of Allergen-Specific Immunotheraphy

    PubMed Central

    Akkoc, Tunc; Akdis, Mübeccel

    2011-01-01

    Allergic diseases represent a complex innate and adoptive immune response to natural environmental allergens with Th2-type T cells and allergen-specific IgE predominance. Allergen-specific immunotherapy is the most effective therapeutic approach for disregulated immune response towards allergens by enhancing immune tolerance mechanisms. The main aim of immunotherapy is the generation of allergen nonresponsive or tolerant T cells in sensitized patients and downregulation of predominant T cell- and IgE-mediated immune responses. During allergen-specific immunotherapy, T regulatory cells are generated, which secrete IL-10 and induce allergen-specific B cells for the production of IgG4 antibodies. These mechanisms induce tolerance to antigens that reduces allergic symptoms. Although current knowledge highlights the role of T regulatory cell-mediated immunetolerance, definite mechanisms that lead to a successful clinical outcomes of allergen-specific immunotherapy still remains an open area of research. PMID:21217920

  7. Analysis of the relations between allergen specific LgG antibody and allergic dermatosis of 14 kinds foods

    PubMed Central

    Yin’e, Hu; Shufang, Dai; Bin, Wang; Wei, Qu; Junling, Gao; Ashraf, Muhammad Aqeel

    2015-01-01

    To use food-specific IgG antibody detection to explore its application in the allergy dermatoses. 181 patients were included from January 2014 to September 2014. Fourteen food-specific IgG antibodies were detected by ELISA. The positive rates of IgG antibody of the patient group and the healthy group were significantly different. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. There was mild and moderate intolerance of food in the allergic dermatitis group while there was no distribution difference of food intolerance in urticaria group and eczema group. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults while there was no significant difference between children and adults among eczema patients with positive antibody. Allergy dermatoses are closely related to food-specific IgG antibody and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  8. Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice.

    PubMed

    Adel-Patient, K; Créminon, C; Bernard, H; Clément, G; Négroni, L; Frobert, Y; Grassi, J; Wal, J M; Chatel, J M

    2000-02-21

    An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.

  9. An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy

    PubMed Central

    Selb, R.; Eckl-Dorna, J.; Vrtala, S.; Valenta, R.; Niederberger, V.

    2017-01-01

    Background It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy. Methods In 18 patients treated by Bet v 1-fragment-specific immunotherapy, the effects of IgG antibodies specific for the fragments on the binding of IgE antibodies to Bet v 1 were measured by ELISA. Blocking and possible enhancing effects on IgE binding were compared with skin sensitivity to Bet v 1 after treatment. Results We found that fragment-specific IgG enhanced IgE binding to Bet v 1 in two patients who also showed an increase of skin sensitivity to Bet v 1. Conclusion Our results indicate that it may be possible to develop serological tests which predict the induction of unfavourable IgG antibodies enhancing the binding of IgE to Bet v 1 during immunotherapy. PMID:23998344

  10. A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help

    PubMed Central

    Narayanan, Meena; Freidl, Raphaela; Focke-Tejkl, Margarete; Baranyi, Ulrike; Wekerle, Thomas; Valenta, Rudolf

    2017-01-01

    More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope–derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope–derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines. PMID:28093528

  11. A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help.

    PubMed

    Narayanan, Meena; Freidl, Raphaela; Focke-Tejkl, Margarete; Baranyi, Ulrike; Wekerle, Thomas; Valenta, Rudolf; Linhart, Birgit

    2017-02-15

    More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope-derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope-derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

  12. Mechanisms of allergen-specific immunotherapy

    PubMed Central

    2012-01-01

    Allergen-specific immunotherapy (allergen-SIT) is a potentially curative treatment approach in allergic diseases. It has been used for almost 100 years as a desensitizing therapy. The induction of peripheral T cell tolerance and promotion of the formation of regulatory T-cells are key mechanisms in allergen-SIT. Both FOXP3+CD4+CD25+ regulatory T (Treg) cells and inducible IL-10- and TGF-β-producing type 1 Treg (Tr1) cells may prevent the development of allergic diseases and play a role in successful allergen-SIT and healthy immune response via several mechanisms. The mechanisms of suppression of different pro-inflammatory cells, such as eosinophils, mast cells and basophils and the development of allergen tolerance also directly or indirectly involves Treg cells. Furthermore, the formation of non-inflammatory antibodies particularly IgG4 is induced by IL-10. Knowledge of these molecular basis is crucial in the understanding the regulation of immune responses and their possible therapeutic targets in allergic diseases. PMID:22409879

  13. Safety of engineered allergen-specific immunotherapy vaccines

    PubMed Central

    Focke-Tejkl, Margarete; Valenta, Rudolf

    2015-01-01

    Purpose of review The purpose of the review is to summarize and comment on recent developments regarding the safety of engineered immunotherapy vaccines. Recent findings In the last 2 years, several studies were published in which allergy vaccines were developed on the basis of chemical modification of natural allergen extracts, the engineering of allergen molecules by recombinant DNA technology and synthetic peptide chemistry, allergen genes, new application routes and conjugation with immune modulatory molecules. Several studies exemplified the general applicability of hypoallergenic vaccines on the basis of recombinant fusion proteins consisting of nonallergenic allergen-derived peptides fused to allergen-unrelated carrier molecules. These vaccines are engineered to reduce both, immunoglobulin E (IgE) as well as allergen-specific T cell epitopes in the vaccines, and thus should provoke less IgE and T-cell-mediated side-effects. They are made to induce allergen-specific IgG antibodies against the IgE-binding sites of allergens with the T-cell help of the carrier molecule. Summary Several interesting examples of allergy vaccines with potentially increased safety profiles have been published. The concept of fusion proteins consisting of allergen-derived hypoallergenic peptides fused to allergen-unrelated proteins that seems to be broadly applicable for a variety of allergens appears to be of particular interest because it promises not only to reduce side-effects but also to increase efficacy and convenience of allergy vaccines. PMID:22885888

  14. Allergen diagnosis microarray with high-density immobilization capacity using diamond-like carbon-coated chips for profiling allergen-specific IgE and other immunoglobulins.

    PubMed

    Suzuki, Koichi; Hiyoshi, Mineyoshi; Tada, Hitomi; Bando, Miwa; Ichioka, Takao; Kamemura, Norio; Kido, Hiroshi

    2011-11-14

    The diagnosis of antibody-mediated allergic disorders is based on clinical findings, skin prick tests and detection of allergen-specific IgE in serum. Here, we present a new microarray technique of high-density antigen immobilization using carboxylated arms on the surface of a diamond-like carbon (DLC)-coated chip. High immobilization capacity of antigen on DLC chip at (0.94-7.82)×10(9) molecules mm(-2) allowed the analysis of allergen-specific immunoglobulins against not only purified proteins but also natural allergen extracts with wide assay dynamic range. The higher sensitivity of the allergen-specific IgE detection on DLC chip was observed for comparison with the UniCAP system: the DLC chip allowed lowering the limit of dilution rate in UniCAP system to further dilution at 4-8-fold. High correlations (ρ>0.9-0.85) of allergen-specific IgE values determined by the DLC chip and UniCAP were found in most of 20 different allergens tested. The DLC chip was useful to determine allergen-induced antibodies of IgA, IgG, IgG1, and IgG4 in sera, apart from IgE, as well as secretory IgA in saliva against the same series of allergens on the chip in a minimal amount (1-2 μL) of sample.

  15. Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

    PubMed Central

    López-Matas, M. Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

    2013-01-01

    Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT. PMID:24222901

  16. Intra and inter-laboratory reproducibility of a monoclonal antibody cocktail based ELISA for detection of allergen specific IgE in dogs: proficiency monitoring of macELISA in six laboratories.

    PubMed

    Lee, Kenneth W; Blankenship, Karen D; McCurry, Zachary M; McKinney, Brennan; Ruffner, Rick; Esch, Robert E; Tambone, Cecilia; Faas, Rebecca; Hermes, Darren; Brazis, Pilar; Drouet, Laurent

    2012-08-15

    The purpose of this study was to evaluate the reproducibility of results yielded using a monoclonal antibody based ELISA for detection of allergen specific IgE when run in six separate affiliated laboratories. On two separate occasions, duplicate samples of 15 different sera pools were independently evaluated by each laboratory in a single blinded fashion. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.2% (range 2.6-18.2%), while the average intra-laboratory inter-assay variance was 12.1% (range 8.0-17.1%). The overall inter-assay inter-laboratory variance was consistent among laboratories and averaged 15.6% (range 15.1-16.6%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose-response profiles observed in each of the laboratories were indistinguishable. Considering positive/negative results, inter-assay inter-laboratory concordance of results exceeded 95%. Correlation of OD values between and among all laboratories was strong (r>0.9, p<0.001). Correlation of OD values between the two separate evaluations was also high for all allergens except olive, which was attributed to lot-to-lot differences of allergen coated wells. Collectively, the results demonstrated that the monoclonal antibody based ELISA for measuring allergen specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but between laboratories using the same monoclonal-based ELISA.

  17. Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

    PubMed

    Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

    1997-02-01

    The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

  18. Allergen Peptides, Recombinant Allergens and Hypoallergens for Allergen-Specific Immunotherapy.

    PubMed

    Marth, Katharina; Focke-Tejkl, Margarete; Lupinek, Christian; Valenta, Rudolf; Niederberger, Verena

    2014-01-01

    Allergic diseases are among the most common health issues worldwide. Specific immunotherapy has remained the only disease-modifying treatment, but it is not effective in all patients and may cause side effects. Over the last 25 years, allergen molecules from most prevalent allergen sources have been isolated and produced as recombinant proteins. Not only are these molecules useful in improved allergy diagnosis, but they also have the potential to revolutionize the treatment of allergic disease by means of immunotherapy. Panels of unmodified recombinant allergens have already been shown to effectively replace natural allergen extracts in therapy. Through genetic engineering, several molecules have been designed with modified immunological properties. Hypoallergens have been produced that have reduced IgE binding capacity but retained T cell reactivity and T cell peptides which stimulate allergen-specific T cells, and these have already been investigated in clinical trials. New vaccines have been recently created with both reduced IgE and T cell reactivity but retained ability to induce protective allergen-specific IgG antibodies. The latter approach works by fusing per se non-IgE reactive peptides derived from IgE binding sites of the allergens to a virus protein, which acts as a carrier and provides the T-cell help necessary for immune stimulation and protective antibody production. In this review, we will highlight the different novel approaches for immunotherapy and will report on prior and ongoing clinical studies.

  19. Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

    PubMed

    Gafvelin, Guro; Parmley, Stephen; Neimert-Andersson, Theresa; Blank, Ulrich; Eriksson, Tove L J; van Hage, Marianne; Punnonen, Juha

    2007-02-09

    Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

  20. STM Images of Cytokeratin and Binding IgG Antibody

    NASA Astrophysics Data System (ADS)

    Vernetti, L. A.; Sarid, D.; Gandolfi, A. J.; Cress, A. E.; Nagle, R. B.; McCuskey, R.; Hameroff, S. R.

    1991-12-01

    In this paper we present scanning tunneling microscopy images of cytokeratin proteins and a monoclonal anti-cytokeratin IgG antibody bound to their carboxyl terminal end. The images are consistent with current models of cytokeratin assembly inferred from biochemical analysis, electron microscopy evaluation of disintegrating cytokeratin filaments, and chemical cross-linking of coiled-coils.

  1. Allergen-Specific Cytokine Polarization Protects Shetland Ponies against Culicoides obsoletus-Induced Insect Bite Hypersensitivity.

    PubMed

    Meulenbroeks, Chantal; van der Lugt, Jaco J; van der Meide, Nathalie M A; Willemse, Ton; Rutten, Victor P M G; Zaiss, Dietmar M W

    2015-01-01

    The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFNγ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFNγ-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder.

  2. Immune mechanisms of allergen-specific sublingual immunotherapy.

    PubMed

    Moingeon, P; Batard, T; Fadel, R; Frati, F; Sieber, J; Van Overtvelt, L

    2006-02-01

    Sublingual immunotherapy has been shown in some clinical studies to modulate allergen-specific antibody responses [with a decrease in the immunoglobulin E/immunoglobulin G4 (IgE/IgG4) ratio] and to reduce the recruitment and activation of proinflammatory cells in target mucosa. Whereas a central paradigm for successful immunotherapy has been to reorient the pattern of allergen-specific T-cell responses in atopic patients from a T helper (Th)2 to Th1 profile, there is currently a growing interest in eliciting regulatory T cells, capable of downregulating both Th1 and Th2 responses through the production of interleukin (IL)-10 and/or transforming growth factor (TGF)-beta. We discuss herein immune mechanisms involved during allergen-specific sublingual immunotherapy (SLIT), in comparison with subcutaneous immunotherapy. During SLIT, the allergen is captured within the oral mucosa by Langerhans-like dendritic cells expressing high-affinity IgE receptors, producing IL-10 and TGF-beta, and upregulating indoleamine dioxygenase (IDO), suggesting that such cells are prone to induce tolerance. The oral mucosa contains limited number of proinflammatory cells, such as mast cells, thereby explaining the well-established safety profile of SLIT. In this context, second-generation vaccines based on recombinant allergens in a native conformation formulated with adjuvants are designed to target Langerhans-like cells in the sublingual mucosa, with the aim to induce allergen-specific regulatory T cells. Importantly, such recombinant vaccines should facilitate the identification of biological markers of SLIT efficacy in humans.

  3. ANTIDOG IgG SECONDARY ANTIBODY SUCCESSFULLY DETECTS IgG IN A VARIETY OF AQUATIC MAMMALS.

    PubMed

    Roehl, Katherine; Jankowski, Mark; Hofmeister, Erik

    2016-12-01

    Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter ( Enhydra lutris ), polar bear ( Ursus maritimus ), grey seal ( Halichoerus grypus ), harbor seal ( Phoca vitulina ), northern elephant seal ( Mirounga angustirostris ), California sea lion ( Zalophus californianus ), Pacific walrus ( Odobenus rosmarus ) and one freshwater mammal: Asian small-clawed otter ( Aonyx cinerea ). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

  4. Hatchery workers' IgG antibody profiles to airborne bacteria.

    PubMed

    Brauner, Paul; Gromöller, Silvana; Pfeifer, Yvonne; Wilharm, Gottfried; Jäckel, Udo

    2017-04-01

    Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, etiology and in particular microorganisms' potential role in pathogenesis still needs to be elucidated. Thus, detection of specific antibodies against occupational microbial antigens may lead to identification of potentially harmful species. For the purpose of IgG titer determination, indirect immunofluorescence on various bacterial isolates from duck hatchery air was combined with image-based quantification of fluorescence intensity. Moreover, in addition to established assays with pure bacterial cultures, a new approach utilized complex bioaerosol samples for detection of anti-microbial antibodies in human sera by determination of percentages of antibody-bound cells in different serum dilutions. Mean titers in sera from hatchery workers and a non-exposed control group did not display significant differences for most tested isolates and application of comprehensive cluster analysis to entire titer data revealed no structure reflecting workers and controls group. Furthermore, determination of immunoreactivity to the complete microbial community in workplace air displayed similar proportions of antibody-bound cells in both groups. Although no general differences in immunoreaction patterns were observed, mean titers to a Proteus mirabilis isolate and to 3 of 4 distinct Acinetobacter baumannii isolates were higher in the group of hatchery workers than in the reference group indicating a potential applicability as exposure markers. We conclude, despite long term bioaerosol exposure, hatchery workers' IgG antibody profiles to tested antigens did not differ substantially from those of the control group. However, increased workers' titers to A. baumannii and clinical relevance of this species should lead to further investigations regarding potential involvement in pathogenesis of occupational respiratory disorders.

  5. Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

    PubMed

    Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje; Grevys, Algirdas; Sandlie, Inger; Michaelsen, Terje E; Hedegaard, Morten; Knudsen, Lisbeth E; Dziegiel, Morten Hanefeld

    2013-08-15

    The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.

  6. IgG subclasses of specific antibodies in Ixodes ricinus-borne borreliosis.

    PubMed Central

    Olsson, I; Hammarström, L; Smith, C I; Hovmark, A; Asbrink, E

    1987-01-01

    Ixodes ricinus-borne borreliosis may run a protracted course. In this study we investigated the different IgG subclasses of antibodies to borreliae at different stages of the disease. In addition to the dominant subclass IgG1 and IgG3 response was found in most cases. This antibody subclass pattern with contributions of IgG2 often persists into the late stage of the disease and may last for decades. The IgG subclass response elicited by this spirochaetosis does not conform to the expected IgG4 restricted response after chronic antigenic stimulation. PMID:3665187

  7. Lung-derived innate cytokines: new epigenetic targets of allergen-specific sublingual immunotherapy

    PubMed Central

    Pishdadian, Abbas; Varasteh, Abdolreza; Gholamin, Mehran; Nasiraie, Leila Roozbeh; Hosseinpour, Mitra; Moghadam, Malihe; Sankian, Mojtaba

    2016-01-01

    Objective(s): Sublingual allergen-specific immunotherapy is a safe and effective method for treatment of IgE-mediated respiratory allergies; however, the underlying mechanisms are not fully understood. This study was planned to test whether sublingual immunotherapy (SLIT) can exert epigenetic mechanisms through which the airway allergic responses can be extinguished. Materials and Methods: BALB/c mice were sensitized intraperitoneally and challenged intranasally. Then, they received sublingual treatment with recombinant Che a 2 (rChe a 2), a major allergen of Chenopodium album. After SLIT, allergen-specific antibodies in sera, cytokine profiles of spleen cell cultures, mRNA and protein expression of lung-derived IL-33, IL-25, and TSLP (thymic stromal lymphopoietin), and histone modifications of these three genes were assessed. Results: Following Immunotherapy, systemic immune responses shifted from Th2 to Th1 profile as demonstrated by significant decrease in IgE and IL-4 and substantial increase in IgG2a and IFN-γ. At local site, mRNA and protein levels of lung-derived pro-inflammatory cytokines IL-33 and TSLP were markedly down-regulated following SLIT that was associated with marked enrichment of trimethylated lysine 27 of histone H3 at promoter regions of these two cytokines. Conclusion: In our study, sublingual immunotherapy with recombinant allergen effectively attenuated allergic immune responses, at least partly, by induction of distinct histone modifications at specific loci. Additionally, the lung-derived pro-allergic cytokines IL-33 and TSLP could be promising mucosal candidates for either monitoring allergic conditions or therapeutic approaches. PMID:27096066

  8. Study of IgG sub-class antibodies in patients with milk intolerance.

    PubMed

    Shakib, F; Brown, H M; Phelps, A; Redhead, R

    1986-09-01

    An ELISA was applied to measure IgG sub-class antibodies to cow's milk beta-lactoglobulin (BLG), alpha-lactalbumin (ALA) and alpha-casein (AC) and to hen's egg ovalbumin (OA) in the sera of nineteen adult patients with milk intolerance causing either asthma, eczema or both. Results were compared with those of forty blood donors and twenty adult patients with either asthma or eczema due to inhalant allergy. Apart from one blood donor, high titres of IgG sub-class antibodies to all three milk proteins were found only in the milk intolerance group. The most frequently detected antibody was AC-specific IgG4; being high (i.e. greater than 9.98 micrograms/ml) in eight milk intolerance cases: six with eczema, one with asthma and one with both. A variable proportion of these eight patients also had high levels of IgG1, IgG2 and IgG3 antibodies to AC and IgG1, IgG2, IgG3 and IgG4 antibodies to BLG and ALA. In contrast, IgG antibody to the egg protein, OA, was remarkably restricted to IgG4 and was present in high titres in 68.4% of milk intolerant patients, 60% of inhalant allergy patients and 30% of blood donors. However, the greater incidence of high titres of IgG4 antibody to OA, compared to AC, was due to the superior coating efficiency of OA resulting in a more sensitive assay. We conclude that some adult cases of milk intolerance, particularly those with eczema, can be diagnosed by detecting raised serum levels of IgG sub-class antibodies to milk proteins.

  9. [Improved IgG Antibody Diagnostics of Feather Duvet Lung by an Antibody Screening Test].

    PubMed

    Sennekamp, J; Lehmann, E

    2015-11-01

    The underdiagnosed feather duvet lung, an extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by duck and goose feathers, can be more frequently diagnosed, if duck and goose feather antibodies are included in the panel of the routinely applied IgG antibody screening test. This does not necessarily require extending the screening test to include duck and goose feather antigens. By analysing 100 sera with duck and goose antibodies we found that the commonly used pigeon and budgerigar antibodies can also screen for feather duvet antibodies. All examined sera lacking pigeon and budgerigar antibodies also lacked clear-cut duck and goose feather antibodies. The examined sera with strong pigeon or budgerigar antibodies always also contained feather duvet antibodies. However, sera with medium or low concentrated pigeon or budgerigar antibodies are not always associated with feather duvet antibodies. In the light of these observations, we find that 71% of the duck and goose antibody analyses would be dispensable without essential loss of quality, if the results of screening for pigeon and budgerigar antibodies were incorporated into the procedure of a step-by- step diagnostics.

  10. “Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation

    PubMed Central

    Chan, Yih-Chih; Ramadani, Faruk; Santos, Alexandra F.; Pillai, Prathap; Ohm-Laursen, Line; Harper, Clare E.; Fang, Cailong; Dodev, Tihomir S.; Wu, Shih-Ying; Ying, Sun; Corrigan, Christopher J.; Gould, Hannah J.

    2014-01-01

    Background Naturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood. Objective Address the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation. Methods IgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI. Results IgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen. Conclusion Naturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma. PMID:25112697

  11. Feeding neonatal rats with IgG antibodies leads to humoral hyporesponsiveness in the adult.

    PubMed Central

    Peppard, J V

    1992-01-01

    Feeding monoclonal IgG2a or IgG1 anti-horseradish peroxidase (HRP) antibodies to 12-16-day-old neonatal rats caused a profound suppression of the humoral anti-HRP response in these rats as adults. The hyporesponsiveness to HRP was specific and long-lasting (up to 5 months). It was shown to be dose dependent, requiring relatively large doses of IgG (100-600 micrograms) for maximum effect. Secondary IgG (IgG1, IgG2a and IgG2b) responses were most depressed. The effect could be reproduced by i.p. injection of antibody. Hyporesponsiveness was not attributable to circulating antiidiotype antibodies directed against the monoclonal IgG, nor to the continued presence of the monoclonal anti-HRP since rats receiving antibody at or some weeks after the time of weaning and gut 'closure' responded well to subsequent HRP challenge. The effect was thus dependent on IgG administered over the identical period during which the neonatal circulation is rich in maternal IgG supplied via the milk. A direct function for maternal IgG in moulding the immune repertoire of the offspring, as well as providing passive protection, is suggested by these results. PMID:1385314

  12. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    PubMed

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  13. Serum and salivary IgE, IgA, and IgG4 antibodies to Dermatophagoides pteronyssinus and its major allergens, Der p1 and Der p2, in allergic and nonallergic children.

    PubMed

    Miranda, Diego O; Silva, Deise A O; Fernandes, Jorge F C; Queirós, Meimei G J; Chiba, Hamilton F; Ynoue, Leandro H; Resende, Rafael O; Pena, Janethe D O; Sung, Sun-Sang J; Segundo, Gesmar R S; Taketomi, Ernesto A

    2011-01-01

    Allergic rhinitis (AR) is a public health problem with high prevalence worldwide. We evaluated levels of specific IgE, IgA, and IgG4 antibodies to the Dermatophagoides pteronyssinus (Dpt) house dust mite and to its major allergens (Der p1 and Der p2) in serum and saliva samples from allergic and nonallergic children. A total of 86 children were analyzed, from which 72 had AR and 14 were nonallergic healthy children. Serum IgE and serum/salivary IgG4 levels to Dpt, Der p1, and Der p2 were higher in allergic children whereas serum/salivary IgA levels to all allergens were higher in nonallergic children. IgE levels positively correlated with IgG4 and IgA to all allergens in allergic children, while IgA levels negatively correlated with IgG4 to Dpt and Der p1 in nonallergic children. In conclusion, mite-specific IgA antibodies predominate in the serum and saliva of nonallergic children whereas mite-specific IgE and IgG4 are prevalent in allergic children. The presence of specific IgA appears to have a key role for the healthy immune response to mucosal allergens. Also, specific IgA measurements in serum and/or saliva may be useful for monitoring activation of tolerance-inducing mechanisms during allergen specific immunotherapeutic procedures, especially sublingual immunotherapy.

  14. IgG antibodies to Aspergillus fumigatus in cystic fibrosis: a laboratory correlate of disease activity.

    PubMed Central

    Forsyth, K D; Hohmann, A W; Martin, A J; Bradley, J

    1988-01-01

    Serum was collected from 50 patients with cystic fibrosis, and IgG antibodies to Aspergillus fumigatus were measured by enzyme linked immunosorbent assay (ELISA). In addition, total IgE and Aspergillus specific IgE antibodies were measured in 41 of the 50. A close association was found between pulmonary function and clinical state, and IgG antibodies to Aspergillus. There was no association between pulmonary function or clinical state and IgE antibodies. It is postulated that in patients with cystic fibrosis, Aspergillus fumigatus may contribute to deterioration in pulmonary function by local pathogenicity, or by hypersensitivity mechanisms mediated by IgG. PMID:3046514

  15. IgG antinuclear antibodies with cross-reactive rheumatoid factor activity.

    PubMed

    Darwin, B S; Grudier, J P; Klatt, C L; Pisetsky, D S

    1986-12-15

    To investigate whether IgG antinuclear antibodies have cross-reactive rheumatoid factor activity, monoclonal IgG antibodies to DNA and Sm from autoimmune MRL-lpr/lpr mice were assayed by ELISA for binding to IgG antigens. Of the nine anti-DNA and anti-Sm monoclonals tested, six showed significant binding to affinity-purified rabbit IgG (RIgG) and human IgG (HIgG). To confirm that cross-reactivities were due to a single antibody, immunoabsorption of a representative polyspecific monoclonal termed C11 (anti-DNA, anti-Sm) on either Sepharose-DNA or Sepharose-RIgG resulted in marked loss of activity to the three antigens DNA, Sm and RIgG compared with immunoabsorption on Sepharose-bovine serum albumin. The monomolecular nature of the cross-reacting antibody was also suggested by inhibition analysis of C11; DNA inhibited C11 binding to RIgG 64%, whereas Sm inhibited binding to RIgG 33%. Aggregated RIgG and HIgG, however, did not inhibit binding of C11 to DNA, Sm, or solid-phase RIgG, probably reflecting the low affinity of this antibody for fluid phase Ig. Together, these findings suggest that antinuclear autoantibodies of the IgG, as well as the IgM, class have polyspecific IgG binding activity and suggest that IgG antinuclear antibodies may emerge from rheumatoid factor responses.

  16. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

    PubMed

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

    2015-03-20

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.

  17. Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

    PubMed Central

    Yine, Hu; Shufang, Dai; Bin, Wang; Wei, Qu; Ashraf, Muhammad Aqeel; Junling, Gao

    2015-01-01

    The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p<0.05) while there was no significant difference between children and adults among eczema patients with positive antibody (p>0.05). Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  18. IgG4 subclass antibodies impair antitumor immunity in melanoma

    PubMed Central

    Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

    2013-01-01

    Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

  19. IgG4 subclass antibodies impair antitumor immunity in melanoma.

    PubMed

    Karagiannis, Panagiotis; Gilbert, Amy E; Josephs, Debra H; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L C; Healy, Ciaran; Harries, Mark; Acland, Katharine M; Blower, Philip J; Mitchell, Tracey; Fear, David J; Spicer, James F; Lacy, Katie E; Nestle, Frank O; Karagiannis, Sophia N

    2013-04-01

    Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

  20. Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain.

    PubMed

    Dimasi, Nazzareno; Fleming, Ryan; Sachsenmeier, Kris F; Bezabeh, Binyam; Hay, Carl; Wu, Jincheng; Sult, Erin; Rajan, Saravanan; Zhuang, Li; Cariuk, Peter; Buchanan, Andrew; Bowen, Michael A; Wu, Herren; Gao, Changshou

    2017-04-01

    We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.

  1. Total and specific IgG4 antibody levels in atopic eczema.

    PubMed Central

    Merrett, J; Barnetson, R S; Burr, M L; Merrett, T G

    1984-01-01

    Total IgG4 and IgG4 antibody levels specific for 10 allergens (three inhaled and seven ingested) were measured by radioimmunoassay of sera taken from three groups of adult patients: (1) 32 cases of atopic eczema, (2) 28 cases of respiratory allergy and (3) 156 normal volunteers. In all three groups IgG4 antibody activity was mainly directed against common foods, and generally the group with atopic eczema had higher total and specific IgG4 levels than the cases of respiratory allergy, who in turn had higher titres than the normal group. There was within each group a tendency for men to have more total IgG4 than women and the difference was statistically significant among the normals. There was evidence of an IgG4 restricted response in atopic eczema because despite the group's elevated total IgG4 its total IgG4 remained within normal limits. Furthermore specific IgG4 was correlated with the corresponding specific IgE level in five of the 10 allergens examined. These results are generally consistent with the view that IgG4 levels are raised in cases of atopic eczema due to prolonged exposure to an allergen which initiated an IgE response. PMID:6744664

  2. In vitro and in vivo modifications of recombinant and human IgG antibodies.

    PubMed

    Liu, Hongcheng; Ponniah, Gomathinayagam; Zhang, Hui-Min; Nowak, Christine; Neill, Alyssa; Gonzalez-Lopez, Nidia; Patel, Rekha; Cheng, Guilong; Kita, Adriana Z; Andrien, Bruce

    2014-01-01

    Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.

  3. In vitro and in vivo modifications of recombinant and human IgG antibodies

    PubMed Central

    Liu, Hongcheng; Ponniah, Gomathinayagam; Zhang, Hui-Min; Nowak, Christine; Neill, Alyssa; Gonzalez-Lopez, Nidia; Patel, Rekha; Cheng, Guilong; Kita, Adriana Z; Andrien, Bruce

    2014-01-01

    Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules. PMID:25517300

  4. IgG and IgG subclass specific antibody responses to diphtheria and tetanus toxoids in newborns and infants given DTP immunization.

    PubMed

    Dengrove, J; Lee, E J; Heiner, D C; St Geme, J W; Leake, R; Baraff, L J; Ward, J I

    1986-08-01

    To evaluate immune responses to diphtheria and tetanus toxoids in infants we used enzyme-linked immunosorbent assays to detect total IgG and specific IgG-1, IgG-2, IgG-3, and IgG-4 antibody. One group of infants received a newborn dose and subsequently received the usual three doses of DTP. A second group of infants received only the routine dosage at 2, 4, and 6 months of age. In sera acquired at birth, 6, and 9 months of age, there were no statistically significant differences between the two vaccine groups in IgG antibody responses to diphtheria or tetanus, or in IgG subclass tetanus-specific antibody responses. In individual children, tetanus-specific subclass responses were similar in pattern to that for total IgG tetanus antibody, i.e. each IgG subclass response appeared to be regulated by similar mechanisms in that child, but the regulation differed between children. In contrast to a prior study of pertussis immunity, maternally acquired antibody did not significantly affect immune responses to diphtheria or tetanus toxoid by 9 months of age. There was no discernible tolerance due to early tetanus or diphtheria immunization or to high levels of maternally acquired antibody.

  5. Serum IgG subclass antibodies to gliadin and other dietary antigens in children with coeliac disease.

    PubMed Central

    Husby, S; Foged, N; Oxelius, V A; Svehag, S E

    1986-01-01

    IgG subclasses of antibodies to the dietary antigens gliadin, glycgli (a gluten component), ovalbumin (OA) and beta-lactoglobulin (BLG) were quantified in children with coeliac disease (CD), nine on a gluten-containing diet, 15 on a gluten-free diet, and in appropriate controls. In addition, total serum IgG subclasses were measured. IgG1 and IgG3 antibodies to gluten and glycgli were detected in 9/9 and 8/9 CD-patient on a gluten-containing diet, respectively, and in 4/15 and 6/15 patients on a gluten-free diet. None of the controls had appreciable levels of IgG1 antibodies and only 1/22 of the controls had IgG3 antibodies to gliadin and glycgli. IgG2 and IgG4 antibodies to the same antigens were found in a few coeliacs and controls. Consecutive samples from coeliac children (8 patients) showed a clear relation between the exposure to gluten and a rise in IgG1 (8/8) and IgG3 antibody levels (7/8). In contrast, IgG antibodies to OA and BLG were almost exclusively of the IgG1 and IgG4 subclasses. The highest levels were found in children with CD, but the differences between the groups were not significant. Total serum IgG subclasses did not differ between the groups, but the IgG2 and IgG4 levels in most coeliac children were low. The production of IgG1 and IgG3 antibodies to gluten components may be an important precondition for the development of coeliac disease in susceptible individuals. PMID:3791689

  6. Development time of IgG antibodies to West Nile virus.

    PubMed

    Papa, Anna; Danis, Kostas; Tsergouli, Katerina; Tsioka, Katerina; Gavana, Elpida

    2011-09-01

    Following an outbreak of West Nile virus (WNV) infections in Greece during summer/autumn 2010, a study was conducted to investigate the patterns of WNV IgG reactivity in 255 patients with respect to the day of illness and the type of clinical syndrome. IgG antibodies were detectable after a mean of 8.1 ± 4.9 and 12.6 ± 11.3 days after onset of illness in neuroinvasive and non-neuroinvasive cases, respectively (p < 0.001), suggesting that a delay in the development of WNV IgG antibodies is seen in non-neuroinvasive cases.

  7. Global Structure of HIV-1 Neutralizing Antibody IgG1 b12 is Asymmetric

    SciTech Connect

    Ashish, F.; Solanki, A; Boone, C; Krueger, J

    2010-01-01

    Human antibody IgG1 b12 is one of the four antibodies known to neutralize a broad range of human immunodeficiency virus-1. The crystal structure of this antibody displayed an asymmetric disposition of the Fab arms relative to its Fc portion. Comparison of structures solved for other IgG1 antibodies led to a notion that crystal packing forces entrapped a 'snap-shot' of different conformations accessible to this antibody. To elucidate global structure of this unique antibody, we acquired small-angle X-ray scattering data from its dilute solution. Data analysis indicated that b12 adopts a bilobal globular structure in solution with a radius of gyration and a maximum linear dimension of {approx}54 and {approx}180 {angstrom}, respectively. Extreme similarity between its solution and crystal structure concludes that non-flexible, asymmetric shape is an inherent property of this rare antibody.

  8. Age related variations of serum concentrations of normally occurring IgG antibodies to Clostridium perfringens.

    PubMed Central

    Zarén, E; Schwan, A; Frenckner, B

    1987-01-01

    In studies using indirect immunofluorescence IgG antibodies to Clostridium perfringens were found in sera from healthy adults. Sera from 236 healthy children were examined. The normally occurring IgG antibodies to C perfringens were found to have an age related variation. Preliminary data suggest that they are not correlated to C perfringens alpha toxin. The antigen(s) against which the antibodies are directed is/are probably part of the cell wall, but its/their exact nature is not known. PMID:2881950

  9. Reverse single radial immunodiffusion for estimation of titre of anti IgG antibody.

    PubMed

    Thakar, Y S; Kulkarni, C; Pande, S; Dhanvijay, A G; Shrikhande, A V; Saoji, A M

    1993-05-01

    Reverse Single Radial Immunodiffusion (SRID) for estimating titre of anti IgG antisera is reported. Unlike the conventional radial immunodiffusion, the antigen (IgG) is held immobile in the gel while the antibody (Anti IgG) diffuses radially from the well (7 microliters) and the diameter of the resulting immuneprecipitates after immunodiffusion at 4 degrees C for 24 hr, represents a linear correlation with the antibody titre. The procedure was standardised by an extensive trial and error employing different concentrations of human IgG in the gel (60-240 micrograms) against varying dilutions of the standard antibody (titre: 3.8 mg/ml). The best results were obtained at 80 micrograms of IgG in the gel. The locally raised rabbit anti IgG antisera displayed a distinctive titre pattern under optimised conditions. Technical reproducibility, high-sensitivity threshold (0.25 mg/ml), simultaneous visual scrutiny of several antibody batches at a glance and ability to assess the shelf life of the stored antisera are its distinct assets.

  10. Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization.

    PubMed

    Phillips, M L; Tao, M H; Morrison, S L; Schumaker, V N

    1994-10-01

    The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).

  11. Seroprevalences of Specific IgG Antibodies to Measles, Mumps, and Rubella in Korean Infants.

    PubMed

    Cho, Hye Kyung; Lee, Hyunju; Kim, Han Wool; Kim, Sung Soon; Kang, Hae Ji; Kim, In Tae; Kim, Kyung Hyo

    2016-12-01

    In this study, the seroprevalences of measles, mumps, and rubella antibodies in infants were determined to assess the immunization strategy and control measures for these infectious diseases. Serum samples from infants < 1 year of age and their mothers were collected to measure the concentrations of specific IgG antibodies to measles, mumps, and rubella by enzyme-linked immunosorbent assay. For selected infant serum samples, measles-specific neutralizing antibody levels were determined by using the plaque reduction neutralization test. The sera from 295 of infants and 80 of their mothers were analyzed. No infants had past measles, mumps, or rubella infections. Almost all infants < 2 months of age were positive for measles and rubella IgG antibodies. However, seroprevalence of measles and rubella antibodies decreased with age, and measles IgG and rubella IgG were barely detectable after 4 months of age. The seroprevalence of mumps antibodies was lower than that of measles and rubella antibodies in infants ≤ 4 months old, and mumps IgG was barely detectable after 2 months of age. The seropositivity of measles-specific neutralizing antibody was 63.6% in infants aged 2 months and undetectable in infants ≥ 6 months old. Because the seropositivity rates of measles, mumps, and rubella antibodies were low after the first few months of age in Korean infants, active immunization with vaccines is strongly recommended for infants aged 6-11 months when measles is epidemic. Timely administration of the first dose of measles-mumps-rubella vaccine at 12 months of age should be encouraged in non-epidemic situations.

  12. Oxidative Stress and IgG Antibody Modify Periodontitis-CRP Association.

    PubMed

    Singer, R E; Moss, K; Kim, S J; Beck, J D; Offenbacher, S

    2015-12-01

    In a previous report, we demonstrated the inverse association of high serum 8-isoprostane levels, a marker for oxidative stress, with decreased serum IgG antibodies to oral bacteria. The association between increased serum IgG with increased plaque and periodontitis (increased probing depths) was attenuated by high systemic oxidative stress. Other investigations have reported a role for systemic oxidative stress as a stimulus of hepatic C-reactive protein (CRP) response. These observations led us to hypothesize that the reported relationship of periodontitis to elevated serum CRP, a systemic inflammatory marker, may be modified by oxidative stress and that the levels of serum antibodies to oral bacteria might be an intermediary explanatory variable linking the association of systemic oxidative stress, periodontal disease, and levels of CRP. This hypothesis was explored as a secondary analysis of the Dental ARIC (Atherosclerosis Risk in Communities) study using serum levels of CRP, serum IgG levels to 16 oral organisms, serum levels of 8-isoprostane, and periodontal status. The findings indicate periodontitis is associated with high CRP in the presence of elevated oxidative stress that serves to suppress the IgG response. Only within the highest 8-isoprostane quartile was periodontitis (pocket depth) associated with increased serum CRP levels (P = 0.0003). Increased serum IgG antibody levels to oral bacteria were associated with lowered serum CRP levels. Thus, systemic oxidative stress, which has been demonstrated to be associated with increased levels of CRP in other studies, appears to be associated with the suppression of bacterial-specific IgG levels, which in the presence of periodontal disease can result in an enhanced systemic CRP response. Conversely, individuals with increased serum IgG antibodies to plaque bacteria exhibit lowered serum CRP levels. These 2 factors, oxidative stress and the serum IgG response, appear to function in opposing directions to

  13. Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

    PubMed

    Edgeworth, Matthew J; Phillips, Jonathan J; Lowe, David C; Kippen, Alistair D; Higazi, Daniel R; Scrivens, James H

    2015-12-07

    Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

  14. IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies.

    PubMed

    Bhat, Neelima M; Kshirsagar, Mihir A; Bieber, Marcia M; Teng, Nelson N H

    2015-01-01

    VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/dysfunction. One of the autoantigens recognized by VH4-34-encoded antibodies are branched/linear poly N-acetyl lactosamine chains. Since the anti-carbohydrate response in humans is dominated by the IgG2 subclass, here we tested whether VH4-34 encoded IgG showed similar subclass segregation. Serum samples from SLE, infectious mononucleosis, nasopharyngeal carcinoma and hepatitis-C were analyzed. Levels of VH4-34-encoded IgM and IgA isotypes were also tested. VH4-34-IgM and IgA were elevated in all four clinical conditions. VH4-34-IgG was detected in the IgG1 and IgG3 subclass but not in the IgG2 and IgG4 subclass. Interestingly, VH4-34-IgG3 was also detected in serum samples of normal healthy adults. These observations are discussed in context of the VH4-34 gene regulation. VH4-34 repertoire development is of interest since it is the only human VH gene profoundly overrepresented in the naïve repertoire but counter-selected for antibody secretion. VH4-34 B-cell could thus become a unique tool to inspect germinal center independent/dependent pathways of subclass and isotype-specific antibody secretion.

  15. Comparison of four commercially available avidity tests for Toxoplasma gondii-specific IgG antibodies.

    PubMed

    Villard, O; Breit, L; Cimon, B; Franck, J; Fricker-Hidalgo, H; Godineau, N; Houze, S; Paris, L; Pelloux, H; Villena, I; Candolfi, E

    2013-02-01

    Toxoplasma infection in pregnant women may cause congenital toxoplasmosis. Diagnosis of infection is based on serological tests aimed at detecting IgM and IgG antibodies against Toxoplasma gondii. However, IgM antibodies are not an accurate marker for discriminating between acute and latent infection. Detection of residual or persistent IgM may occur months or even years after primary infection, while the IgG avidity test is a rapid means of identifying latent infections in pregnant women who exhibit both IgG and IgM anti-Toxoplasma antibodies on initial testing during pregnancy. In this study, we assessed and compared the performances of four commercially available Toxoplasma IgG avidity tests in immunocompetent and immunocompromised patients with acute and latent toxoplasmosis. The positive predictive value of high avidity to confirm latent toxoplasmosis was 100% for all the assays, indicating that high avidity is a hallmark of latent infection. However, the negative predictive value of high avidity ranged from 99.2% (bioMérieux) to 95.3% (Abbott), indicating that acute toxoplasmosis could not be reliably diagnosed based on low IgG avidity alone. Thus, the avidity test provides a rapid means for identifying latent Toxoplasma infection in immunocompetent pregnant women presenting both IgG and IgM anti-Toxoplasma antibodies on initial testing. In terms of cost-effectiveness, avidity testing is a powerful tool that optimizes screening and follow-up of pregnant women while minimizing the costs of screening by avoiding subsequent costly maternal and fetal investigation and unnecessary treatment. The cheapest assay, Vidas Toxo IgG Avidity, also had the best performance for the diagnosis of latent toxoplasmosis.

  16. Comparison of Four Commercially Available Avidity Tests for Toxoplasma gondii-Specific IgG Antibodies

    PubMed Central

    Breit, L.; Cimon, B.; Franck, J.; Fricker-Hidalgo, H.; Godineau, N.; Houze, S.; Paris, L.; Pelloux, H.; Villena, I.

    2013-01-01

    Toxoplasma infection in pregnant women may cause congenital toxoplasmosis. Diagnosis of infection is based on serological tests aimed at detecting IgM and IgG antibodies against Toxoplasma gondii. However, IgM antibodies are not an accurate marker for discriminating between acute and latent infection. Detection of residual or persistent IgM may occur months or even years after primary infection, while the IgG avidity test is a rapid means of identifying latent infections in pregnant women who exhibit both IgG and IgM anti-Toxoplasma antibodies on initial testing during pregnancy. In this study, we assessed and compared the performances of four commercially available Toxoplasma IgG avidity tests in immunocompetent and immunocompromised patients with acute and latent toxoplasmosis. The positive predictive value of high avidity to confirm latent toxoplasmosis was 100% for all the assays, indicating that high avidity is a hallmark of latent infection. However, the negative predictive value of high avidity ranged from 99.2% (bioMérieux) to 95.3% (Abbott), indicating that acute toxoplasmosis could not be reliably diagnosed based on low IgG avidity alone. Thus, the avidity test provides a rapid means for identifying latent Toxoplasma infection in immunocompetent pregnant women presenting both IgG and IgM anti-Toxoplasma antibodies on initial testing. In terms of cost-effectiveness, avidity testing is a powerful tool that optimizes screening and follow-up of pregnant women while minimizing the costs of screening by avoiding subsequent costly maternal and fetal investigation and unnecessary treatment. The cheapest assay, Vidas Toxo IgG Avidity, also had the best performance for the diagnosis of latent toxoplasmosis. PMID:23239801

  17. Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS.

    PubMed

    Nascimento, Fernanda S; Suzuki, Lisandra A; Branco, Nilson; Franco, Regina M B; Andrade, Paula D; Costa, Sandra C B; Pedro, Marcelo N; Rossi, Cláudio L

    2015-01-01

    Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

  18. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    DTIC Science & Technology

    2006-04-01

    pentavalent toxoid; Fab, antibody fragment; HRP, horseradish peroxidase; LCκ, kappa light chain ; scFv, single- chain antibody fragments; VL, variable light ...The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified...from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated

  19. Does allergen-specific immunotherapy induce contact allergy to aluminium?

    PubMed

    Netterlid, Eva; Hindsén, Monica; Siemund, Ingrid; Björk, Jonas; Werner, Sonja; Jacobsson, Helene; Güner, Nuray; Bruze, Magnus

    2013-01-01

    Persistent, itching nodules have been reported to appear at the injection site after allergen-specific immuno-therapy with aluminium-precipitated antigen extract, occasionally in conjunction with contact allergy to aluminium. This study aimed to quantify the development of contact allergy to aluminium during allergen-specific immunotherapy. A randomized, controlled, single-blind multicentre study of children and adults entering allergen-specific immunotherapy was performed using questionnaires and patch-testing. A total of 205 individuals completed the study. In the 3 study groups all subjects tested negative to aluminium before allergen-specific immunotherapy and 4 tested positive after therapy. In the control group 4 participants tested positive to aluminium. Six out of 8 who tested positive also had atopic dermatitis. Positive test results were found in 5/78 children and 3/127 adults. Allergen-specific immunotherapy was not shown to be a risk factor for contact allergy to aluminium. Among those who did develop aluminium allergy, children and those with atopic dermatitis were more highly represented.

  20. Human IgG1 antibodies suppress angiogenesis in a target-independent manner

    PubMed Central

    Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

    2016-01-01

    Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies. PMID:26918197

  1. Neutralizing antibodies in mucosal secretions: IgG or IgA?

    PubMed

    Alexander, Rashada; Mestecky, Jiri

    2007-11-01

    The mucosal immune response to HIV weighs in heavily on the battle against it, as the majority of infections occur via the mucosal route. The antibody response in the mucosae, specifically the genital tract, is characterized by binding and, in some studies, neutralizing HIV-specific IgG and IgA antibodies. Ample evidence, however, points to discrepancies and difficulties in the detection of HIV-specific IgA in HIV-positive subjects, and an even more pronounced divide surfaces in studies done with individuals exposed to HIV, but uninfected. Reports in the literature detail HIV-specific (in some cases, neutralizing) IgA antibodies, in the absence of specific IgG, in the serum and mucosal secretions of virus-exposed, seronegative subjects; this has given rise to speculation that HIV-specific IgA provides a protective immune response to the virus in high-risk individuals who remain seronegative. Contradictory results, however, describe the absence of both IgA and IgG HIV antibodies in the mucosal secretions of similar cohorts. Considering the importance of the antibody response to ascertaining the correlates of HIV immunity, as well as on vaccine research and development, this review addresses the relevant studies and their implications.

  2. Multilevel ensemble model for prediction of IgA and IgG antibodies.

    PubMed

    Khanna, Divya; Rana, Prashant Singh

    2017-04-01

    Identification of antigen for inducing specific class of antibody is prime objective in peptide based vaccine designs, immunodiagnosis, and antibody productions. It's urge to introduce a reliable system with high accuracy and efficiency for prediction. In the present study, a novel multilevel ensemble model is developed for prediction of antibodies IgG and IgA. Epitope length is important in training the model and it is efficient to use variable length of epitopes. In this ensemble approach, seven different machine learning models are combined to predict variable length of epitopes (4 to 50). The proposed model of IgG specific epitopes achieves 94.43% of accuracy and IgA specific epitopes achieves 97.56% of accuracy with repeated 10-fold cross validation. The proposed model is compared with the existing system i.e. IgPred model and outcome of proposed model is improved.

  3. Competition between Serum IgG, IgM, and IgA Anti-Glycan Antibodies

    PubMed Central

    Muthana, Saddam M.; Xia, Li; Campbell, Christopher T.; Zhang, Yalong; Gildersleeve, Jeffrey C.

    2015-01-01

    Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies. PMID:25807519

  4. IgG antibody response to toxins A and B in patients with Clostridium difficile infection.

    PubMed

    Wullt, M; Norén, T; Ljungh, A; Åkerlund, T

    2012-09-01

    IgG antibodies against Clostridium difficile toxins A and B were followed in controls and in patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies.

  5. Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

    PubMed

    Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-10-20

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

  6. Allergen-Specific Immunotherapy in Food Anaphylaxis

    PubMed Central

    2008-01-01

    Specific immunotherapy (SIT) protocols for nutritional allergens have only recently been established with a focus on oral allergy syndrome because of pollen cross-reacting antibodies. For these patients, a substantial number of studies have been published suggesting benefits from SIT. The situation in true anaphylaxis to food allergens such as peanut allergy is more complex, and therapeutic strategies are based on individual protocols rather than controlled studies. However, in defined cases, SIT represents a promising approach for a durable protection from life-threatening risks after accidental ingestion. PMID:23283385

  7. Regulation of secondary antibody responses in rodents. I. Potentiation of IgG production by cyclophosphamide.

    PubMed Central

    Gagnon, R F; MacLennan, I C

    1979-01-01

    This paper describes the effects of a single dose of cyclophosphamide on specific IgG production in rats during an established secondary immune response. (PVG X Agus)F1 rats were immunized twice (days 0 and 28) with chicken erythrocytes (CRBC), received cyclophosphamide (100 mg/m2 of body surface area) on day 33 and were killed 8 days later. The production of anti-CRBC IgG antibodies was assessed by testing the supernatants of spleen cell cultures in a cytotoxicity assay with 51Cr-labelled CRBC as target cells and normal rat spleen cells as effector cells. In observations of fifty-nine pairs of treated and untreated rats from eight separate experiments, the administration of cyclophosphamide resulted in: (1) a decrease in the number of spleen cell to a median of 10(8.63) from a median of 10(8.7) (P less than 0.0025); (2) an increase in the anti-CRBC IgG antibody titre of the supernatants of cultured spleen cells to a median of 10(0.67) from a median of 10(0.27 (P less than 0.0025); and (3) the calculated anti-CRBC. IgG antibody production per spleen to be increased in the drug-treated rats to a median of 10(2.26) from a median of 10(2.0) (P less than 0.005). In a cyclophosphamide dose-response study, it was shown that some enhancement of antibody production was induced by doses between 12.5 and 50 mg/m2 and consistently elevated levels of antibody production were associated with doses between 100 and 400 mg/m2. PMID:487659

  8. IgG1 antibodies to acetylcholine receptors in ‘seronegative’ myasthenia gravis†

    PubMed Central

    Leite, Maria Isabel; Jacob, Saiju; Viegas, Stuart; Cossins, Judy; Clover, Linda; Morgan, B. Paul; Beeson, David; Willcox, Nick

    2008-01-01

    Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients’ thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new

  9. Extraction of monoclonal antibodies (IgG1) using anionic and anionic/nonionic reverse micelles.

    PubMed

    George, Daliya A; Stuckey, David C

    2010-01-01

    Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels and many current research initiatives are focused on finding alternatives to chromatography for the purification of Monoclonal antibodies (MAbs). In this article, we have investigated an alternative separation technique based on liquid-liquid extraction called the reverse micellar extraction. We extracted MAb (IgG1) using reverse micelles of an anionic surfactant, sodium bis 2-ethyl-hexyl sulfosuccinate (AOT) and a combination of anionic (AOT) and nonionic surfactants (Brij-30, Tween-85, Span-85) using isooctane as the solvent system. The extraction efficiency of IgG1 was studied by varying parameters, such as pH of the aqueous phase, cation concentration, and type and surfactant concentration. Using the AOT/Isooctane reverse micellar system, we could achieve good overall extraction of IgG1 (between 80 and 90%), but only 30% of the bioactivity of IgG1 could be recovered at the end of the extraction by using its binding to affinity chromatography columns as a surrogate measure of activity. As anionic surfactants were suspected as being one of the reasons for the reduced activity, we decided to combine a nonionic surfactant with an anionic surfactant and then study its effect on the extraction efficiency and bioactivity. The best results were obtained using an AOT/Brij-30/Isooctane reverse micellar system, which gave an overall extraction above 90 and 59% overall activity recovery. An AOT/Tween-85/Isooctane reverse micellar system gave an overall extraction of between 75 and 80% and overall activity recovery of around 40-45%. The results showed that the activity recovery of IgG1 can be significantly enhanced using different surfactant combination systems, and if the recovery of IgG1 can be further enhanced, the technique shows considerable promise for the downstream purification of MAbs.

  10. Plant-based Production of Two Chimeric Monoclonal IgG Antibodies Directed against Immunodominant Epitopes of Vibrio cholerae Lipopolysaccharide

    PubMed Central

    Levinson, Kara J.; Giffen, Samantha R.; Pauly, Michael H.; Kim, Do H.; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J.; Zeitlin, Larry; Mantis, Nicholas J.

    2015-01-01

    We have produced and characterized two chimeric IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest. PMID:25865265

  11. Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide.

    PubMed

    Levinson, Kara J; Giffen, Samantha R; Pauly, Michael H; Kim, Do H; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J; Zeitlin, Larry; Mantis, Nicholas J

    2015-07-01

    We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.

  12. Effects of Nasal Corticosteroids on Boosts of Systemic Allergen-Specific IgE Production Induced by Nasal Allergen Exposure

    PubMed Central

    Egger, Cornelia; Lupinek, Christian; Ristl, Robin; Lemell, Patrick; Horak, Friedrich; Zieglmayer, Petra; Spitzauer, Susanne; Valenta, Rudolf; Niederberger, Verena

    2015-01-01

    Background Allergen exposure via the respiratory tract and in particular via the nasal mucosa boosts systemic allergen-specific IgE production. Intranasal corticosteroids (INCS) represent a first line treatment of allergic rhinitis but their effects on this boost of allergen-specific IgE production are unclear. Aim Here we aimed to determine in a double-blind, placebo-controlled study whether therapeutic doses of an INCS preparation, i.e., nasal fluticasone propionate, have effects on boosts of allergen-specific IgE following nasal allergen exposure. Methods Subjects (n = 48) suffering from grass and birch pollen allergy were treated with daily fluticasone propionate or placebo nasal spray for four weeks. After two weeks of treatment, subjects underwent nasal provocation with either birch pollen allergen Bet v 1 or grass pollen allergen Phl p 5. Bet v 1 and Phl p 5-specific IgE, IgG1–4, IgM and IgA levels were measured in serum samples obtained at the time of provocation and one, two, four, six and eight weeks thereafter. Results Nasal allergen provocation induced a median increase to 141.1% of serum IgE levels to allergens used for provocation but not to control allergens 4 weeks after provocation. There were no significant differences regarding the boosts of allergen-specific IgE between INCS- and placebo-treated subjects. Conclusion In conclusion, the application of fluticasone propionate had no significant effects on the boosts of systemic allergen-specific IgE production following nasal allergen exposure. Trial Registration http://clinicaltrials.gov/ NCT00755066 PMID:25705889

  13. Seed-based oral vaccines as allergen-specific immunotherapies.

    PubMed

    Takaiwa, Fumio

    2011-03-01

    Plant-based vaccines have advantages over conventional vaccines in terms of scalability, lack of requirement for cold chain logistics, stability, safety, cost-effectiveness and needle-free administration. In particular, when antigen is expressed in seeds, high production is possible and immunogenicity is not lost even if stocked at ambient temperature for several years. Induction of immune tolerance (desensitization) to allergen is a principle strategy for controlling allergic diseases, and is generally carried out by subcutaneous injection. Seed-based oral administration offers a straightforward and inexpensive alternative approach to deliver vaccines effectively to the GALT without loss of activity. Consumption of transgenic seeds containing modified hypo-allergenic tolerogen or T-cell epitope peptides derived from allergens has no or very few severe side effects and can induce immune tolerance leading to reduction of allergen-specific IgE production, T-cell proliferation and release of histamine. Suppression of allergen-specific clinical symptoms results. Thus, seed-based allergy vaccines offer an innovative and convenient allergen-specific immunotherapeutic approach as an alternative to conventional allergen-specific immunotherapy.

  14. Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain of Functions

    PubMed Central

    Huang, Wei; Giddens, John; Fan, Shu-Quan; Toonstra, Christian; Wang, Lai-Xi

    2012-01-01

    The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG. Two new glycosynthase mutants (EndoS-D233A and D233Q) were generated by site-directed mutagenesis of EndoS (an endoglycosidase from Streptococcus pyogenes), and were found to be capable of effficiently transferring pre-defined N-glycans from corresponding glycan oxazolines to the Fc-deglycosylated intact IgGs without product hydrolysis. As a model study, rituximab (a therapeutic monoclonal antibody) was successfully transformed from mixtures of G0F, G1F and G2F glycoforms to well-defined homogeneous glycoforms, including fully sialylated (S2G2F) glycoform that may gain anti-inflammatory activity, a non-fucosylated G2 glycoform that showed significantly enhanced FcγIIIa receptor binding activity, and an azido-tagged glycoform that can be further transformed into other glycoforms. We also found that EndoS could selectively remove the Fc N-glycans in the presence of FAB glycosylation. This finding, coupled with the remarkable transglycosylation activity of the EndoS glycosynthase mutants, permitted a highly selective glycoengineering of the IVIG’s Fc glycans into a fully sialylated Fc glycoform, which may possess significantly enhanced anti-inflammatory activity. The glycoengineering approach described here provides a general platform to modulate the effector functions of IgG antibodies, enabling the optimization of therapeutic efficacy and gain of new functions of monoclonal antibodies and IVIG. PMID:22747414

  15. Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called anti-keratin antibodies) in rheumatoid arthritis.

    PubMed Central

    Vincent, C; Serre, G; Basile, J P; Lestra, H C; Girbal, E; Sebbag, M; Soleilhavoup, J P

    1990-01-01

    Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic diseases. The serum level of IgG1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rheumatoid sera, the serum level of IgG1 was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgG1,2,3, and 4 AKA were detected in 27 (87%), 6 (19%), 4 (13%) and 11 (35%) of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of inter-individual heterogeneity, two predominant profiles were distinguished: IgG1 (alone) and IgG(1 + 4), which together represented 18 sera (58%). The large predominance of IgG1 AKA and the quasi-absence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis. Images Fig. 1 PMID:1696185

  16. Monoclonal antibodies specific for equine IgG sub-isotypes including an antibody which recognizes B lymphocytes.

    PubMed

    Lunn, D P; Holmes, M A; Schram, B; Duffus, W P

    1995-08-01

    Equine immunoglobulin G is currently classified as consisting of five sub-isotypes: IgGa, b, and c, IgG(T), and IgG(B). The study of the role of these immunoglobulins in antigen-specific responses, and the examination of their functional properties would be greatly facilitated by the availability of monoclonal antibodies (Mabs) that distinguish between them. The production and characterization of two Mabs that recognize an IgG sub-isotype with the characteristics of IgG(ab) is described. The immunoglobulin identified by these Mabs had a heavy chain weight of 53 kDa, was of rapid cathodal electrophoretic mobility in immuno-electrophoretic analysis, and reacted only with anti-sera to IgG, and not with anti-sera to IgG(T), IgA, or IgM in radial-immunodiffusion analysis. In addition, one of these two Mabs (CVS1) also recognized the majority of peripheral blood B lymphocytes in indirect immunofluorescent staining analysis, suggesting either that equine IgD may share a common antigenic epitope with an IgG sub-isotype, or that a large proportion of equine B lymphocytes may express an IgG sub-isotype on their surface.

  17. IgG antibodies in patients with pemphigus vulgaris before and after diagnosing with immunofluorescence.

    PubMed

    Azimi, Hamideh; Majidi, Jafar; Estakhri, Rasul; Goldust, Mohamad

    2013-06-15

    Pemphigus is defined as a group of chronic self-immune vesicular diseases histologically recognized by inter-epidermic vesicles resulting from acantholysis. The aim of this study was to evaluate the precipitant and circulative IgG antibodies in patients with pemphigus vulgaris before and after treating with immunofluorescence. Sixty-two patients (34 females and 28 males) with clinically and pathologically confirmed P.V. were studied prospectively over a one year period of time during which direct and indirect immunofluorescent tests were performed before and after treatment. They had mild or moderate forms of disease. All patients received prednisolon 1-2 mg/kg/day and Azathioprine 2-3 mg/kg/day or methylpredisolon (1 g day(-1) for 4 days) and cyclophosphamide (500 mg/first day) pulse therapy due to general condition. Thirty- four females and 28 males enrolled, the mean age were 39.5 years (SD = 12.7). Before treatment, 10 and 52 cases were positive for skin depositing + or ++) and circulatory IgG (1/20 -1/60), respectively. Two to 3 month later, 37 were IgG positive with titers 1/20 to 1/160. The correlation between circulatory IgG before and after treatment was weakly positive (p = 0.05, r = 0.415). In the present study, treatment methods used for patients suffering from pemphigus vulgaris were not successful in significantly decreasing the circulative autoantibodies levels.

  18. Detection of Impaired IgG Antibody Formation Facilitates the Decision on Early Immunoglobulin Replacement in Hypogammaglobulinemic Patients

    PubMed Central

    Wolf, Hermann M.; Thon, Vojtech; Litzman, Jiri; Eibl, Martha M.

    2015-01-01

    Hypogammaglobulinemia (serum IgG lower than 2 SD below the age-matched mean) and clinical symptoms such as increased susceptibility to infection, autoimmune manifestations, granulomatous disease, and unexplained polyclonal lymphoproliferation are considered to be diagnostic hallmarks in patients with common variable immunodeficiency (CVID), the most frequent clinically severe primary immunodeficiency syndrome. In the present study, we investigated patients with hypogammaglobulinemia and no clinical or immunological signs of defective cell-mediated immunity and differentiated two groups on the basis of their IgG antibody formation capacity against a variety of different antigens (bacterial toxins, polysaccharide antigens, viral antigens). Patients with hypogammaglobulinemia and intact antibody production (HIAP) displayed no or only mild susceptibility to infections, while CVID patients showed marked susceptibility to bacterial infections that normalized following initiation of IVIG or subcutaneous immunoglobulin replacement therapy. There was a substantial overlap in IgG serum levels between the asymptomatic HIAP group and the CVID patients examined before immunoglobulin treatment. HIAP patients showed normal levels of switched B-memory cells (CD19+CD27+IgD−), while both decreased and normal levels of switched B-memory cells could be found in CVID patients. IgG antibody response to a primary antigen, tick-borne encephalitis virus (TBEV), was defective in CVID patients, thus confirming their substantial defect in IgG antibody production. Defective IgG antibody production against multiple antigens could also be demonstrated in an adult patient with recurrent infections but normal IgG levels. To facilitate early treatment before recurrent infections may lead to organ damage, the antibody formation capacity should be examined in hypogammaglobulinemic patients and the decision to treat should be based on the finding of impaired IgG antibody production. PMID:25699049

  19. Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases.

    PubMed Central

    Ogawa, T; Kusumoto, Y; Hamada, S; McGhee, J R; Kiyono, H

    1990-01-01

    The level of serum IgM, IgG and IgA antibodies including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 subclass-specific antibodies to Bacteroides (Porphyromonas) gingivalis fimbriae and to lipopolysaccharide (LPS) were analysed in patients with different forms of periodontal disease (PD) and control subjects by ELISA. Among PD subjects, sera obtained from adult periodontitis (AP), rapidly progressive periodontitis (RPP) and gingivitis contained high titres of fimbriae-specific IgG antibodies (7500-15,000 ELISA units) followed by IgA (90-700 units) and IgM (30-90 units). In contrast, sera from localized juvenile periodontitis (LJP) subjects exhibited much lower titres of fimbriae-specific IgG (89 +/- 11 units), IgA (31 +/- 5 units) and IgM (17 +/- 3 units) antibodies. A similar response pattern was also seen in sera from normal subjects aged 35-41 years who practice normal oral hygiene, while sera of younger adults (aged 18-24) with superior hygiene did not have any antigen-specific antibodies. Analysis of IgG subclass anti-fimbriae responses revealed that the major response was IgG3 followed by IgG1, IgG2 and IgG4 in AP, RPP and gingivitis. Although lower, a similar pattern of IgG subclass titre was seen in LJP and normal subjects aged 35-41 years. When IgA subclass responses were measured in AP and RPP, higher titres of the fimbriae-specific response were noted with IgA1 when compared with IgA2. However, lower but approximately equal levels of fimbriae-specific IgA1 and IgA2 titres were seen in other PD groups. When anti-B. gingivalis LPS-specific responses were measured, the sera of AP patients contained high levels of IgG antibodies (2265 +/- 224 units) followed by IgA (411 +/- 90 units) and IgM (214 +/- 56 units). Further, IgG anti-LPS responses were mainly IgG2 followed by IgG4, IgG3 and IgG1. For IgA subclass responses, higher titres of anti-LPS-specific antibodies were noted in IgA2 subclass over IgA1. These results showed that higher anti-B. gingivalis antibody

  20. Induction of allergen-specific tolerance via mucosal routes.

    PubMed

    Mascarell, Laurent; Zimmer, Aline; Van Overtvelt, Laurence; Tourdot, Sophie; Moingeon, Philippe

    2011-01-01

    Allergen-specific immunotherapy is the only curative treatment of allergies against insect venom, house dust mites, tree/grass pollens, or cat dander. Subcutaneous immunotherapy is successful to reorient the immune system and re-establish long-term tolerance. However, major drawbacks for using this route include: repeated injections, as well as the risk of anaphylaxis. In this context, alternative mucosal routes of administration are being considered together with the combined use of adjuvants/vector systems and recombinant allergens or peptide fragments. Herein, we review the current status in the use of mucosal routes (i.e., sublingual, oral, intranasal) for allergen-specific immunotherapy, as well as the latest understanding with respect to underlying mechanisms of action.

  1. Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches

    PubMed Central

    Valenta, R.; Campana, R.; Marth, K.; van Hage, M.

    2015-01-01

    Immunoglobulin E-mediated allergies affect more than 25% of the population. Allergen exposure induces a variety of symptoms in allergic patients, which include rhinitis, conjunctivitis, asthma, dermatitis, food allergy and life-threatening systemic anaphylaxis. At present, allergen-specific immunotherapy (SIT), which is based on the administration of the disease-causing allergens, is the only disease-modifying treatment for allergy. Current therapeutic allergy vaccines are still prepared from relatively poorly defined allergen extracts. However, with the availability of the structures of the most common allergen molecules, it has become possible to produce well-defined recombinant and synthetic allergy vaccines that allow specific targeting of the mechanisms of allergic disease. Here we provide a summary of the development and mechanisms of SIT, and then review new forms of therapeutic vaccines that are based on recombinant and synthetic molecules. Finally, we discuss possible allergen-specific strategies for prevention of allergic disease. PMID:22640224

  2. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  3. Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein

    PubMed Central

    Kakalacheva, Kristina; Regenass, Stephan; Wiesmayr, Silke; Azzi, Tarik; Berger, Christoph; Dale, Russell C.; Brilot, Fabienne; Münz, Christian; Rostasy, Kevin; Nadal, David; Lünemann, Jan D.

    2016-01-01

    A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM. PMID:26907324

  4. Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein.

    PubMed

    Kakalacheva, Kristina; Regenass, Stephan; Wiesmayr, Silke; Azzi, Tarik; Berger, Christoph; Dale, Russell C; Brilot, Fabienne; Münz, Christian; Rostasy, Kevin; Nadal, David; Lünemann, Jan D

    2016-02-12

    A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM.

  5. Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS*

    PubMed Central

    Fornelli, Luca; Damoc, Eugen; Thomas, Paul M.; Kelleher, Neil L.; Aizikov, Konstantin; Denisov, Eduard; Makarov, Alexander; Tsybin, Yury O.

    2012-01-01

    The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure–function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules. PMID:22964222

  6. 3D structural fluctuation of IgG1 antibody revealed by individual particle electron tomography

    DOE PAGES

    Zhang, Xing; Zhang, Lei; Tong, Huimin; ...

    2015-05-05

    Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, wemore » derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.« less

  7. 3D structural fluctuation of IgG1 antibody revealed by individual particle electron tomography

    SciTech Connect

    Zhang, Xing; Zhang, Lei; Tong, Huimin; Peng, Bo; Rames, Matthew J.; Zhang, Shengli; Ren, Gang

    2015-05-05

    Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

  8. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

  9. Serum IgA and IgG gliadin antibodies and small intestinal mucosal damage in children.

    PubMed

    Lindberg, T; Nilsson, L A; Borulf, S; Cavell, B; Fällström, S P; Jansson, U; Stenhammar, L; Stintzing, G

    1985-12-01

    Serum immunoglobulin (Ig) A and IgG gliadin antibodies were determined with a simple, rapid, and inexpensive method--diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA)--and the results were related to small intestinal mucosal morphology in 234 children suspected of having malabsorption. Fifty-six of 58 children with flat intestinal mucosa had increased IgA and/or IgG gliadin antibody levels (sensitivity 97%). Fifty-four of the 58 children had celiac disease (CD) (n = 25) or probable CD (n = 29). Four children with flat mucosa had cow's milk protein and/or soy protein intolerance and three of these had increased gliadin antibody levels. Seventeen percent of 132 children with normal intestinal mucosa had increased IgA and/or IgG gliadin antibody levels. IgA and IgG gliadin antibody levels decreased significantly in the celiac children on a gluten-free diet and increased significantly after gluten challenge. Determination of serum IgA and IgG gliadin antibodies by means of DIG-ELISA is a sensitive test for small intestinal mucosal damage in children. When malabsorption is suspected, we suggest that this assay be used to select children for a small intestinal biopsy. It is also very useful for the follow-up of adherence to a gluten-free diet and to determine the effect of gluten challenge in celiac children.

  10. Nonenzymatic glycosylation of serum IgG and its effect on antibody activity in patients with diabetes mellitus.

    PubMed

    Kaneshige, H

    1987-07-01

    Susceptibility to infection is assumed to be increased in diabetic patients, although its mechanism is unknown. The purpose of this study was to determine whether glycosylation of circulating immunoglobulins is related to the decrease of antibody activity in diabetic patients. Thirty-five patients with type II (non-insulin-dependent) diabetes and 14 age-matched normal controls were examined. Nonenzymatic glycosylation of serum immunoglobulin G (IgG) in vivo was measured by two different techniques, colorimetry and affinity chromatography. The levels of glycosylated IgG were significantly higher in diabetic patients than in normal controls. To evaluate the antibody activity of glycosylated IgG, anti-streptolysin O (ASO) titers after in vitro glycosylation of IgG and antibody titers before and after in vivo immunization with influenza vaccine were determined. IgG specific for streptolysin O purified by affinity chromatography decreased ASO titers after in vitro glycosylation. In diabetic patients, serum titers of hemagglutinin-inhibiting antibody against influenza viruses 4 wk after initial immunization were significantly lower than those in normal controls. These results indicate that serum IgG in diabetic patients was nonenzymatically glycosylated, and this modification in vivo might be associated with its functional alteration.

  11. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    PubMed

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  12. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

    PubMed

    Liu, Y Diana; Goetze, Andrew M; Bass, Randal B; Flynn, Gregory C

    2011-04-01

    Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

  13. Evaluation of cysticercus-specific IgG (total and subclasses) and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies.

    PubMed

    Suzuki, Lisandra Akemi; Rossi, Cláudio Lúcio

    2013-02-01

    In the present study, an enzyme-linked immunosorbent assay (ELISA) standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses) and IgE antibodies in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA)=1.17) and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49) and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46) and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12) and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85) and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60) and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

  14. Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

    PubMed

    Kramski, Marit; Lichtfuss, Gregor F; Navis, Marjon; Isitman, Gamze; Wren, Leia; Rawlin, Grant; Center, Rob J; Jaworowski, Anthony; Kent, Stephen J; Purcell, Damian F J

    2012-10-01

    Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission.

  15. A rapid assay for Hendra virus IgG antibody detection and its titre estimation using magnetic nanoparticles and phycoerythrin.

    PubMed

    Gao, Yuan; Pallister, Jackie; Lapierre, Florian; Crameri, Gary; Wang, Lin-Fa; Zhu, Yonggang

    2015-09-15

    Detection of Hendra viral IgG antibody in animal sera is useful for surveillance following a virus outbreak. The commonly used enzyme-linked immunosorbent assay and fluorescence-based Luminex assay typically consist of three steps and take at least several hours to complete. We have simplified the procedure to two steps in an effort to develop a rapid procedure for IgG antibody, but not IgM antibody, detection. This is achieved by conjugating the fluorescence label R-phycoerythrin directly onto the IgG binding protein Protein G. The use of magnetic nanoparticles, due to their large specific surface area, has helped reduce each of the binding steps to 20 min. As a result, the whole assay can be completed in 60 min. We also demonstrate a method to quickly estimate IgG antibody titres by assaying the sera at only two dilutions (i.e. 1:20 and 1:1000) and using the fluorescence ratio at these dilutions as an indicator of antibody titre. The results of this approach correlated well with the well-regarded serum neutralization test in virus antibody assays. This protocol reported here can be adopted in Luminex assays, fluorescence-linked immunosorbent assays and assays on microfluidics platforms for rapid antibody surveillance of Hendra and other viruses.

  16. Specific IgG subclass antibody pattern to Aspergillus fumigatus in patients with cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA)

    PubMed Central

    Skov, M; Pressler, T; Jensen, H; Hoiby, N; Koch, C

    1999-01-01

    BACKGROUND—IgG and IgG subclass antibodies to Aspergillus fumigatus (A fumigatus) were measured in a large population of patients with cystic fibrosis to elucidate a putative antibody pattern specific for allergic bronchopulmonary aspergillosis (ABPA).
METHODS—An ELISA technique using water soluble somatic hyphal (WSSH) A fumigatus antigens and subclass specific monoclonal antibodies was used for cross sectional quantification of IgG and IgG1-4 subclass antibody levels in the serum of 238 patients with cystic fibrosis and 107 healthy controls.
RESULTS—In patients with cystic fibrosis persistently colonised with A fumigatus the subclass antibody levels were significantly increased compared with patients with cystic fibrosis never or rarely colonised (p<0.001). The group of patients persistently colonised with A fumigatus with ABPA (+Af+ABPA) had significantly increased levels of IgG antibodies to A fumigatus (Af-IgG) (median 69 ELISA units (EU) versus 31) and of subclasses Af-IgG1 (91 versus 27), Af-IgG2 (143 versus 56), and Af-IgG4 antibodies (72 versus 20), but not of IgG3 (17 versus 15), compared with the colonised patients without ABPA (+Af-ABPA). Patients with cystic fibrosis with no or only rare isolates of A fumigatus without ABPA (-Af-ABPA) also had significantly increased subclass antibody levels (Af-IgG1 9 versus 3, Af-IgG2 28 versus 5, Af-IgG4 16 versus 4; p<0.001) compared with healthy controls. Low, although detectable, levels of antibodies were demonstrated in healthy controls. ABPA seemed to occur independently of Pseudomonas aeruginosa infection. Using diagnostic cut off levels for ABPA, sensitivity and specificity were calculated. The highest specificity was found for IgG4 (88%); sensitivity was between 65% and 73%. The positive predictive values (PPV) were moderate, whereas the negative predictive values (NPV) were high (96% in all subclasses except IgG3 with 94%). PPV increased to 50% if IgG1 as well as IgG2 and IgG4 were included

  17. Shaken, not stirred: mechanical stress testing of an IgG1 antibody.

    PubMed

    Kiese, Sylvia; Papppenberger, Astrid; Friess, Wolfgang; Mahler, Hanns-Christian

    2008-10-01

    Protein aggregation is known to occur under different stress conditions and displays a wide variety of morphologies. In this work, the aggregation behavior of a monoclonal antibody (IgG1) was investigated using two different mechanical stress methods namely stirring and shaking at two temperatures, various fill volumes and headspaces and different amounts of polysorbate present in the formulation. The detection of aggregates in terms of size and number was carried out using various analytical techniques including visible particle inspection, turbidity, sub-visible particle analysis, size exclusion chromatography and dynamic light scattering. The data showed that shaking and stirring resulted in different species of aggregates both qualitatively and quantitatively, where stirring was found more stressful than shaking on the IgG1 formulation. Mechanical stress testing performed at 5 and 25 degrees C only showed a difference on samples stressed by shaking and not by stirring. The headspace in the vials had great influence on the stability of the protein formulation when stressed by shaking. The presence of polysorbate had a protective effect on the antibody, however certain polysorbate concentrations even resulted in increased protein aggregation. An array of analytical methods was essential in order to cover the vast aggregate morphologies, which occurred during agitation.

  18. Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment.

    PubMed

    Solano-Gallego, L; Riera, C; Roura, X; Iniesta, L; Gallego, M; Valladares, J E; Fisa, R; Castillejo, S; Alberola, J; Ferrer, L; Arboix, M; Portús, M

    2001-04-19

    The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and

  19. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    PubMed

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.

  20. Study of natural IgG antibodies against vascular endothelial growth factor receptor 1 in hepatocellular carcinoma

    PubMed Central

    Wang, Yilai; Yan, Zhaoping; Huang, Yile; Qiu, Cailing; Chen, Xiangyun; Hu, Ying; Meng, Qingyong; Wei, Jun

    2017-01-01

    Natural antibodies have been found to have anti-tumorigenic function. This study was designed to investigate whether natural IgG antibodies against vascular endothelial growth factor receptor 1 (VEGFR1) could suppress the growth of hepatocellular carcinoma (HCC) cells. Three HCC cell lines and A549 lung cancer cells were used for this study. They were grown, respectively, with human plasma positive or negative for anti-VEGFR1 IgG. Cell viability, apoptosis and VEGFR1 gene expression were examined. Three patients with HCC were recruited for a case study. The results showed that plasma anti-VEGFR1 IgG significantly inhibited the proliferation of all three HCC cell lines but not A549 cell line; the proportions of apoptotic cells were significantly higher in HCC cells treated with anti-VEGFR1 IgG positive plasma than those treated with IgG negative plasma. The expression of the VEGFR1 gene was significantly higher in HCC cells than A549 cells. Of three HCC patients who received transfusion of anti-VEGFR1 IgG positive plasma, two cases with stage B showed a good response to the treatment but one with distant metastasis did not. Human plasma IgG against VEGFR1 may be a promising agent for anti-HCC therapy.

  1. Does the antibody production ability affect the serum anti-Helicobacter pylori IgG titer?

    PubMed Central

    Chung, Hyun Ah; Lee, Sun-Young; Moon, Hee Won; Kim, Jeong Hwan; Sung, In-Kyung; Park, Hyung Seok; Shim, Chan Sup; Han, Hye Seung

    2016-01-01

    AIM To investigate the relationship between serum titers of anti-Helicobacter pylori (H. pylori) immunoglobulin G (IgG) and hepatitis B virus surface antibody (HBsAb). METHODS Korean adults were included whose samples had positive Giemsa staining on endoscopic biopsy and were studied in the hepatitis B virus surface antigen (HBsAg)/HBsAb serologic assay, pepsinogen (PG) assay, and H. pylori serologic test on the same day. Subjects were excluded if they were positive for HBsAg, had a recent history of medication, or had other medical condition(s). We analyzed the effects of the following factors on serum titers of HBsAb and the anti-H. pylori IgG: Age, density of H. pylori infiltration in biopsy samples, serum concentrations of PG I and PG II, PG I/II ratio, and white blood cell count. RESULTS Of 111 included subjects, 74 (66.7%) exhibited a positive HBsAb finding. The serum anti-H. pylori IgG titer did not correlate with the serum HBsAb titer (P = 0.185); however, it correlated with the degree of H. pylori infiltration on gastric biopsy (P < 0.001) and serum PG II concentration (P = 0.042). According to the density of H. pylori infiltration on gastric biopsy, subjects could be subdivided into those with a marked (median: 3.95, range 0.82-4.00) (P = 0.458), moderate (median: 3.37, range 1.86-4.00), and mild H. pylori infiltrations (median: 2.39, range 0.36-4.00) (P < 0.001). Subjects with a marked H. pylori infiltration on gastric biopsy had the highest serological titer, whereas in subjects with moderate and mild H. pylori infiltrations titers were correspondingly lower (P < 0.001). After the successful eradication, significant decreases of the degree of H. pylori infiltration (P < 0.001), serum anti-H. pylori IgG titer (P < 0.001), and serum concentrations of PG I (P = 0.028) and PG II (P = 0.028) were observed. CONCLUSION The anti-H. pylori IgG assay can be used to estimate the burden of bacteria in immunocompetent hosts with H. pylori infection, regardless

  2. Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector-Borne Diseases: A Hypothesis

    PubMed Central

    Londono-Renteria, Berlin; Cardenas, Jenny C.; Troupin, Andrea; Colpitts, Tonya M.

    2016-01-01

    Chronic exposure to antigens may favor the production of IgG4 antibodies over other antibody types. Recent studies have shown that up to a 30% of normal human IgG4 is bi-specific and is able to recognize two antigens of different nature. A requirement for this specificity is the presence of both eliciting antigens in the same time and at the same place where the immune response is induced. During transmission of most vector-borne diseases, the pathogen is delivered to the vertebrate host along with the arthropod saliva during blood feeding and previous studies have shown the existence of IgG4 antibodies against mosquito salivary allergens. However, there is very little ongoing research or information available regarding IgG4 bi-specificity with regard to infectious disease, particularly during immune responses to vector-borne diseases, such as malaria, filariasis, or dengue virus infection. Here, we provide background information and present our hypothesis that IgG4 may not only be a useful tool to measure exposure to infected mosquito bites, but that these bi-specific antibodies may also play an important role in modulation of the immune response against malaria and other vector-borne diseases in endemic settings. PMID:27746778

  3. Development of stage-dependent glycans on the Fc domains of IgG antibodies of ALS animals.

    PubMed

    Edri-Brami, Meital; Sharoni, Hila; Hayoun, Dana; Skutelsky, Linor; Nemirovsky, Ana; Porgador, Angel; Lichtenstein, Rachel G

    2015-05-01

    We recently revealed a unique glycan on the Fc domain of IgG antibodies in ALS patients that mediates antibody-dependent cell cytotoxicity (ADCC). This glycan has a bi-antennary structure that lacks the core fucose and sialic acid residues but contains a bisecting GlcNAc (A2BG2). Little is known, however, about the incidence of A2BG2 expression and IgG cytotoxicity under ALS conditions within well-defined clinical stages. Here, we characterize the IgG antibodies produced in ALS Tg mice by detecting intra- and extra-cellular antigens of motor neurons that express different glycan patterns during the disease. The increased number of innate immune cells found at the disease onset was insufficient to induce an optimal systemic T-cell response. Nevertheless, IgG antibodies were produced against intracellular antigens at the pre-symptomatic stage in the secondary lymphoid organs under the conditions of a poor systemic immune response. Moreover, while the glycosyltransferases of plasma B-cells that synthesize the Fc-glycans were regulated by IL-2 or IL-4, the observed glycosyltransferase pattern did not match that found in ALS Tg mice. We further found that A2BG2 glycan is specific for ALS, its quantity increased with disease progression and that the IgG antibodies identifying extracellular motor neuron antigens were developed at the final stage of the disease. Therefore, the most effective ADCC of motor neurons was observed at the end stage of the disease. We conclude that in ALS, IgG antibodies are produced despite the poor systemic immune response and that the frequency and quantity of A2BG2 glycan expression on the Fc domain depends on the clinical stage. Therefore, A2BG2 is a potential prognostic biomarker for ALS.

  4. T cell responses induced by allergen-specific immunotherapy

    PubMed Central

    Maggi, E

    2010-01-01

    Allergen-specific immunotherapy is recognized as a highly effective practice in the treatment of patients with severe allergic rhinitis and/or asthma and is recommended by World Health Organization as an integrated part of allergy management strategy. Several studies have shown that allergen-specific immunotherapy, based on the administration of increasing doses of allergen, achieves a hyposensitization and reduces both early and late responses occurring during the natural exposure to the allergen itself. This is the unique antigen-specific immunomodulatory treatment in current use for human diseases. Successful immunotherapy is associated with reductions in symptoms and medication scores and improved quality of life. After interruption it usually confers long-term remission of symptoms and prevents the onset of new sensitizations in children up to a number of years. Subcutaneous immunotherapy usually suppresses the allergen-induced late response in target organs, likely due to the reduction of the infiltration of T cells, eosinophils, basophils, mast cells and neutrophils. In addition to the reduction of cells of allergic inflammation, immunotherapy also decreases inflammatory mediators at the site of allergen exposure. This review provides an update on the immunological T cell responses induced by conventional subcutaneous and sublingual immunotherapy, and gives a unifying view to reconciling the old dualism between immunoredirecting and immunoregulating mechanisms. PMID:20408857

  5. Antibody response of Echinococcus granulosus infected mice: protoscolex specific response during infection is associated with decreasing specific IgG1/IgG3 ratio as well as decreasing avidity.

    PubMed

    Severi, M A; Ferragut, G; Nieto, A

    1997-12-01

    The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity.

  6. Human polyreactive IgM monoclonal antibodies with blocking activity against self-reactive IgG.

    PubMed

    Melero, J; Tarragó, D; Núñez-Roldán, A; Sánchez, B

    1997-04-01

    Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')2 fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.

  7. Myeloma with xanthoderma due to an IgG lambdamonoclonal anti-flavin antibody.

    PubMed

    Farhangi, M; Osserman, E F

    1976-01-22

    When yellow skin and yellow hair developed in an elderly patient with multiple myeloma, we ruled out the usual causes of such pigmentation but identified a monoclonal IgGlambda (lgGGar) with anti-flavin antibody activity. Purified IgGGar was bright yellow, and the acid-dissociated chromophore was identified as riboflavin by chromatography and absorption spectroscopy. Native IgGGar contained 1.45 moles of flavin per mole of IgG, and increased to 2 moles with addition of riboflavin to saturation. The flavin was localized to the Fab fragment and was bound to IgGGar with high affinity. IgGGar showed strongest affinities for riboflavin, flavin mononucleotide and flavin adenine dinucleotide, and lower affinities for dinitrophenyl derivatives and naphthoquinone. The demonstration of hapten bound to the circulating monoclonal immunoglobulin in this case suggests the possibility of bound but colorless haptens on other myeloma proteins as well as on normal immunoglobulins.

  8. The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein.

    PubMed

    Bird, P; Reyburn, H T; Blacklaws, B A; Allen, D; Nettleton, P; Yirrell, D L; Watt, N; Sargan, D; McConnell, I

    1995-11-01

    Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.

  9. The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein.

    PubMed Central

    Bird, P; Reyburn, H T; Blacklaws, B A; Allen, D; Nettleton, P; Yirrell, D L; Watt, N; Sargan, D; McConnell, I

    1995-01-01

    Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence. Images Fig. 1 Fig. 4 PMID:7586678

  10. Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

    PubMed

    Nishiya, Casey T; Boxx, Gayle M; Robison, Kerry; Itatani, Carol; Kozel, Thomas R; Zhang, Mason X

    2015-11-16

    Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

  11. Specific recognition pattern of IgM and IgG antibodies produced in the course of experimental paracoccidioidomycosis.

    PubMed Central

    Vaz, C A; Mackenzie, D W; Hearn, V M; Camargo, Z P; Singer-Vermes, L M; Burger, E; Calich, V L

    1992-01-01

    Specific IgM and IgG responses to Paracoccidioides brasiliensis produced in resistant and susceptible mice during experimental paracoccidioidomycosis were examined by the immunoblotting procedure. Sera from infected mice recognized 51 antigen bands with apparent molecular masses from 8 to 86 kD. Sixteen of these were defined as major antigen bands because of almost universal presence of antibodies to them, and their intense staining. All sera, including those from normal control mice, tested for both IgM and IgG antibody reacted with the major E antigen which appeared as a large diffuse band from 43 to 47 kD. Comparisons between resistant and susceptible mice showed some significant differences in IgM responses to many antigen bands. While IgG responses were quite similar for both strains, differences were apparent in the response to the antigens at 62 and 68 kD. Images Fig. 1 Fig. 2 Fig. 3 PMID:1563097

  12. Time course of antibodies against IgG and type II collagen in adjuvant arthritis. Role of mycobacteria administration in antibody production.

    PubMed

    Franch, A; Cassany, S; Castellote, C; Castell, M

    1994-02-01

    The aim of this study was to elucidate, during the time course of adjuvant arthritis, the existence of antibodies directed to IgG (rheumatoid factor-like) and antibodies against type II collagen. In a second study, we also studied the relation between antibody production, arthritic process and mycobacteria administration. We have demonstrated the presence of antibodies to IgG and type II collagen by means of ELISA techniques. This reactivity appeared on day 7 post-induction, decreased later, and increased progressively from day 21 until last day studied (day 56 post-induction). We have also quantified antibodies against a soluble fraction of Mycobacterium butyricum, the inductor of the disease. Anti-mycobacteria antibodies appeared during the first seven days after induction, but from day 14, when systemic inflammation began, their levels suddenly increased. There is a positive correlation between anti-mycobacteria antibody levels and articular swelling. Anti-IgG and anti-collagen antibody production was not directly linked to arthritic process since these antibodies were synthesized when M. butyricum was administered intraperitoneally, which does not induce arthritis. Anti-mycobacteria antibody concentration was higher when arthritis induction by mycobacterial was successful than when it was unsuccessful.

  13. Liquid high concentration IgG1 antibody formulations by precipitation.

    PubMed

    Matheus, Susanne; Friess, Wolfgang; Schwartz, Daniel; Mahler, Hanns-Christian

    2009-09-01

    A manufacturing approach for liquid high concentration antibody formulations based on precipitation and subsequent re-dissolution was investigated. IgG1 antibody solutions were concentrated from 20 to 100 mg/mL by intermediate precipitation, with a recovery exceeding 95%, retention of the native secondary structure and binding activity as well as adequate stability. Quantitative, reproducible precipitation was performed using 1.45 M ammonium sulphate (pH 5.5 and 8.0), 0.67 M sodium citrate (pH 8.0) and 9% (w/v) PEG 4000 (pH 5.5 and 8.0). Scalability was confirmed from 1 to 100 mL. The concentrations achievable in the re-dissolution step were less affected by the re-dissolution medium, but limited by the residual precipitant. Both, improved removal of remaining precipitant liquid and larger precipitation scales were successful in increasing the final protein concentration. SEC and turbidity analysis directly after re-dissolution indicated that similar protein qualities were obtained, independent from the precipitant used. However, increased aggregate formation was observed after short term storage of the precipitated protein particles at either 2-8 degrees C or ambient temperature. An accelerated mechanical and thermal stability program verified comparable stability of the re-dissolved liquid 100 mg/mL formulations produced by intermediate precipitation to a control formulation obtained by standard ultrafiltration.

  14. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    PubMed

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs.

  15. IgG1 and IgG4 Antibody Responses to the Anopheles gambiae Salivary Protein gSG6 in the Sympatric Ethnic Groups Mossi and Fulani in a Malaria Hyperhendemic Area of Burkina Faso

    PubMed Central

    Lombardo, Fabrizio; Mangano, Valentina; Sirima, Sodiomon Bienvenu; Nèbiè, Issa; Fiorentino, Gabriella; Troye-Blomberg, Marita; Modiano, David; Arcà, Bruno

    2014-01-01

    Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP) exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response. PMID:24760038

  16. A historical look at the first reported cases of Lassa fever: IgG antibodies 40 years after acute infection.

    PubMed

    Bond, Nell; Schieffelin, John S; Moses, Lina M; Bennett, Andrew J; Bausch, Daniel G

    2013-02-01

    Lassa fever is an acute and sometimes severe viral hemorrhagic illness endemic in West Africa. One important question regarding Lassa fever is the duration of immunoglobulin G (IgG) antibody after infection. We were able to locate three persons who worked in Nigeria dating back to the 1940s, two of whom were integrally involved in the early outbreaks and investigations of Lassa fever in the late 1960s, including the person from whom Lassa virus was first isolated. Two persons had high titers of Lassa virus-specific IgG antibody over 40 years after infection, indicating the potential for long-term duration of these antibodies. One person was likely infected in 1952, 17 years before the first recognized outbreak. We briefly recount the fascinating stories of these three pioneers and their important contribution to our understanding of Lassa fever.

  17. A Historical Look at the First Reported Cases of Lassa Fever: IgG Antibodies 40 Years After Acute Infection.

    PubMed

    Bond, Nell; Schieffelin, John S; Moses, Lina M; Bennett, Andrew J; Bausch, Daniel G

    2012-12-31

    Lassa fever is an acute and sometimes severe viral hemorrhagic illness endemic in West Africa. One important question regarding Lassa fever is the duration of immunoglobulin G (IgG) antibody after infection. We were able to locate three persons who worked in Nigeria dating back to the 1940s, two of whom were integrally involved in the early outbreaks and investigations of Lassa fever in the late 1960s, including the person from whom Lassa virus was first isolated. Two persons had high titers of Lassa virus-specific IgG antibody over 40 years after infection, indicating the potential for long-term duration of these antibodies. One person was likely infected in 1952, 17 years before the first recognized outbreak. We briefly recount the fascinating stories of these three pioneers and their important contribution to our understanding of Lassa fever.

  18. Serum pepsinogen I and II concentrations and IgG antibody to Helicobacter pylori in dyspeptic patients.

    PubMed Central

    Biasco, G; Paganelli, G M; Vaira, D; Holton, J; Di Febo, G; Brillanti, S; Miglioli, M; Barbara, L; Samloff, I M

    1993-01-01

    AIMS--To investigate the association between histologically confirmed gastritis, carriage of Helicobacter pylori and pepsinogen (PG) I and PG II concentrations. METHODS--Prospective study of 81 dyspeptic patients undergoing upper gastrointestinal endoscopy was made. The extent of gastric mucosal inflammation and the presence of H pylori was determined, and serology to evaluate PG I and II concentrations and IgG titres to H pylori was carried out. RESULTS--The presence of H pylori was strongly correlated with high IgG antibody titres to H pylori and gastritis. Patients who were H pylori positive had significantly higher PG I and PG II concentrations and a significantly lower PG I:PG II ratio than patients who were negative for H pylori. In 13 patients with duodenal ulcer and H pylori positive gastritis serum PG I concentrations were significantly higher than in H pylori positive patients without duodenal ulcer. Significant correlations were found between the age of patients and serum PG II, the PG I:PG II ratio, IgG antibodies to H pylori, the severity of body gastritis and H pylori infection, and between the degree of gastritis in the body of the stomach and the PG II concentration. CONCLUSIONS--Serum PG I and II concentrations, together with a fall in the PG I:PG II ratio, could be used as predictors of H pylori infection as well as serum IgG antibody response to H pylori. PMID:8227432

  19. Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes

    PubMed Central

    Nicholas, Dequina A.; Salto, Lorena M.; Boston, Ava M.; Kim, Nan Sun; Larios, Marco; Beeson, W. Lawrence; Firek, Anthony F.; Casiano, Carlos A.; Langridge, William H. R.; Cordero-MacIntyre, Zaida; De Leon, Marino

    2015-01-01

    High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1β. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1β, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1β secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients. PMID:26633920

  20. Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes.

    PubMed

    Nicholas, Dequina A; Salto, Lorena M; Boston, Ava M; Kim, Nan Sun; Larios, Marco; Beeson, W Lawrence; Firek, Anthony F; Casiano, Carlos A; Langridge, William H R; Cordero-MacIntyre, Zaida; De Leon, Marino

    2015-01-01

    High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1β. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1β, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1β secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.

  1. Maternal autism-associated IgG antibodies delay development and produce anxiety in a mouse gestational transfer model.

    PubMed

    Braunschweig, Daniel; Golub, Mari S; Koenig, Claire M; Qi, Lihong; Pessah, Isaac N; Van de Water, Judy; Berman, Robert F

    2012-11-15

    A murine passive transfer model system was employed to ascertain the effects of gestational exposure to a single, intravenous dose of purified, brain-reactive IgG antibodies from individual mothers of children with autism (MAU) or mothers with typically developing children (MTD). Growth and behavioral outcomes in offspring were measured from postnatal days 8 to 65 in each group. Comparisons revealed alterations in early growth trajectories, significantly impaired motor and sensory development, and increased anxiety. This report demonstrates for the first time the effects of a single, low dose gestational exposure of IgG derived from individual MAU on their offspring's physical and social development.

  2. Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris.

    PubMed

    Aw, Rochelle; McKay, Paul F; Shattock, Robin J; Polizzi, Karen M

    2017-12-01

    The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 μg mL(-1) in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly, using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.

  3. Search for Anti-EA(D) Antibodies in Subjects with an "Isolated VCA IgG" Pattern.

    PubMed

    De Paschale, Massimo; Cagnin, Debora; Cerulli, Teresa; Manco, Maria Teresa; Agrappi, Carlo; Mirri, Paola; Gatti, Arianna; Rescaldani, Cristina; Clerici, Pierangelo

    2010-01-01

    The presence of an "isolated viral capsid antigen (VCA) IgG" pattern in serum is not easy to interpret without the aid of further tests, such as specific immunoblotting or a virus genome search, that often give rise to organisational and economic problems. However, one alternative is to use an enzyme-linked immunosorbent assay (ELISA) to detect anti-early antigen (EA) antibodies, which can be found in about 85% of subjects with acute Epstein-Barr virus (EBV) infections. The purpose of this work was to search for anti-EA(D) antibodies in 130 samples with an isolated VCA IgG pattern at ELISA screening and classified as being indicative of past (102 cases) or acute (28 cases) infection on the basis of the immunoblotting results. Thirty-seven samples (28.5%) were positive for anti-EA(D), of which 25 (89.3%) had been classified by immunoblotting as indicating acute and 12 (11.8%) past EBV infection. This difference was statistically significant (P < .01). The results of our search for anti-EA(D) antibodies correctly identified nearly 90% of acute (presence) or past EBV infections (absence). When other tests are not available, the search for anti-EA antibodies may therefore be helpful in diagnosing patients with an isolated VCA IgG pattern at screening tests.

  4. Allergen-specific immunotherapy in pediatric allergic asthma

    PubMed Central

    2016-01-01

    Allergen-specific immunotherapy (AIT) is the only curative way that can change the immunologic response to allergens and thus can modify the natural progression of allergic diseases. There are some important criteria which contributes significantly on efficacy of AIT, such as the allergen extract used for treatment, the dose and protocol, patient selection in addition to the severity and control of asthma. The initiation of AIT in allergic asthma should be considered in intermittent, mild and moderate cases which coexisting with other allergic diseases such as allergic rhinitis, and in case of unacceptable adverse effects of medications. Two important impact of AIT; steroid sparing effect and preventing from progression to asthma should be taken into account in pediatric asthma when making a decision on starting of AIT. Uncontrolled asthma remains a significant risk factor for adverse events and asthma should be controlled both before and during administration of AIT. The evidence concerning the efficacy of subcutaneous (SCIT) and sublingual immunotherapy (SLIT) for treatment of pediatric asthma suggested that SCIT decreases asthma symptoms and medication scores, whereas SLIT can ameliorate asthma symptoms. Although the effectiveness of SCIT has been shown for both seasonal and perennial allergens, the data for SLIT is less convincing for perennial allergies in pediatric asthma. PMID:27489785

  5. Skin as a novel route for allergen-specific immunotherapy.

    PubMed

    Li, Wei; Zhang, Zhuo; Tian, Rong; Zhang, Ke

    2014-01-01

    Subcutaneous allergen specific immunotherapy is an effective treatment of IgE-mediated allergies, but it requires repeated allergen injections with a risk of systemic allergic reactions. Other routes of immunization had been explored to improve patient compliance and safety. Skin is not only a physical barrier between the body and outside world, but also an important immune organ eliciting innate and adaptive immune function. Skin has been used as an ideal site for vaccination of infectious diseases. Food allergen topically applied onto disrupted skin can induce sensitization and food allergy would develop subsequently. However, immune tolerance would be induced if the skin barrier is kept intact. Several mice and human studies on epicutaneous immunotherapy showed successful treatment on IgE-induced allergy models or allergic diseases. Migratory Langerhans cells might play a decisive role in the induction of different immune responses. Further research on the underlying mechanism of the crosstalk between skin and gut or airway is helpful for the understanding of many protective or sensitizing immune responses induced via skin, and also is helpful for the development of new strategy for the treatment of allergic disease.

  6. IgA and IgG antineutrophil cytoplasmic antibody engagement of Fc receptor genetic variants influences granulomatosis with polyangiitis.

    PubMed

    Kelley, James M; Monach, Paul A; Ji, Chuanyi; Zhou, Yebin; Wu, Jianming; Tanaka, Sumiaki; Mahr, Alfred D; Johnson, Sharleen; McAlear, Carol; Cuthbertson, David; Carette, Simon; Davis, John C; Dellaripa, Paul F; Hoffman, Gary S; Khalidi, Nader; Langford, Carol A; Seo, Phillip; St Clair, E William; Specks, Ulrich; Stone, John H; Spiera, Robert F; Ytterberg, Steven R; Merkel, Peter A; Edberg, Jeffrey C; Kimberly, Robert P

    2011-12-20

    Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR (FCAR) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ∼30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced (P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).

  7. Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.

    PubMed

    Rajendra, Yashas; Peery, Robert B; Hougland, Maria D; Barnard, Gavin C; Wu, Xiufeng; Fitchett, Jonathan R; Bacica, Michael; Demarest, Stephen J

    2016-12-15

    IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  8. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    PubMed

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  9. IgG, IgM, and IgA antinuclear antibodies in discoid and systemic lupus erythematosus patients.

    PubMed

    Jost, Sheridan A; Tseng, Lin-Chiang; Matthews, Loderick A; Vasquez, Rebecca; Zhang, Song; Yancey, Kim B; Chong, Benjamin F

    2014-01-01

    IgG antinuclear antibodies (ANAs) are elevated in patients with systemic lupus erythematosus (SLE) compared with patients with discoid lupus erythematosus (DLE). To provide an expanded immunologic view of circulating ANAs in lupus patients, we compared the expressions of IgG, IgM, and IgA ANAs in DLE and SLE patients. In this cross-sectional study, sera from age-, gender-, and ethnic-matched SLE (N = 35), DLE (N = 23), and normal patients (N = 22) were tested for IgG, IgM, and IgA ANAs using enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence (IIF) with monkey esophagus as substrate. ELISAs showed elevated levels of IgG ANA, IgM ANA, and IgG/IgM ANA ratios in SLE patients compared with DLE and normal patients. IgA ANA expression was higher in SLE and DLE patients versus normal patients. IIF studies showed higher percentages of patients positive for IgG, IgM, and IgA ANAs in the SLE group. Higher IgG/IgM ANA ratios in SLE than DLE show enhanced class-switching and a more sustained humoral response in SLE. They also suggest a potential connection of IgM ANAs with disease containment.

  10. Detection of phase I IgG antibodies to Coxiella burnetii with EIA as a screening test for blood donations.

    PubMed

    van der Hoek, W; Wielders, C C H; Schimmer, B; Wegdam-Blans, M C A; Meekelenkamp, J; Zaaijer, H L; Schneeberger, P M

    2012-11-01

    The presence of a high phase I IgG antibody titre may indicate chronic infection and a risk for the transmission of Coxiella burnetii through blood transfusion. The outbreak of Q fever in the Netherlands allowed for the comparison of an enzyme immunoassay (EIA) with the reference immunofluorescence assay (IFA) in a large group of individuals one year after acute Q fever. EIA is 100 % sensitive in detecting high (≥1:1,024) phase I IgG antibody titres. The cost of screening with EIA and confirming all EIA-positive results with IFA is much lower than screening all donations with IFA. This should be taken into account in cost-effectiveness analyses of screening programmes.

  11. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    PubMed Central

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  12. Agglutination of Histoplasma capsulatum by IgG Monoclonal Antibodies against Hsp60 Impacts Macrophage Effector Functions▿

    PubMed Central

    Guimarães, Allan Jefferson; Frases, Susana; Pontes, Bruno; de Cerqueira, Mariana Duarte; Rodrigues, Marcio L.; Viana, Nathan Bessa; Nimrichter, Leonardo; Nosanchuk, Joshua Daniel

    2011-01-01

    Histoplasma capsulatum can efficiently survive within macrophages, facilitating H. capsulatum translocation from the lung into the lymphatics and bloodstream. We have recently generated monoclonal antibodies (MAbs) to an H. capsulatum surface-expressed heat shock protein of 60 kDa (Hsp60) that modify disease in a murine histoplasmosis model. Interestingly, the MAbs induced different degrees of yeast cell agglutination in vitro. In the present study, we characterized the agglutination effects of the antibodies to Hsp60 on H. capsulatum yeast cells by light microscopy, flow cytometry, dynamic light scattering, measuring zeta potential, and using optical tweezers. We found that immunoglobulin Gs (IgGs) to Hsp60 cause H. capsulatum aggregation dependent on the (i) concentration of MAbs, (ii) MAb binding constant, and (iii) IgG subclass. Furthermore, infection of macrophages using agglutinates of various sizes after incubation with different Hsp60-binding MAbs induced association to macrophages through distinct cellular receptors and differentially affected macrophage antifungal functions. Hence, the capacity of IgG MAbs to agglutinate H. capsulatum significantly impacted pathogenic mechanisms of H. capsulatum during macrophage infection, and the effect was dependent on the antibody subclass and antigen epitope. PMID:21134968

  13. Factors affecting allergen-specific IgE serum levels in cats

    PubMed Central

    Belova, S.; Wilhelm, S.; Linek, M.; Beco, L.; Fontaine, J.; Bergvall, K.; Favrot, C.

    2012-01-01

    Pruritic skin diseases are common in cats and demand rigorous diagnostic workup for finding an underlying etiology. Measurement of a serum allergen-specific IgE in a pruritic cat is often used to make or confirm the diagnosis of a skin hypersensitivity disease, although current evidence suggests that elevated allergen-specific IgE do not always correlate with a clinical disease and vice versa. The aim of the study was to to assess the possible influence of age, deworming status, lifestyle, flea treatment, and gender on allergen-specific IgE levels and to evaluate the reliability of IgE testing in predicting the final diagnosis of a pruritic cat. For this purpose sera of 179 cats with pruritus of different causes and 20 healthy cats were evaluated for allergen-specific IgE against environmental, food and flea allergens using the Fc-epsilon receptor based enzyme-linked immunosorbent assay (ELISA) test. The results of the study showed positive correlation between age, outdoor life style, absence of deworming, absence of flea control measures and levels of allergen-specific IgE. Gender and living area (urban versus rural) did not seem to affect the formation of allergen-specific IgE. According to these findings, evaluating allergen-specific IgE levels, is not a reliable test to diagnose hypersensitivity to food or environmental allergens in cats. On the contrary, this test can be successfully used for diagnosing feline flea bite hypersensitivity. PMID:22754094

  14. The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands*

    PubMed Central

    Rayner, Lucy E.; Hui, Gar Kay; Gor, Jayesh; Heenan, Richard K.; Dalby, Paul A.; Perkins, Stephen J.

    2015-01-01

    The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s20,w0 of 6.3–6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration Rg increased with salt concentration, whereas the neutron Rg values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications. PMID:25659433

  15. Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients.

    PubMed

    Algaar, Fairoz; Eltzov, Evgeni; Vdovenko, Marina M; Sakharov, Ivan Yu; Fajs, Luka; Weidmann, Manfred; Mirazimi, Ali; Marks, Robert S

    2015-08-18

    Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, the Middle East, and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on real-time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore, there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive than colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect a small amount of antibodies at an early stage of infection and can be integrated as a point-of-care diagnostic system of CCHF.

  16. Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

    PubMed Central

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J.; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S.; Burton, Dennis R.

    2013-01-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  17. Allergen-specific immunoglobulin E and allergic rhinitis severity

    PubMed Central

    Corsico, Angelo G.; De Amici, Mara; Ronzoni, Vanessa; Giunta, Vania; Mennitti, Maria Chiara; Viscardi, Arianna; Marseglia, Gian Luigi

    2017-01-01

    Background: Allergic rhinitis (AR) is a common disorder. The diagnosis is based on the concordance between allergy sensitization and history. Serum allergen specific immunoglobulin E (sIgE) assessment allows characterization of the relevant sensitizing allergens. Presently, Allergic Rhinitis and its Impact on Asthma (ARIA) classification subdivides AR based on symptoms severity and duration. However, the relationship between sIgE levels and symptom severity is still a matter of debate. Objective: Therefore, this study aimed at relating sIgE levels with symptom severity assessed by ARIA classification in a group of patients with AR. Methods: We enrolled 217 patients with AR (123 women; median age, 39.5 years). The sIgE levels (expressed in kUA/L) to house-dust mite were detected by the fluorescence enzyme immunoassay in peripheral blood samples. The IgE calibrators were traceable to the second international reference preparation 75/502 of human serum IgE from the World Health Organization. Symptom severity was assessed by ARIA classification. Results: We found a significant difference in sIgE levels in patients with mild intermittent versus mild persistent symptoms (p < 0.05), mild intermittent versus moderate-to-severe persistent symptoms (p < 0.001), moderate-to-severe intermittent versus moderate-to-severe persistent symptoms (p < 0.01), and mild persistent versus moderate-to-severe persistent symptoms (p < 0.05). Conclusion: Analysis of these findings indicated that the sIgE level to house-dust mite might be a reliable biomarker for symptom severity in patients with AR. This outcome might be clinically relevant, particularly in candidates for immunotherapy. PMID:28381320

  18. Production and Purification of a Polyclonal Antibody Against Purified Mouse IgG2b in Rabbits Towards Designing Mouse Monoclonal Isotyping Kits

    PubMed Central

    Eivazi, Sadeq; Majidi, Jafar; Aghebati Maleki, leili; Abdolalizadeh, Jalal; Yousefi, Mehdi; Ahmadi, Majid; Dadashi, Somayeh; Moradi, Zahra; Zolali, Elmira

    2015-01-01

    Purpose: Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Methods: Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. Results: The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. Conclusion: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies. PMID:25789227

  19. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    PubMed Central

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  20. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

    PubMed

    Gilbert, Amy E; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H; Takhar, Pooja; Geh, Jenny L C; Healy, Ciaran; Harries, Mark; Acland, Katharine M; Rudman, Sarah M; Beavil, Rebecca L; Blower, Philip J; Beavil, Andrew J; Gould, Hannah J; Spicer, James; Nestle, Frank O; Karagiannis, Sophia N

    2011-04-29

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  1. Comparison of Total and IgG ABO Antibody Titers in Healthy Individuals by Using Tube and Column Agglutination Techniques

    PubMed Central

    Park, Eun Su; Jo, Kyung Il; Park, Rojin; Choi, Tae Yoon; Bang, Hae In; Chai, Gum Ran; Yun, Soon Gyu

    2014-01-01

    Background Most immune reactions related to transfusion and transplantation are caused by IgM ABO antibodies. However, IgG also plays an important role in these reactions. Therefore, a method to measure antibodies, including IgG, is necessary. We investigated ABO antibody titers of healthy individuals using a column agglutination technique (CAT) with or without dithiothreitol (DTT) and compared them with titers obtained using a conventional tube method. Methods Among healthy adults who underwent a medical examination, 180 individuals (60 with blood group A, 60 with group B, and 60 with group O) were selected. Antibody titrations were performed using the immediate spin (IS) tube, anti-human globulin (AHG) tube, and CAT with or without DTT methods. Results Higher median values of anti-B and anti-A titers in groups A and B individuals, respectively, were obtained using the IS method than using the AHG method. Higher values for group O individuals were obtained using the AHG method. Higher median titers of anti-B and anti-A in group O individuals were obtained using CAT without DTT than using the AHG method. Median titers of anti-B and anti-A in all blood groups were higher in CAT without DTT than in CAT with DTT, especially for group O individuals. Conclusions We recommend CAT with and without DTT for titration of anti-A and anti-B, especially in group O individuals, to provide more sensitive results that include IgG data. Adjustment of insurance coverage of fees associated with antibody titration might be necessary, considering the actual cost of reagents and personnel. PMID:24790910

  2. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    SciTech Connect

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  3. Polyclonal Activation of the Murine Immune System by an Antibody to IgD. 2. Generation of Polyclonal Antibody Production and Cells with Surface IgG

    DTIC Science & Technology

    1982-08-01

    TO IGD , II. GENERATION OF POLYCLONAL ANTIBODY PRODUCTION AND CELLS WITH SURFACE IGG. F.DFINKELMANi I.SCHER, J,JoMOND, S.KESSLER, J.T.KUNG, AND E...1982 by The Ameuican Assoclari of irturlogists Prined in U.S.A POLYCLONAL ACTIVATION OF THE MURINE IMMUNE SYSTEM BY AN ANTIBODY TO IgD II. Generation...of the A linking of B cell surface IgD by ligand leads to 1) direct second wave of lymphocyte proliferation suggested that the in * activation of B

  4. Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.

    PubMed Central

    Patel, T P; Parekh, R B; Moellering, B J; Prior, C P

    1992-01-01

    A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it. Images Fig. 1. PMID:1497622

  5. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    PubMed

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

  6. Leukocyte procoagulant activity: enhancement of production in vitro by IgG and antigen-antibody complexes.

    PubMed Central

    Rothberger, H; Zimmerman, T S; Spiegelberg, H L; Vaughan, J H

    1977-01-01

    In a variety of immunologic diseases, fibrin-fibrinogen and immune complexes deposit in areas of tissue damage. However, the mechanisms which initiate fibrin-fibrinogen deposition have not been clarified. We find that the procoagulant activity of human leukocytes is markedly increased after incubation with immunoglobulin and immune complexes. This procoagulant activity is evident after 4-24 h incubation in the presence of as little as 0.1 mg/ml of autologous, isologous, or heterologous IgG. At least three of the four subclasses of IgG myeloma proteins are effective. Experiments with purified rabbit and rat antibodies demonstrate that enhancement of procoagulant activity is significantly greater with soluble antigen-antibody complexes than with immunoglobulin alone. In contrast, insoluble complexes are less affective than immunoglobulin alone. Artifacts due to endotoxin contamination of the IgG preparations were excluded on the basis of the differential sensitivities of immunoglobulin and endotoxin to heat and polymyxin B. Evidence is also presented which shows that enhancement of procoagulant activity involves the production, rather than a simple release, of leukocyte procoagulant activity in vitro. PMID:190271

  7. Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations.

    PubMed

    Gronski, P; Haas, T; Kanzy, E J; Lang, W; Röder, J; Ruhwedel, K; Simshäuser, K

    2003-09-01

    Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.

  8. High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

    PubMed

    Lunn, M P; Johnson, L A; Fromholt, S E; Itonori, S; Huang, J; Vyas, A A; Hildreth, J E; Griffin, J W; Schnaar, R L; Sheikh, K A

    2000-07-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

  9. Predominant role for activation-induced cytidine deaminase in generating IgG anti-nucleosomal antibodies of murine SLE.

    PubMed

    Detanico, Thiago; Guo, Wenzhong; Wysocki, Lawrence J

    2015-04-01

    Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLE-associated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda(-/-) mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.

  10. Suitability of bio-based ionic liquids for the extraction and purification of IgG antibodies.

    PubMed

    Mondal, Dibyendu; Sharma, Mukesh; Quental, Maria V; Tavares, Ana P M; Prasad, Kamalesh; Freire, Mara G

    2016-11-21

    In the past decade, remarkable advances in the production and use of antibodies as therapeutic drugs and in research/diagnostic fields have led to their recognition as value-added proteins. These biopharmaceuticals have become increasingly important, reinforcing the current demand for the development of more benign, scalable and cost-effective techniques for their purification. Typical polymer-polymer and polymer-salt aqueous biphasic systems (ABS) have been studied for such a goal; yet, the limited polarity range of the coexisting phases and their low selective nature still are their major drawbacks. To overcome this limitation, in this work, ABS formed by bio-based ionic liquids (ILs) and biocompatible polymers were investigated. Bio-based ILs composed of ions derived from natural sources, namely composed of the cholinium cation and anions derived from plants natural acids, have been designed, synthesized, characterized and used for the creation of ABS with polypropyleneglycol (PPG 400). The respective ternary phase diagrams were initially determined at 25 °C to infer on mixture compositions required to form aqueous systems of two phases, further applied in the extraction of pure immunoglobulin G (IgG) to identify the most promising bio-based ILs, and finally employed in the purification of IgG from complex and real matrices of rabbit serum. Remarkably, the complete extraction of IgG to the IL-rich phase was achieved in a single-step. With pure IgG a recovery yield of 100% was obtained, while with rabbit serum this value slightly decreased to ca. 85%. Nevertheless, a 58% enhancement in the IgG purity was achieved when compared with its purity in serum samples. The stability of IgG before and after extraction was also evaluated by size exclusion high-performance liquid chromatography (SE-HPLC), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR). In most ABS formed by bio-based ILs, IgG retained

  11. Patients with papular urticaria have IgG antibodies to bedbug (Cimex lectularius) antigens.

    PubMed

    Abdel-Naser, Mohamed Badawy; Lotfy, Ranya Adel; Al-Sherbiny, Maged Mustafa; Sayed Ali, Nehad Mahmoud

    2006-05-01

    IgG and IgE against salivary gland proteins of bedbug (Cimex lectularius) were assessed in comparison with mosquito (Culex pipiens) and flea (Pulex irritans) antigens in the sera of papular urticaria patients (group I), siblings without papular urticaria (group IIa), patients' parents (group IIb), and healthy controls (group III) (Immunoblotting). Anti-C. lectularius IgG was significantly recognized at 66 and 10 kDa in 40% of group I, besides others ranging from 45 to 107 kDa. Group IIa significantly reacted with 70 kDa (57.1%). Group IIb reacted with 21 and 8.5 kDa (26.7%). Sixty percent of group IIb and 100% of group III significantly identified a band of 12.5 kDa. IgG against C. pipiens was significantly recognized at a range of 18-105 kDa in group I, IIb (115, 7 kDa), and III (58, 50 kDa). Anti-P. irritans IgG was significantly recognized by group I (100, 70 kDa) and group IIa (60, 35 kDa). IgE response was confined to C. pipiens at 115 and 54 kDa in groups I and III, respectively, besides 68 and 58 kDa in group IIa. It is concluded that IgG is present against C. lectularius, C. pipiens, and P. irritans in papular urticaria and may contribute to its pathogenesis.

  12. Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA

    PubMed Central

    Humphreys, Alan F.; Tan, Jie; Peng, RongSheng; Benton, Susan M.; Qin, Xiang; Worley, Kim C.; Mikulski, Rose L.; Chow, Dar-Chone; Palzkill, Timothy G.; Ling, Paul D.

    2015-01-01

    Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV. PMID:25658336

  13. Persistence of IgE-Associated Allergy and Allergen-Specific IgE despite CD4+ T Cell Loss in AIDS

    PubMed Central

    Marth, Katharina; Wollmann, Eva; Gallerano, Daniela; Ndlovu, Portia; Makupe, Ian; Valenta, Rudolf; Sibanda, Elopy

    2014-01-01

    The infection of CD4+ cells by HIV leads to the progressive destruction of CD4+ T lymphocytes and, after a severe reduction of CD4+ cells, to AIDS. The aim of the study was to investigate whether HIV-infected patients with CD4 cell counts <200 cells/µl can suffer from symptoms of IgE-mediated allergy, produce allergen-specific IgE antibody responses and show boosts of allergen-specific IgE production. HIV-infected patients with CD4 counts ≤200 cells/µl suffering from AIDS and from IgE-mediated allergy were studied. Allergy was diagnosed according to case history, physical examination, skin prick testing (SPT), and serological analyses including allergen microarrays. HIV infection was confirmed serologically and the disease was staged clinically. The predominant allergic symptoms in the studied patients were acute allergic rhinitis (73%) followed by asthma (27%) due to IgE-mediated mast cell activation whereas no late phase allergic symptoms such as atopic dermatitis, a mainly T cell-mediated skin manifestation, were found in patients suffering from AIDS. According to IgE serology allergies to house dust mites and grass pollen were most common besides IgE sensitizations to various food allergens. Interestingly, pollen allergen-specific IgE antibody levels in the patients with AIDS and in additional ten IgE-sensitized patients with HIV infections and low CD4 counts appeared to be boosted by seasonal allergen exposure and were not associated with CD4 counts. Our results indicate that secondary allergen-specific IgE production and IgE-mediated allergic inflammation do not require a fully functional CD4+ T lymphocyte repertoire. PMID:24896832

  14. Persistence of IgE-associated allergy and allergen-specific IgE despite CD4+ T cell loss in AIDS.

    PubMed

    Marth, Katharina; Wollmann, Eva; Gallerano, Daniela; Ndlovu, Portia; Makupe, Ian; Valenta, Rudolf; Sibanda, Elopy

    2014-01-01

    The infection of CD4+ cells by HIV leads to the progressive destruction of CD4+ T lymphocytes and, after a severe reduction of CD4+ cells, to AIDS. The aim of the study was to investigate whether HIV-infected patients with CD4 cell counts <200 cells/µl can suffer from symptoms of IgE-mediated allergy, produce allergen-specific IgE antibody responses and show boosts of allergen-specific IgE production. HIV-infected patients with CD4 counts ≤ 200 cells/µl suffering from AIDS and from IgE-mediated allergy were studied. Allergy was diagnosed according to case history, physical examination, skin prick testing (SPT), and serological analyses including allergen microarrays. HIV infection was confirmed serologically and the disease was staged clinically. The predominant allergic symptoms in the studied patients were acute allergic rhinitis (73%) followed by asthma (27%) due to IgE-mediated mast cell activation whereas no late phase allergic symptoms such as atopic dermatitis, a mainly T cell-mediated skin manifestation, were found in patients suffering from AIDS. According to IgE serology allergies to house dust mites and grass pollen were most common besides IgE sensitizations to various food allergens. Interestingly, pollen allergen-specific IgE antibody levels in the patients with AIDS and in additional ten IgE-sensitized patients with HIV infections and low CD4 counts appeared to be boosted by seasonal allergen exposure and were not associated with CD4 counts. Our results indicate that secondary allergen-specific IgE production and IgE-mediated allergic inflammation do not require a fully functional CD4+ T lymphocyte repertoire.

  15. Characteristics of HPV-Specific Antibody Responses Induced by Infection and Vaccination: Cross-Reactivity, Neutralizing Activity, Avidity and IgG Subclasses

    PubMed Central

    Scherpenisse, Mirte; Schepp, Rutger M.; Mollers, Madelief; Meijer, Chris J. L. M.; Berbers, Guy A. M.; van der Klis, Fiona R. M.

    2013-01-01

    Objectives In order to assess HPV-specific IgG characteristics, we evaluated multiple aspects of the humoral antibody response that will provide insight in the HPV humoral immune response induced by HPV infection and vaccination. Methods Cross-reactivity of HPV-specific antibodies induced by infection or vaccination was assessed with VLP16 or 18 inhibition using a VLP-based multiplex immunoassay (MIA) for HPV16, 18, 31, 33, 45, 52 and 58. HPV16/18 specific IgG1-4 subclasses and avidity were determined with the VLP-MIA in sera after HPV infection and after vaccination. Neutralizing antibodies were determined in a small subset of single-seropositive and multi-seropositive naturally derived antibodies. Results Naturally derived antibodies from single-positive sera were highly genotype-specific as homologue VLP-inhibition percentages varied between 78-94%. In multi-positive sera, cross-reactive antibodies were observed both within and between α7 and α9 species. After vaccination, cross-reactive antibodies were mainly species-specific. Avidity of vaccine-derived HPV-specific antibodies was 3 times higher than that of antibodies induced by HPV infection (p<0.0001). IgG1 and IgG3 were found to be the predominant subclasses observed after HPV infection and vaccination. In the small subset tested, the number of single-positive sera with neutralizing capacity was higher than of multi-positive sera. Conclusion Naturally derived HPV-specific antibodies from single-positive samples showed different characteristics in terms of cross-reactivity and neutralizing capacity compared with antibodies from multi-positive sera. Post-vaccination, HPV antibody avidity was approximately 3 times higher than antibody avidity induced by HPV infection. Therefore, antibody avidity might be a potential surrogate of protection. PMID:24058629

  16. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the development aplastic crises.

    PubMed

    Sant'Anna, Anadayr L M; Garcia, Rita de Cássia N Cubel; Marzoche, Mônica; da Rocha, Heloisa Helena A Gallo; Paula, Maria Tereza M; Lobo, Clarisse C; Nascimento, Jussara P

    2002-01-01

    The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  17. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    PubMed

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  18. Detection of systemic and mucosal HPV-specific IgG and IgA antibodies in adolescent girls one and two years after HPV vaccination

    PubMed Central

    Scherpenisse, Mirte; Mollers, Madelief; Schepp, Rutger M.; Meijer, Chris J.L.M.; de Melker, Hester E.; Berbers, Guy A.M.; van der Klis, Fiona R.M.

    2013-01-01

    The bivalent HPV16/18 vaccine induces high antibody concentrations in serum while data about antibody responses in the cervix are limited. In this study, we investigated pre- and post-vaccination antibody responses against seven high-risk HPV types by detection of IgG and IgA HPV-specific antibodies in cervical secretion samples (CVS) and serum. From an HPV vaccine monitoring study CVS and serum samples were available (pre-vaccination (n = 297), one year (n = 211) and two years (n = 141) post-dose-one vaccination) from girls aged 14–16 y. The girls were vaccinated with the bivalent HPV vaccine at months 0, 1 and 6. CVS was self-sampled using a tampon. Samples were tested for HPV-specific antibodies (HPV16/18/31/33/45/52/58) by a VLP-based multiplex immunoassay. Post-vaccination, IgG and IgA antibody levels for HPV16/18 were detectable in CVS and amounted to 2% and 1% of the IgG and IgA antibody levels observed in serum, respectively. The antibody levels remained constant between one and two years after vaccination. The correlation between CVS and serum was similar for IgG and IgA vaccine-derived antibody levels for HPV16 (rs = 0.58, rs = 0.54) and HPV18 (rs = 0.50, rs = 0.55). Vaccine-derived IgG antibody levels against cross-reactive HPV types in CVS and in serum were highest for HPV45. No IgA cross-reactive antibody responses could be detected in CVS. Post-vaccination, HPV16/18 IgG and IgA antibodies are not only detectable in serum but also in CVS. The correlation of HPV16/18 IgG antibody levels between serum and CVS suggests that vaccine induced HPV antibodies transudate and/or exudate from the systemic circulation to the cervical mucosa to provide protection against HPV infections. PMID:23149693

  19. Detection of systemic and mucosal HPV-specific IgG and IgA antibodies in adolescent girls one and two years after HPV vaccination.

    PubMed

    Scherpenisse, Mirte; Mollers, Madelief; Schepp, Rutger M; Meijer, Chris J L M; de Melker, Hester E; Berbers, Guy A M; van der Klis, Fiona R M

    2013-02-01

    The bivalent HPV16/18 vaccine induces high antibody concentrations in serum while data about antibody responses in the cervix are limited. In this study, we investigated pre- and post-vaccination antibody responses against seven high-risk HPV types by detection of IgG and IgA HPV-specific antibodies in cervical secretion samples (CVS) and serum. From an HPV vaccine monitoring study CVS and serum samples were available (pre-vaccination (n = 297), one year (n = 211) and two years (n = 141) post-dose-one vaccination) from girls aged 14-16 y. The girls were vaccinated with the bivalent HPV vaccine at months 0, 1 and 6. CVS was self-sampled using a tampon. Samples were tested for HPV-specific antibodies (HPV16/18/31/33/45/52/58) by a VLP-based multiplex immunoassay. Post-vaccination, IgG and IgA antibody levels for HPV16/18 were detectable in CVS and amounted to 2% and 1% of the IgG and IgA antibody levels observed in serum, respectively. The antibody levels remained constant between one and two years after vaccination. The correlation between CVS and serum was similar for IgG and IgA vaccine-derived antibody levels for HPV16 (rs = 0.58, rs = 0.54) and HPV18 (rs = 0.50, rs = 0.55). Vaccine-derived IgG antibody levels against cross-reactive HPV types in CVS and in serum were highest for HPV45. No IgA cross-reactive antibody responses could be detected in CVS. Post-vaccination, HPV16/18 IgG and IgA antibodies are not only detectable in serum but also in CVS. The correlation of HPV16/18 IgG antibody levels between serum and CVS suggests that vaccine induced HPV antibodies transudate and/or exudate from the systemic circulation to the cervical mucosa to provide protection against HPV infections.

  20. An investigation into the effect of the IgG antibody system on the susceptibility of IgA-deficient patients to respiratory tract infections.

    PubMed Central

    French, M A; Harrison, G

    1986-01-01

    Serum IgG subclass concentrations and IgG-tetanus toxoid antibody (IgG-TTab) responses were measured in IgA-deficient patients with severe respiratory tract infections (n = 11), mild respiratory tract infections (n = 5) or no increased susceptibility to respiratory tract infections (n = 15). The severe infection group had lower IgG2 concentrations than the patients without infections (P less than 0.02) and was the only group with IgG2-deficient patients (36%). The number of sera in which IgG4 was not detected was higher in patients with severe infections than in both normal controls (45% vs 10%, P less than 0.01) and the other IgA-deficient patients (45% vs 20%), in part explained by a strong association with IgG2 deficiency. Subnormal IgG-TTab responses were demonstrated in 45% of patients with severe infections but in only one patient from each of the other two groups. Five patients with IgG2 deficiency and/or subnormal IgG-TTab responses were treated with gammaglobulin and apparently improved. There was a high serum concentration of IgG1 in 35% and IgG3 in 19% of the 31 patients, predominantly in those without severe infections. Thus a proportion of IgA-deficient patients have additional defects of IgG; IgG1 and IgG3 antibody responses may compensate for the IgA deficiency in asymptomatic patients. PMID:3568451

  1. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.

    PubMed

    Irani, Vashti; Guy, Andrew J; Andrew, Dean; Beeson, James G; Ramsland, Paul A; Richards, Jack S

    2015-10-01

    Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity.

  2. 2-Hydroxyethyl methacrylate (HEMA) promotes IgG but not IgM antibody production in vivo in mice.

    PubMed

    Andersson, Jennie; Dahlgren, Ulf

    2011-08-01

    Individuals working in a dental clinic are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the immune system, including acting as an adjuvant in mice and stimulating the production of human IgG1 in vitro. In this study we continued to explore the immunomodulatory properties of HEMA in mice. Mice were co-injected subcutaneously with the following: HEMA + ovalbumin (OVA) in bicarbonate buffer, OVA in bicarbonate buffer, HEMA in bicarbonate buffer, or bicarbonate buffer alone. Mice immunized with OVA were killed 2 wk after a booster injection. Mice exposed to HEMA only were killed 6 d after the last injection with HEMA. Serum and spleens were collected. The activities of anti-OVA IgG and anti-OVA IgM were determined using ELISAs, as was the in vitro production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by splenocytes after 2 d of incubation. Splenocyte proliferation was analyzed using [(3) H]thymidine decomposition. Mice exposed twice to HEMA in vivo had a higher baseline and a higher concanavalin A-stimulated proliferation of splenocytes, and produced less TNF-α in relation to IL-6, compared with controls. Immunization of mice with OVA/HEMA resulted in a higher anti-OVA IgG activity, relative to anti-OVA IgM activity, compared with controls. In conclusion, HEMA has selective effects on cytokine and antibody production in mice.

  3. Standardization of Assays That Detect Anti-Rubella Virus IgG Antibodies

    PubMed Central

    Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle

    2015-01-01

    SUMMARY Rubella virus usually causes a mild infection in humans but can cause congenital rubella syndrome (CRS). Vaccination programs have significantly decreased primary rubella virus infection and CRS; however, vaccinated individuals usually have lower levels of rubella virus IgG than those with natural infections. Rubella virus IgG is quantified with enzyme immunoassays that have been calibrated against the World Health Organization (WHO) international standard and report results in international units per milliliter. It is recognized that the results reported by these assays are not standardized. This investigation into the reasons for the lack of standardization found that the current WHO international standard (RUB-1-94) fails by three key metrological principles. The standard is not a pure analyte but is composed of pooled human immunoglobulin. It was not calibrated by certified reference methods; rather, superseded tests were used. Finally, no measurement uncertainty estimations have been provided. There is an analytical and clinical consequence to the lack of standardization of rubella virus IgG assays, which leads to misinterpretation of results. The current approach to standardization of rubella virus IgG assays has not achieved the desired results. A new approach is required. PMID:26607813

  4. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  5. Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assays based on recombinant TBEV subviral particles.

    PubMed

    Levanov, Lev; Vera, Cristina Pérez; Vapalahti, Olli

    2016-07-01

    Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

  6. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

    PubMed Central

    Liu, A Y; Robinson, R R; Hellström, K E; Murray, E D; Chang, C P; Hellström, I

    1987-01-01

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man. PMID:3106970

  7. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.

    PubMed

    Nogales-Gadea, Gisela; Saxena, Abhishek; Hoffmann, Carolin; Hounjet, Judith; Coenen, Daniëlle; Molenaar, Peter; Losen, Mario; Martinez-Martinez, Pilar

    2015-06-05

    Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

  8. Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen.

    PubMed Central

    Challacombe, S J; Rahman, D; Jeffery, H; Davis, S S; O'Hagan, D T

    1992-01-01

    Intragastric immunization may lead to the induction of antibodies in the secretory immune system including saliva. The antibody response is usually short-lived. The objectives of this study were to see whether oral immunization with biodegradable microparticles containing antigen might lead to enhanced mucosal responses. Ovalbumin (OVA) was entrapped in a novel antigen delivery system comprising poly (D,L-lactide-co-glycolide) (PLGA) microparticles. Salivary IgA and serum IgG responses after three daily oral immunizations in BALB/c mice were assayed by ELISA at weekly intervals and compared with those to soluble antigen. Low levels of salivary IgA antibodies were detected at Weeks 2 and 3 in both groups and no significant differences were found. After a secondary series of intragastric immunizations at Week 4, marked differences were apparent between the groups. The mean salivary IgA titre at Week 6 was 959 +/- 494 U compared with 30 +/- 5 in the soluble OVA group (P less than 0.0001). Significant differences were still apparent at Weeks 7-8 through the value was falling. Serum IgG antibodies were detectable and were significantly greater in the particle group (at Weeks 4 and 8) than in controls (P less than 0.001). These results suggest that microparticles are taken up by antigen-presenting cells in Peyer's patches, then slowly degrade in vivo and release entrapped antigens, and thus can function as potent antigen delivery systems giving rise to both mucosal and systemic responses. Microparticles have considerable potential as a controlled released antigen delivery system for the induction of longer-term immune responses at mucosal surfaces. PMID:1628895

  9. Immunogenic and antigenic epitopes of immunoglobulins I. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species.

    PubMed Central

    Jefferis, R; Lowe, J; Ling, N R; Porter, P; Senior, S

    1982-01-01

    Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-C gamma 3, fourteen anti-C gamma 2, one anit-kappa and one anti-lambda) were selected for study of their cross-reactivity with the IgG of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgG expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fc gamma and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human IgG. Four clones demonstrated specificity for bovine IgG subclass gamma 1 and gamma 2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents. PMID:6173313

  10. Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma.

    PubMed

    Pinlaor, Porntip; Pongsamart, Porntip; Hongsrichan, Nuttanan; Sangka, Arunnee; Srilunchang, Thitima; Mairiang, Eimorn; Sithithaworn, Paiboon; Pinlaor, Somchai

    2012-03-01

    Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels-against these antigens from subjects with O. viverrini-positive HBD-higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA>severe HBD>mild HBD>healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.

  11. Aggregation and pH-temperature phase behavior for aggregates of an IgG2 antibody.

    PubMed

    Sahin, Erinc; Weiss, William F; Kroetsch, Andrew M; King, Kevin R; Kessler, R Kendall; Das, Tapan K; Roberts, Christopher J

    2012-05-01

    Monomer unfolding and thermally accelerated aggregation kinetics to produce soluble oligomers or insoluble macroscopic aggregates were characterized as a function of pH for an IgG2 antibody using differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC). Aggregate size was quantified via laser light scattering, and aggregate solubility via turbidity and visual inspection. Interestingly, nonnative oligomers were soluble at pH 5.5 above approximately 15°C, but converted reversibly to visible/insoluble particles at lower temperatures. Lower pH values yielded only soluble aggregates, whereas higher pH resulted in insoluble aggregates, regardless of the solution temperature. Unlike the growing body of literature that supports the three-endotherm model of IgG1 unfolding in DSC, the results here also illustrate limitations of that model for other monoclonal antibodies. Comparison of DSC with monomer loss (via SEC) from samples during thermal scanning indicates that the least conformationally stable domain is not the most aggregation prone, and that a number of the domains remain intact within the constituent monomers of the resulting aggregates. This highlights continued challenges with predicting a priori which domain(s) or thermal transition(s) is(are) most relevant for product stability with respect to aggregation.

  12. Association of WT1 IgG antibody against WT1 peptide with prolonged survival in glioblastoma multiforme patients vaccinated with WT1 peptide

    PubMed Central

    Hashimoto, Naoya; Tsuboi, Akihiro; Murakami, Yui; Iwai, Miki; Kagawa, Naoki; Chiba, Yasuyoshi; Izumoto, Shuichi; Elisseeva, Olga; Ichinohasama, Ryo; Sakamoto, Junichi; Morita, Satoshi; Nakajima, Hiroko; Takashima, Satoshi; Nakae, Yoshiki; Nakata, Jun; Kawakami, Manabu; Nishida, Sumiyuki; Hosen, Naoki; Fujiki, Fumihiro; Morimoto, Soyoko; Adachi, Mayuko; Iwamoto, Masahiro; Oka, Yoshihiro; Yoshimine, Toshiki; Sugiyama, Haruo

    2016-01-01

    We previously evaluated Wilms’ tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA‐A*24:02 restricted, 9‐mer WT1‐235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1‐235‐specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1‐235 peptide (WT1‐235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1‐235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1‐235 IgG antibody subclass was Th1‐type, IgG1 and IgG3. WT1‐235 IgG antibody production was significantly and positively correlated with both progression‐free survival (PFS) and overall survival (OS). Importantly, the combination of WT1‐235 IgG antibody production and positive delayed type‐hypersensitivity (DTH) to the WT1‐235 peptide was a better prognostic marker for long‐term OS than either parameter alone. These results suggested that WT1‐235 peptide vaccination induces not only WT1‐235‐specific CTLs as previously described but also WT1‐235‐specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine‐treated patients. PMID:27170523

  13. Hidden IgG Antibodies to the Tumor-Associated Thomsen-Friedenreich Antigen in Gastric Cancer Patients: Lectin Reactivity, Avidity, and Clinical Relevance

    PubMed Central

    Kurtenkov, Oleg

    2017-01-01

    Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients (P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed (P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors (P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival. PMID:28316982

  14. Heteroantibody-mediated cytotoxicity: antibody to the high affinity Fc receptor for IgG mediates cytotoxicity by human monocytes that is enhanced by interferon-gamma and is not blocked by human IgG.

    PubMed

    Shen, L; Guyre, P M; Anderson, C L; Fanger, M W

    1986-12-01

    An IgG1 monoclonal antibody, 32.2, raised against the 72,000 dalton monocyte high affinity Fc receptor, was used to examine the role of this receptor in ADCC. This antibody did not inhibit the binding of human IgG1 to monocytes or to the U937 cell line, nor did it block or stimulate their killing of IgG-coated chicken erythrocytes (CE). Whole 32.2 or its Fab fragments were cross-linked to Fab fragments of rabbit anti-CE by using the agent SPDP. The resulting heteroantibodies (32.2 X Fab anti-CE) mediated monocyte and U937 cytotoxicity against CE, whereas an anti-HLA X anti-CE reagent did not. Both FcR expression and heteroantibody-mediated cytotoxicity were increased by culturing monocytes or U937 with IFN-gamma. Although IgG-mediated ADCC was significantly inhibited by 40 micrograms/ml human IgG1, cytotoxicity mediated by 32.2 X Fab anti-CE was not blocked by 2 mg/ml human IgG1, suggesting that such cytotoxicity might not be blocked by IgG in vivo. These data indicate the potential of 32.2 heteroantibodies in analysis of FcR function and in therapy.

  15. Liver-X-receptor activator prevents homocysteine-induced production of IgG antibodies from murine B lymphocytes via the ROS-NF-{kappa}B pathway

    SciTech Connect

    Chang Lina; Zhang, Zhenmin; Li Wenjing; Dai Jing; Guan Youfei; Wang Xian . E-mail: xwang@bjmu.edu.cn

    2007-06-08

    Our previous study showed that homosysteine (Hcy) promotes proliferation of mouse splenic B lymphocytes. In this study, we investigated whether Hcy could stimulate the production of IgG antibodies. Hcy significantly increased the production of IgG antibodies from resting B lymphocytes. B lymphocytes from ApoE-knockout mice with hyperhomocysteinemia showed elevated IgG secretion at either the basal Hcy level or in response to lipopolysaccharide. Hcy promoted reactive oxygen species (ROS) formation, and free radical scavengers, MnTMPyP decreased Hcy-induced IgG secretion. The inhibitor of NF-{kappa}B (MG132) also significantly reduced Hcy-induced IgG secretion. Furthermore, Hcy-induced formation of ROS, activation of NF-{kappa}B, and secretion of IgG could be inhibited by the liver-X-receptor (LXR) agonist TO 901317. Thus, our data provide strong evidence that HHcy induces IgG production from murine splenic B lymphocytes both in vitro and in vivo. The mechanism might be through the ROS-NF-{kappa}B pathway and can be attenuated by the activation of LXR.

  16. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa.

    PubMed

    Damgaard, Johanne; Meyrowitsch, Dan W; Rwegoshora, Rwehumbiza T; Magesa, Stephen M; Mukoko, Dunstan A; Simonsen, Paul E

    2016-09-01

    A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection). The antibodies are commonly regarded as markers of infection and/or exposure to filarial larvae, but a direct association between the antibodies and these indices has not been well documented. The present study assessed the role and relative effect of potential drivers of the human IgG4 antibody reactivity to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4 antibodies to Bm14, and associations between antibody reactivity and the different variables were statistically analyzed. IgG4 reactivity to Bm14 was highly positively associated with CFA, and to a lesser extent with age. However, an expected association with household exposure to infective filarial larvae was not found. Bm14 antibody reactivity thus appeared mainly to reflect actual infection of individuals with adult filarial worms rather than ongoing exposure to transmission. The analyses moreover suggested that many of the CFA negative but Bm14 positive individuals had early or low level infections where antibodies had been induced but where CFA was not (yet?) measurable. Although the study indicated that IgG4 reactivity to Bm14 is a marker of filarial infection, assessment of this reactivity, especially in children, will still be useful for indirect monitoring of changes in transmission intensity, including break of transmission and post-elimination surveillance, in LF control.

  17. CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

    PubMed

    Miescher, S; Zahn-Zabal, M; De Jesus, M; Moudry, R; Fisch, I; Vogel, M; Kobr, M; Imboden, M A; Kragten, E; Bichler, J; Mermod, N; Stadler, B M; Amstutz, H; Wurm, F

    2000-10-01

    Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

  18. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    PubMed Central

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgGantibodies. PMID:23567210

  19. Prevalence of IgG Antibodies to Toxoplasma gondii in Veterinary and Undergraduate Students at Virginia Tech, Blacksburg, Virginia.

    PubMed

    Rosypal, A C; Houk, A E; Zajac, A M; Lindsay, D S

    2015-11-01

    Toxoplasma gondii is a globally distributed parasitic protozoan that infects humans and other warm-blooded vertebrates. Felids are the only definitive host for T. gondii, and they excrete oocysts in their faeces. The national prevalence in humans is declining in the United States. This zoonotic organism is of particular interest due to its importance in pregnant women, in individuals with altered immune systems, and in reactivated ocular infections. Exposure to the parasite in humans is usually associated with consumption of raw or undercooked meat or by accidental ingestion of oocysts. It was hypothesized that veterinary students would have a greater chance at exposure to the parasite than an average population of undergraduate students due to increased contact with cats who are infected. A commercially available ELISA was used to examine serum samples from 336 students (252 veterinary students and 84 undergraduate students) at Virginia Polytechnic Institute and State University and the Virginia-Maryland Regional College of Veterinary Medicine for serum IgG antibodies to T. gondii antigen. The prevalence of T. gondii in these subjects was 5.6% in veterinary school students (n = 252) and 2.4% in undergraduates (n = 84). There was no significant difference (P > 0.05) in the prevalence of T. gondii antibodies in veterinary versus undergraduate students. The overall prevalence of 4.8% in all students in this study reflects the continuing decline of antibodies to T. gondii in humans in the United States.

  20. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

    PubMed

    de Jong, Rob N; Beurskens, Frank J; Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J; Oostindie, Simone C; Wang, Guanbo; Heck, Albert J R; Schuurman, Janine; Parren, Paul W H I

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

  1. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

    PubMed Central

    Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D.; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J.; Oostindie, Simone C.; Wang, Guanbo; Heck, Albert J. R.; Schuurman, Janine; Parren, Paul W. H. I.

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen. PMID:26736041

  2. First steps in the standardization of immunoglobulin IgG myeloperoxidase-anti-neutrophil cytoplasmic antibody measurements.

    PubMed

    Hutu, D P; Tuddenham, E; Monogioudi, E; Meroni, P; Schimmel, H; Sheldon, J; Zegers, I

    2016-02-01

    The standardization of immunoassays for immunoglobulin (Ig)G myeloperoxidase-anti-neutrophil cytoplasmic antibodies (MPO-ANCA) could contribute to a more accurate diagnosis and follow-up of small vessels-associated vasculitis, a systemic autoimmune disorder that leads to necrosis of blood vessel walls. Despite significant efforts by different groups, the level of comparability of results from commercially available immunoassays used for IgG MPO-ANCA detection is still poor. Therefore, the potential for improvement using reference materials was assessed. The evaluation of a set of 30 patient samples with 11 assays showed that differences between assays result in different interpretations for individual patients. Only 10 of 30 patient samples had the same clinical interpretation among 11 assays applying the cut-off values provided by each respective manufacturer. The correlation between results from 13 different assays was assessed in a pairwise manner. The correlation between results from patient samples was systematically very good for combinations of seven of those assays. The correlation of results ranged from reasonable to good for combinations with four other assays, therefore it should be possible to improve the comparability of results using a commutable reference material for calibration. Feasibility studies were conducted in order to find a reference material format most suitable for a calibrator. Two sets of candidate reference materials were produced from different raw materials, and assessed according to their suitability. A final format was selected, and a candidate reference material was produced.

  3. Role of IgA, IgG, and IgM antibodies against Haemophilus parainfluenzae antigens in IgA nephropathy.

    PubMed

    Suzuki, S; Gejyo, F; Nakatomi, Y; Odani, S; Sato, H; Arakawa, M

    1996-11-01

    We have recently demonstrated glomerular deposition of outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and the presence of IgA antibody against OMHP in patients with IgA nephropathy (IgA-N). In this study, we analyzed IgA-, IgG-, and IgM-classes of antibodies against OMHP, and the relationship between these antibodies and renal lesions in IgA-N. The subjects included 44 patients with IgA-N and 62 patients with outer glomerular diseases (OGD); the latter group consisted of 23 patients with predominantly IgG or IgM deposits and small amounts of IgA in the mesangium (group A), and 39 with IgG or IgM deposits without IgA (group B). IgA, IgG, and IgM antibodies against OMHP in patients sera were detected by enzyme-linked immunosorbent assay (ELISA). Immunoblotting demonstrated that the IgA, IgG, and IgM antibodies against OMHP in the sera of IgA-N patients bound to components of OMHP with molecular weights of 19.5, 30, and 40.5 kD. The amino acid compositions of these three OMHP components were similar to those reported for the outer membrane protein (OMP) P6 precursor, OMP P5, and OMP P2 (porin) of Haemophilus influenzae. Both IgA-N and group A patients, (i.e. those with IgA-related renal disease), demonstrated a significantly higher level of IgA antibodies against OMHP than did group B patients. However, only IgA-N patients revealed a significant correlation between the IgA-antibody titer and degree of glomerular changes. IgA-N patients with macroscopic hematuria or arterio(lo)sclerosis had a significantly higher IgA antibody titer than other IgA-N patients. There was no relationship between renal lesions and IgG or IgM antibody titers in any group. These findings suggest that IgA antibodies against OMHP are significantly increased in patients with IgA-related renal disease compared to those without mesangial IgA deposits and that a significant relationship between these antibodies and renal lesions exists only in patients with IgA-N.

  4. Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

    PubMed Central

    Vojdani, Aristo

    2009-01-01

    Background Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. PMID:19435515

  5. Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

    PubMed

    Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

    2014-07-17

    Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs.

  6. Detection of anti-infliximab antibodies is impacted by antibody titer, infliximab level and IgG4 antibodies: a systematic comparison of three different assays

    PubMed Central

    Afonso, Joana; Lopes, Susana; Gonçalves, Raquel; Caldeira, Paulo; Lago, Paula; Tavares de Sousa, Helena; Ramos, Jaime; Gonçalves, Ana Rita; Ministro, Paula; Rosa, Isadora; Vieira, Ana Isabel; Coelho, Rosa; Tavares, Patrícia; Soares, João; Sousa, Ana Lúcia; Carvalho, Diana; Sousa, Paula; da Silva, João Pereira; Meira, Tânia; Silva Ferreira, Filipa; Dias, Cláudia Camila; Chowers, Yehuda; Ben-Horin, Shomron; Magro, Fernando

    2016-01-01

    Background: There is scant information on the accuracy of different assays used to measure anti-infliximab antibodies (ADAs), especially in the presence of detectable infliximab (IFX). We thus aimed to evaluate and compare three different assays for the detection of IFX and ADAs and to clarify the impact of the presence of circulating IFX on the accuracy of the ADA assays. Methods: Blood samples from 79 ulcerative colitis (UC) patients treated with infliximab were assessed for IFX levels and ADAs using three different assays: an in-house assay and two commercial kits, Immundiagnostik and Theradiag. Sera samples with ADAs and undetectable levels of IFX were spiked with exogenous IFX and analyzed for ADAs. Results: The three assays showed 81–96% agreement for the measured IFX level. However, the in-house assay and Immundiagnostik assays detected ADAs in 34 out of 79 samples, whereas Theradiag only detected ADAs in 24 samples. Samples negative for ADAs with Theradiag, but ADA-positive in both the in-house and Immundiagnostik assays, were positive for IFX or IgG4 ADAs. In spiking experiments, a low concentration of exogenous IFX (5 µg/ml) hampered ADA detection with Theradiag in sera samples with ADA levels of between 3 and 10 µg/ml. In the Immundiagnostik assay detection interference was only observed at concentrations of exogenous IFX higher than 30 µg/ml. However, in samples with high levels of ADAs (>25 µg/ml) interference was only observed at IFX concentrations higher than 100 µg/ml in all three assays. Binary (IFX/ADA) stratification of the results showed that IFX+/ADA- and IFX-/ADAs+ were less influenced by the assay results than the double-positive (IFX+/ADAs+) and double-negative (IFX-/ADAs-) combination. Conclusions: All three methodologies are equally suitable for measuring IFX levels. However, erroneous therapeutic decisions may occur when patients show double-negative (IFX-/ADAs-) or double-positive (IFX+/ADAs+) status, since agreement between

  7. Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

    PubMed

    Armour, Kathryn L; Smith, Cheryl S; Ip, Natasha C Y; Ellison, Cara J; Kirton, Christopher M; Wilkes, Anthony M; Williamson, Lorna M; Clark, Michael R

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

  8. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    PubMed

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  9. Evaluation of the diagnostic value of measuring IgG, IgM, and IgA antibodies to mycobacterial A60 antigen in active tuberculosis.

    PubMed

    Ben-selma, Walid; Harizi, Hedi; Marzouk, Manel; Ben Kahla, Imen; Ben Lazreg, Foued; Ferjeni, Asma; Boukadida, Jalel

    2010-09-01

    The purpose of the present study was to evaluate the clinical usefulness of detection of serum immunoglobulin A (IgA), IgG, and IgM antibodies raised against the mycobacterial A60 antigen for the diagnosis and discrimination of active tuberculosis (TB) from other pulmonary diseases. Three commercially available ELISA kits (IgA, IgG, and IgM) (ANDA Biologicals, Strasbourg, France) were evaluated simultaneously in 246 serum samples from 3 groups of patients: group I, 171 patients with active TB (128 pulmonary TB and 43 extrapulmonary TB); group II, 73 patients with pulmonary non-TB diseases; and group III, 2 leprosies patients. The sensitivities of tests ranged from 31.3% (IgA) to 94% (IgG) in pulmonary TB patients and from 21% (IgA) to 84% (IgG) in extrapulmonary TB patients. The specificities of assays varied from 92% (IgG) to 96% (IgA) in the pulmonary non-TB group. Combination of IgG with IgA and/or IgM does not improve its sensitivity. Clinical use of the A60-based serodiagnostic IgG assay is of great value for the rapid diagnosis and discrimination between active TB and pulmonary non-TB diseases. Moreover, this test could be used to increase diagnostic accuracy, especially for smear-negative TB cases, which are difficult to diagnose.

  10. Specific IgG Antibodies React to Mimotopes of BK Polyomavirus, a Small DNA Tumor Virus, in Healthy Adult Sera

    PubMed Central

    Pietrobon, Silvia; Bononi, Ilaria; Mazzoni, Elisa; Lotito, Francesca; Manfrini, Marco; Puozzo, Andrea; Destro, Federica; Guerra, Giovanni; Nocini, Pier Francesco; Martini, Fernanda; Tognon, Mauro G.

    2017-01-01

    BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon after its identification, BKPyV was characterized as a kidney-tropic virus, which is responsible of a significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental conditions, BKPyV is able to transform different types of animal and human cells and to induce tumors of different histotypes in experimental animals. BKPyV DNA sequences have been detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot hybridization methods. Serum antibodies against this polyomavirus were revealed using immunological techniques, which, however, cross-react with other polyomaviruses such as JC (JCPyV) and Simian Virus 40. These non-specific data indicate the need of novel immunological methods and new investigations to check in a specific manner, BKPyV spread in humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme-linked immunosorbent assay with synthetic peptides mimicking immunogenic epitopes of BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this innovative immunological assay indicate that serum antibodies against BKPyV VP1 mimotopes are detectable in healthy subjects ranging from 18 to 90 years old. The overall prevalence of serum samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation are the novelty of the immunological method, its simplicity of the approach, and the specificity of BKPyV antibody reaction to VP1 mimotopes. PMID:28321224

  11. Study on the production of IgG-, IgA- and IgM-antibodies to somatic antigens of Salmonella typhi in humans

    PubMed Central

    Chernokhvostova, Elena; Luxemburg, K. I.; Starshinova, Valentina; Andreeva, Natalia; German, Galina

    1969-01-01

    The immune response to O- and Vi-antigens of Salmonella typhi in humans was studied under a variety of conditions. In sera of persons immunized with various typhoid vaccines and with chemically purified Vi-antigen of S. typhi, anti-Vi-antibodies of three main immunoglobulin types (IgG, IgA and IgM) were found, but anti-O-antibodies were of IgM-type only. In sera of typhoid patients anti-O-antibodies of IgG-, IgA- and IgM-types were detected. Anti-Vi-antibodies appearing in the course of typhoid fever were heterogeneous to the same extent as anti-O-antibodies. The antibody response to Vi-antigen administered subcutaneously was quite similar in typhoid patients and in healthy individuals. Both anti-O- and anti-Vi-antibodies in sera of chronic typhoid carriers were usually of IgG-type only. Immunization of typhoid carriers with Vi-antigen was followed by the significant augmentation of IgG-antibody level, not preceded by IgM-antibody production. The possible reasons of IgM-deficiency in typhoid carrier state are discussed. ImagesFIG. 1 PMID:4182404

  12. Production and Characterization of a Set of Mouse-Human Chimeric Immunoglobulin G (IgG) Subclass and IgA Monoclonal Antibodies with Identical Variable Regions Specific for Pseudomonas aeruginosa Serogroup O6 Lipopolysaccharide

    PubMed Central

    Preston, Michael J.; Gerçeker, A. Alev; Reff, Mitchell E.; Pier, Gerald B.

    1998-01-01

    The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy. PMID

  13. [Diagnostic value of IgG antibody levels against 38 kDa mycobacterial antigen].

    PubMed

    Demkow, U; Zielonka, T M; Strzałkowski, J; Michałowska-Mitczuk, D; Augustynowicz-Kopeć, E; Białas-Chromiec, B; Kuś, J; Skopińska-Rózewska, E; Zwolska, Z

    1998-01-01

    Tuberculosis diagnosis bases on clinical and radiological symptoms and identification of mycobacteria. Accuracy of both methods is limited. Therefore reliable serological test would have considerable advantage. The present study was aimed at evaluating IgG-mediated immune response against specific mycobacterial antigens 38 kDa in group of 200 patients and control subjects. Our material consisted of 104 tuberculosis patients, 25 with sarcoidosis, 24 with lung cancer, 13 with bacterial or fungal pulmonary infection, 8 with mycobacterial infections other than tuberculosis and 26 healthy persons. We used commercially available ELISA based kits (Pathozyme TB-complex). Specificity of 100% and sensitivity of 49% was achieved. Sensitivity increased to 59% in chronic cases and to 52% in culture positive cases. Sensitivity decreased to only 14% in group of new culture negative cases. Measurement of IgG serum level against 38 kDa can be helpful in tuberculosis diagnosis. As the test lacks falsely positive results it indicates its high positive predictive value.

  14. Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody

    PubMed Central

    Sinha, Sandipan; Zhang, Lei; Duan, Shaofeng; Williams, Todd D; Vlasak, Josef; Ionescu, Roxana; Topp, Elizabeth M

    2009-01-01

    The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (∼50 kD) (“intact protein”), or corresponding synthetic peptides (“peptide”) were stored in Tris buffer at 37°C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N382 to form the isoaspartate (isoD382) and aspartate (D382) products in the ratio of ∼4:1, with a half-life of ∼3.4 days. The succinimide intermediate (Su382) was also detected; deamidation was not observed for the other two sites (N387 and N388) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of ∼108 days to produce the isoD382 and D387 products, together with minor amounts of D382. Surprisingly, the D382 and isoD387 products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N388. The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution. PMID:19544580

  15. [A case of IgM paraproteinemic neuropathy associated with anti-sulfated glucuronic paragloboside (SGPG) IgG antibody without anti-myelin-associated glycoprotein (MAG) activity].

    PubMed

    Nakamura, Haruko; Endo, Masanao; Sugawara, Eriko; Kuwahara, Motoi; Kusunoki, Susumu; Tanaka, Fumiaki; Takahashi, Tatsuya

    2013-01-01

    We report a case of IgM paraproteinemic neuropathy associated with anti-sulfated glucuronic paragloboside (SGPG) IgG antibody. An 84-year old man complained of numbness on the left side of the face and in the distal portions of the limbs. Neurological examination showed mild sensory ataxia. The laboratory tests revealed the presence of IgM lambda paraproteinemia and anti-SGPG IgG antibody without anti-myelin-associated glycoprotein (MAG) activity and anti-MAG/SGPG IgM antibody. Results of nerve conduction study showed decreased sensory nerve action potential (SNAP) amplitude, indicating the presence of sensory-dominant axonal polyneuropathy, and the prolongation of distal latency was not observed. Treatment with corticosteroids resulted in a rapid improvement in neurological abnormalities. In IgM paraproteinemic neuropathy associated with anti-MAG/SGPG antibody, distal acquired demyelinating sensory neuropathy and resistance to immunological treatments are the characteristic pathologic and clinical features, respectively. On the other hand our rare case of IgM paraproteinemic neuropathy positive for anti-SGPG IgG antibody presented with axonal sensory polyneuropathy and a good responsiveness to corticosteroids.

  16. Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection.

    PubMed

    Astronomo, Rena D; Santra, Sampa; Ballweber-Fleming, Lamar; Westerberg, Katharine G; Mach, Linh; Hensley-McBain, Tiffany; Sutherland, Laura; Mildenberg, Benjamin; Morton, Georgeanna; Yates, Nicole L; Mize, Gregory J; Pollara, Justin; Hladik, Florian; Ochsenbauer, Christina; Denny, Thomas N; Warrier, Ranjit; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Ferrari, Guido; Shaw, George M; Xia, Shi-Mao; Liao, Hua-Xin; Montefiori, David C; Tomaras, Georgia D; Haynes, Barton F; McElrath, Juliana M

    2016-12-01

    HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1JR-CSF and HIV-1Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development.

  17. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis.

    PubMed

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35-76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants' health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0-0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1-57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0-88.2) and body mass index (OR = 3.0; 95%CI 1.7-5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is suggested.

  18. Nucleotide sequence of the variable region of the heavy and light chains of a monoclonal IgG antibody reactive to herbicides, terbutryn and prometryn.

    PubMed

    Kreissig, S B; Ward, V K; Hammock, B D; Choudary, P V

    1995-01-01

    Members of the triazine family of herbicides are reliable indicators of contamination of the ground water or soil with pesticide residues. To facilitate better detection of the chemical residues using improved immunoassay procedures, several monoclonal antibodies against triazine herbicides have been developed. K1F4 is a hybridoma secreting monoclonal (IgG) antibody reactive to terbutryn and prometryn, two members of the triazine family. We have cloned the genes encoding the variable regions of the heavy and light chains of this monoclonal antibody and report the nucleotide sequence here.

  19. Detection of IgG1 antibodies against Mycobacterium tuberculosis DosR and Rpf antigens in tuberculosis patients before and after chemotherapy.

    PubMed

    Mattos, Ana Márcia Menezes; Chaves, Alexandre Silva; Franken, Kees L M C; Figueiredo, Bárbara Bruna Muniz; Ferreira, Ana Paula; Ottenhoff, Tom H M; Teixeira, Henrique Couto

    2016-01-01

    Diagnosis of tuberculosis (TB) remains challenging. Serum IgG1 antibodies against Mycobacterium tuberculosis active growth phase antigens (ESAT-6/CFP-10, Rv0717 and Rv3353), DosR regulon-encoded proteins (Rv1733, Rv1737, Rv2628 and Rv2029), and resuscitation-promoting factors (Rv0867 and Rv2389) were evaluated in TB patients using ELISA. Active TB patients showed elevated levels of IgG1 antibodies against ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867 in comparison to healthy controls (p < 0.001). These levels remained high after the initiation of treatment, while responses to Rv0717 and Rv1733 peaked early during treatment. IgG1 responses to ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029 and Rv0867 declined to control levels after the completion of 6 months chemotherapy. ROC analysis confirmed the good diagnostic performance of Rv0717, Rv1733, Rv3353, Rv2628, Rv2029 and Rv0867antigens. These data suggest that detecting IgG1 antibodies against M. tuberculosis antigens, including DosR and Rpf proteins, may represent an additional tool in the diagnosis of tuberculosis.

  20. Peptide-based allergen specific immunotherapy for the treatment of allergic disorders.

    PubMed

    El-Qutob, David; Reche, Pedro; Subiza, José L; Fernández-Caldas, Enrique

    2015-01-01

    Allergen specific immunotherapy (ASIT) and environmental control are the only etiologic treatments of allergic rhino-conjunctivitis, asthma and atopic dermatitis. The clinical benefit of ASIT relies on the selection of the patients and the identification and administration of the allergen, or allergens. Different routes of administration have been investigated, including subcutaneous, intradermal, epicutaneous, sublingual, inhaled, or intra-lymphatic. While subcutaneous and sublingual allergen specific immunotherapy may require from 3 to 5 years of treatment, clinical efficacy with intra-lymphatic treatment can be achieved after 3 injections. The most severe side effect of ASIT is anaphylaxis. Novel approaches are being investigated to reduce the allergenicity of immunotherapy vaccines, maintaining immunogenicity. Peptide immunotherapy has been directed mostly against autoimmune diseases, but the use of synthetic peptides for ASIT is a promising field in basic science, applied immunology and in clinical development. Short synthetic peptides bear allergen-specific CD4 T-cell epitopes which induce tolerance by stimulating regulatory (Treg) and Th1 cells. In the present patent review, we describe new trends in allergen immunotherapy using peptides, which, from a clinical point of view, are promising.

  1. Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

    PubMed

    Gebe, John A; Yadava, Koshika; Ruppert, Shannon M; Marshall, Payton; Hill, Paul; Falk, Ben A; Sweere, Johanna M; Han, Hongwei; Kaber, Gernot; Medina, Carlos; Mikecz, Katalin; Ziegler, Steven F; Balaji, Swathi; Keswani, Sundeep G; Perez, Vinicio A de Jesus; Butte, Manish J; Nadeau, Kari; Altemeier, William A; Fanger, Neil; Bollyky, Paul L

    2017-01-01

    The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

  2. Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with Hevylite™ antibodies. comparison with the free light chain oligoclonal pattern

    PubMed Central

    2012-01-01

    Background Oligoclonal IgG bands in cerebrospinal fluid that are absent in serum indicate intrathecal IgG synthesis and are a sensitive marker of CNS inflammatory diseases, in particular multiple sclerosis. It may be of interest to determine whether these bands are predominantly IgGκ or IgGλ. Methods We have used Hevylite™ antibodies and developed a technique for detection of oligoclonal IgGκ and IgGλ bands by means of isoelectric focusing followed by immunoblotting. The same technique was used for oligoclonal free κ and free λ detection. Among several techniques tested, affinity immunoblotting appears to be the most sensitive; it can detect less than 1 ng of IgGκ or IgGλ paraprotein. We compared oligoclonal IgG profiles with those of oligoclonal IgGκ and IgGλ. There was good agreement concerning the presence or absence of intrathecal synthesis. We observed the ratios between oligoclonal IgGκ and IgGλ bands, and they did not always match the ratios between free κ and free λ bands. We were also able to detect antigen-specific CSF-restricted oligoclonal IgGκ and IgGλ bands in neuroborreliosis. It remains to be determined subsequently by a clinically-oriented prospective study, whether predominant IgGκ/IgGλ or free κ/free λ can be observed more frequently in particular diseases with oligoclonal IgG synthesis. Discussion Very sensitive detection of oligoclonal IgGκ and IgGλ bands in cerebrospinal fluid with Hevylite antibodies is feasible; detection of antigen-specific IgGκ or IgGλ is possible as well. In particular situations, e.g. when difficulties arise in distinguishing between oligoclonal and monoclonal pattern, the test may be of considerable clinical value. PMID:22360869

  3. Passive transfer with serum and IgG antibodies of irradiated cercaria-induced resistance against Schistosoma mansoni in mice

    SciTech Connect

    Mangold, B.L.; Dean, D.A.

    1986-04-01

    The role of humoral immunity to Schistosoma mansoni infection in C57BL/6J mice was examined by employing a passive transfer system. Sera from highly resistant mice that had been exposed to two or three immunizations with 50-kilorad-gamma-irradiated cercariae were tested for their ability to transfer protection against S. mansoni challenge. All five batches of serum tested were observed to have protective activity. Immune serum recipients exhibited statistically significant reductions in challenge worm burdens of 20 to 50% compared with recipients of normal serum or no serum. The most consistent level of resistance was obtained when immune serum was administered several days post-challenge, i.e., at a time coincident with schistosomulum residence in the lungs. Furthermore, it was shown that the protective activity in immune serum was associated with factors that bind to staphylococcal protein A and that are precipitated by 50% ammonium sulfate; thus it appears that the protective factors in immune serum are IgG antibodies.

  4. Effect of ambient light on IgG1 monoclonal antibodies during drug product processing and development.

    PubMed

    Sreedhara, Alavattam; Yin, Jian; Joyce, Michael; Lau, Kimberly; Wecksler, Aaron T; Deperalta, Galahad; Yi, Li; John Wang, Y; Kabakoff, Bruce; Kishore, Ravuri S K

    2016-03-01

    Photostability studies are standard stress testing conducted during drug product development of various pharmaceutical compounds, including small molecules and proteins. These studies as recommended by ICH Q1B are carried out using no less than 1.2× 10(6)lux-hours in the visible region and no less than 200Wh/m(2) in UV light. However, normal drug product processing is carried out under fluorescent lamps that emit white light almost exclusively in the >400nm region with a small UV quotient. We term these as ambient or mild light conditions. We tested several IgG1 monoclonal antibodies (mAbs 1-5) under these ambient light conditions and compared them to the ICH light conditions. All the mAbs were significantly degraded under the ICH light but several mAbs (mAbs 3-5) were processed without impacting any product quality attributes under ambient or mild light conditions. Interestingly we observed site-specific Trp oxidation in mAb1, while higher aggregation and color change were observed for mAb2 under mild light conditions. The recommended ICH light conditions have a high UV component and hence may not help to rank order photosensitivity under normal protein DP processing conditions.

  5. Detection of IgM and IgG antibodies against Brucella by dot-ELISA in humans.

    PubMed

    Barbuddhe, S B; Yadava, V K; Singh, D K

    1994-03-01

    Dot-enzyme linked immunosorbent assay (dot-ELISA) with autoclaved extract of B. abortus S99 was used for the detection of Brucella antibodies in human sera. Results were compared with those of STAT, RBPT and CFT. Out of 80 sera 8(10 per cent), 15(18.7 per cent), 20(25 per cent) and 13(16.25 per cent) were found positive by RBPT, STAT, IgM dot-ELISA and IgG dot-ELISA respectively. Out of 74 sera samples tested with CFT 9(12.16 per cent) proved positive. The relative sensitivity and relative specificity in comparison with CFT was found to be 33.33 per cent, 96.92 percent for RBPT, 33.33 per cent and 84.61 per cent for STAT and 88.88 per cent and 76.92 per cent for dot-ELISA, respectively. The dot-ELISA was found to be a more sensitive, economical and a rapid test for screening of human brucellosis under field conditions.

  6. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

    PubMed

    Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

    2006-10-03

    Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.

  7. Correlation between the Amount of Anti-D Antibodies and IgG Subclasses with Severity of Haemolytic Disease of Foetus and Newborn

    PubMed Central

    Velkova, Emilija

    2015-01-01

    AIM: The aim of this study was to investigate the influence of subclasses to IgG anti-D on the intensity of hemolytic disease of fetus and newborn (HDFN) at 45 fetuses/newborns with symptoms of mild and severe HDFN in Republic of Macedonia. MATERIAL AND METHODS: In retrospective and prospective studies, in a period of 10 years, from 2004 to 2014, there have been immunohemathology tests performed on 22 009 samples on serums of pregnant women. RESULTS: At 37.78% of the total number of tested patients, IgG1 and IgG3 was the reason for severe HDFN. At 17.77% of the total number of tested patients, which had only IgG1detected, was the reason for serious intensity of HDFN. The correlation of the titer to anti-D antibodies in the mother’s serum and the intensity of HDFN were researched in 48 newborns. The titers between 1:8 and 1:32 resulted in 3 cases of HDFN with symptoms of severe disease and in 4 cases there were no signs of HDFN. At 12 women that had a titre between 1:32 and 1:512, five of the newborns developed severe HDFN, and seven had symptoms of mild and weak intensity form. In 3 cases the titer was higher than 512, and out of them one newborn had weak symptoms of HDFN, one developed severe HDFN and one ended with foetal death. Only in one case the titer reached a value higher than 1000, and it ended with a fetal death. CONCLUSIONS: The titers of the pregnant women serum those are lower than 32 and those higher than 1000 can well predict HDFN. The titers of anti-D antibodies between 64 and 512 have no exact predictive value. IgG1 and IgG3 subclasses of anti-D have no predictive value by themselves, and cannot foresee the outcome of HDFN. The research study results suggest that IgG1 and IgG3 should be included in a multi – parameter protocol for evaluation of the HDFN intensity. They can give a real assessment of the expected HDFN intensity in combination with the titer hight and the significance of the antibodies. PMID:27275238

  8. Aggregation Kinetics for IgG1-Based Monoclonal Antibody Therapeutics.

    PubMed

    Singla, A; Bansal, R; Joshi, Varsha; Rathore, Anurag S

    2016-05-01

    Monoclonal antibodies (mAbs) as a class of therapeutic molecules are finding an increasing demand in the biotechnology industry for the treatment of diseases like cancer and multiple sclerosis. A key challenge associated to successful commercialization of mAbs is that from the various physical and chemical instabilities that are inherent to these molecules. Out of all probable instabilities, aggregation of mAbs has been a major problem that has been associated with a change in the protein structure and is a hurdle in various upstream and downstream processes. It can stimulate immune response causing protein misfolding having deleterious and harmful effects inside a cell. Also, the extra cost incurred to remove aggregated mAbs from the rest of the batch is huge. Size exclusion chromatography (SEC) is a major technique for characterizing aggregation in mAbs where change in the aggregates' size over time is estimated. The current project is an attempt to understand the rate and mechanism of formation of higher order oligomers when subjected to different environmental conditions such as buffer type, temperature, pH, and salt concentration. The results will be useful in avoiding the product exposure to conditions that can induce aggregation during upstream, downstream, and storage process. Extended Lumry-Eyring model (ELE), Lumry-Eyring Native Polymerization model (LENP), and Finke-Watzky model (F-W) have been employed in this work to fit the aggregation experimental data and results are compared to find the best fit model for mAb aggregation to connect the theoretical dots with the reality.

  9. Polymorphisms in B Cell Co-Stimulatory Genes Are Associated with IgG Antibody Responses against Blood–Stage Proteins of Plasmodium vivax

    PubMed Central

    Cassiano, Gustavo C.; Furini, Adriana A. C.; Capobianco, Marcela P.; Storti-Melo, Luciane M.; Cunha, Maristela G.; Kano, Flora S.; Carvalho, Luzia H.; Soares, Irene S.; Santos, Sidney E.; Póvoa, Marinete M.; Machado, Ricardo L. D.

    2016-01-01

    The development of an effective immune response can help decrease mortality from malaria and its clinical symptoms. However, this mechanism is complex and has significant inter-individual variation, most likely owing to the genetic contribution of the human host. Therefore, this study aimed to investigate the influence of polymorphisms in genes involved in the costimulation of B-lymphocytes in the naturally acquired humoral immune response against proteins of the asexual stage of Plasmodium vivax. A total of 319 individuals living in an area of malaria transmission in the Brazilian Amazon were genotyped for four SNPs in the genes CD40, CD40L, BLYS and CD86. In addition, IgG antibodies against P. vivax apical membrane antigen 1 (PvAMA–1), Duffy binding protein (PvDBP) and merozoite surface protein 1 (PvMSP–119) were detected by ELISA. The SNP BLYS –871C>T was associated with the frequency of IgG responders to PvAMA–1 and PvMSP–119. The SNP CD40 –1C>T was associated with the IgG response against PvDBP, whereas IgG antibody titers against PvMSP–119 were influenced by the polymorphism CD86 +1057G>A. These data may help to elucidate the immunological aspects of vivax malaria and consequently assist in the design of malaria vaccines. PMID:26901523

  10. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects.

    PubMed

    Fachiroh, Jajah; Schouten, Tabitha; Hariwiyanto, Bambang; Paramita, Dewi K; Harijadi, Ahmad; Haryana, Sofia M; Ng, Mun H; Middeldorp, Jaap M

    2004-07-01

    Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.

  11. First Attempt To Validate Human IgG Antibody Response to Nterm-34kDa Salivary Peptide as Biomarker for Evaluating Exposure to Aedes aegypti Bites

    PubMed Central

    Elanga Ndille, Emmanuel; Doucoure, Souleymane; Damien, Georgia; Mouchet, François; Drame, Papa Makhtar; Cornelie, Sylvie; Noukpo, Herbert; Yamadjako, Sandra; Djenontin, Armel; Moiroux, Nicolas; Misse, Dorothee; Akogbeto, Martin; Corbel, Vincent; Henry, Marie-Claire; Chandre, Fabrice; Baldet, Thierry; Remoue, Franck

    2012-01-01

    Background Much effort is being devoted for developing new indicators to evaluate the human exposure to Aedes mosquito bites and the risk of arbovirus transmission. Human antibody (Ab) responses to mosquito salivary components could represent a promising tool for evaluating the human-vector contact. Methodology/Principal findings To develop a specific biomarker of human exposure to Aedes aegypti bites, we measured IgG Ab response to Ae. aegypti Nterm-34 kDa salivary peptide in exposed children in 7 villages of Southern Benin (West Africa). Results showed that specific IgG response presented high inter-individual heterogeneity between villages. IgG response was associated with rainfall and IgG level increased from dry (low exposure) to rainy (high exposure) seasons. These findings indicate that IgG Ab to Nterm-34 kDa salivary peptide may represent a reliable biomarker to detect variation in human exposure to Ae. aegypti bites. Conclusion/Significance This preliminary study highlights the potential use of Ab response to this salivary peptide for evaluating human exposure to Ae. aegypti. This biomarker could represent a new promising tool for assessing the risk of arbovirus transmission and for evaluating the efficacy of vector control interventions. PMID:23166852

  12. Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

    PubMed Central

    Waldmeier, Lorenz; Hellmann, Ina; Gutknecht, Chantal K.; Wolter, Fabian I.; Cook, Skylar C.; Reddy, Sai T.; Grawunder, Ulf; Beerli, Roger R.

    2016-01-01

    ABSTRACT In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates. PMID:26986818

  13. Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries.

    PubMed

    Waldmeier, Lorenz; Hellmann, Ina; Gutknecht, Chantal K; Wolter, Fabian I; Cook, Skylar C; Reddy, Sai T; Grawunder, Ulf; Beerli, Roger R

    2016-01-01

    In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

  14. IgG2 deficiency in a healthy blood donor. Concomitant lack of IgG2, IgA and IgE immunoglobulins and specific anti-carbohydrate antibodies.

    PubMed Central

    Hammarström, L; Smith, C I

    1983-01-01

    Lack of serum IgG2, IgA and IgE was found in a healthy male adult blood donor. No secretory IgA could be demonstrated. In vitro activation of lymphocytes did not induce IgA secreting cells although no class specific suppressor cells could be found. Normal or slightly subnormal titres to a variety of bacterial and viral antigens were demonstrated whereas anti-carbohydrate antibodies (anti-teichoic acid, anti-dextran and anti-pneumococcal polysaccharide) were virtually absent. Isoagglutinins and heteroagglutinins were present in somewhat lower concentrations than normal. PMID:6189654

  15. Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation.

    PubMed

    Minanov, O P; Itescu, S; Neethling, F A; Morgenthau, A S; Kwiatkowski, P; Cooper, D K; Michler, R E

    1997-01-27

    We have previously demonstrated that hyperacute rejection does not occur in a pig-to-newborn baboon heart transplant model, presumably because of low levels of cytotoxic antipig antibodies present in the serum of newborn baboons. Cytotoxic antipig antibodies are primarily directed to alpha-1,3-galactosyl (alpha Gal) residues on endothelial cell surface structures Twenty-one full-term humans and 5 full-term baboons were tested for complement mediated lysis (CML) of pig kidney (PK-15) cells and anti-alpha Gal activity with an ELISA using BSA-conjugated alpha Gal residues as target. To evaluate the significance of the anti-alpha Gal titers in vivo 5 newborn baboons underwent heterotopic pig cardiac xenotransplantation. Six of 21 human samples and 1 of 5 baboon samples demonstrated significant cytotoxicity to PK-15 cells. Twelve of 21 newborn humans had anti-alpha Gal IgG antibodies at titers of 1:80 or greater. None of the samples had anti-alpha Gal IgM. In newborn baboons, 1 of 5 sera had anti-alpha Gal IgG antibodies at titers greater than 1:80 and none of these samples had anti-alpha Gal IgM. Xenografts survived for an average of 3.6 days, even in the baboon with high anti-alpha Gal IgG titers. Analysis of the explanted grafts showed minimal evidence of complement-mediated hyperacute rejection (HAR), but prominent mononuclear cell infiltrates. In serum tested posttransplant there was an induced anti-alpha Gal response with cytotoxicity against PK-15 cells. These results show that anti-alpha Gal IgM is absent in newborn human and baboon sera, allowing pig grafts to avoid HAR. However, the presence of anti-alpha Gal IgG may be associated with mononuclear cell infiltration of the xenograft and its subsequent rejection.

  16. Reactivation of IgG-switched memory B cells by BCR-intrinsic signal amplification promotes IgG antibody production

    PubMed Central

    Lutz, Johannes; Dittmann, Kai; Bösl, Michael R; Winkler, Thomas H; Wienands, Jürgen; Engels, Niklas

    2015-01-01

    Secondary antibody responses are marked by faster kinetics, improved antibody affinity and a switch from IgM to other immunoglobulin isotypes, most notably IgG, compared with primary responses. These changes protect from reinfection and represent the principle of most vaccination strategies. Yet, the molecular mechanisms that underlie B-cell memory responses are unclear. Here we show, by inactivating the immunoglobulin tail tyrosine (ITT) signalling motif of membrane-bound IgG1 in the mouse, that the ITT facilitates maintenance and reactivation of IgG-switched memory B cells in vivo. The ITT motif equips IgG-switched cells with enhanced BCR signalling capacity, which supports their competitiveness in secondary immune reactions and drives the formation of IgG-secreting plasma cells even in the absence of T-cell help. Our results demonstrate that ITT signalling promotes the vigorous production of IgG antibodies and thus provide a molecular basis for humoral immunological memory. PMID:26815242

  17. Community Rates of IgG4 Antibodies to Ascaris Haemoglobin Reflect Changes in Community Egg Loads Following Mass Drug Administration

    PubMed Central

    Vlaminck, Johnny; Supali, Taniawati; Geldhof, Peter; Hokke, Cornelis H.; Fischer, Peter U.; Weil, Gary J.

    2016-01-01

    Background Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA). Methodology/Principal findings We developed and evaluated an ELISA to detect human IgG4 antibodies to AsHb. We tested 1066 plasma samples collected at different times from 599 subjects who lived in a village in rural Indonesia that was highly endemic for ascariasis. The community received 6 rounds of MDA for lymphatic filariasis with albendazole plus diethylcarbamazine between 2002 and 2007. While the AsHb antibody assay was not sensitive for detecting all individuals with Ascaris eggs in their stools, the percentage of seropositive individuals decreased rapidly following MDA. Reductions in antibody rates reflected decreased mean egg output per person both at the community level and in different age groups. Two years after the last round of MDA the community egg output and antibody prevalence rate were reduced by 81.6% and 78.9% respectively compared to baseline levels. Conclusion/Significance IgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission. PMID:26991326

  18. Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach.

    PubMed

    Zhang, Yuling; Bailey, Robert; Nightlinger, Nancy; Gillespie, Alison; Balland, Alain; Rogers, Richard

    2015-08-01

    In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.

  19. Genetic homogeneity but IgG subclass-dependent clinical variability of alloimmune membranous nephropathy with anti-neutral endopeptidase antibodies.

    PubMed

    Vivarelli, Marina; Emma, Francesco; Pellé, Thimothée; Gerken, Christopher; Pedicelli, Stefania; Diomedi-Camassei, Francesca; Klaus, Günter; Waldegger, Siegfried; Ronco, Pierre; Debiec, Hanna

    2015-03-01

    Alloimmune antenatal membranous nephropathy (MN) during pregnancy results from antibodies produced by a neutral endopeptidase (NEP)-deficient mother. Here we report two recent cases that provide clues to the severity of renal disease. Mothers of the two children had circulating antibodies against NEP showing the characteristic species-dependent pattern by immunofluorescence on kidney slices. A German mother produced predominantly anti-NEP IgG4 accompanied by a low amount of IgG1. Her child recovered renal function within a few weeks. In sharp contrast, an Italian mother mainly produced complement-fixing anti-NEP IgG1, which also inhibits NEP enzymatic activity, whereas anti-NEP IgG4 has a weak inhibitory potency. Her child was dialyzed for several weeks. A kidney biopsy performed at 12 days of age showed MN, ischemic glomeruli, and arteriolar and tubular lesions. A second biopsy performed at 12 weeks of age showed aggravation with an increased number of collapsed capillary tufts. Both mothers were homozygous for the truncating deletion mutation 466delC and were thus NEP deficient. The 466delC mutation, identified in three previously described families, suggests a founder effect. Because of the potential severity of alloimmune antenatal MN, it is essential to identify families at risk by the detection of anti-NEP antibodies and NEP antigen in urine. On the basis of the five families identified to date, we propose an algorithm for the diagnosis of the disease and the prevention of complications.

  20. Assessment of IgG Antibodies Against HSV1, HSV2, CMV and EBV in Patients with Pemphigus Vulgaris Versus Healthy People

    PubMed Central

    Ghalayani, Parichehr; Rashidi, Fateme; Saberi, Zahra

    2015-01-01

    Objectives: Regarding the implication of viruses particularly herpes in pemphigus vulgaris, we sought to assess and compare the level of immunoglobulin G (IgG) antibodies against herpes simplex virus types 1 and 2 (HSV1 and HSV2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in patients with pemphigus vulgaris and healthy people. Materials and Methods: In this cross-sectional study, 25 patients with pemphigus vulgaris and 27 healthy individuals comprised the experimental and control groups, respectively. Serum samples were taken from both groups; the levels of IgG antibodies against HSV1, HSV2, CMV and EBV were measured using ELISA. Results: Immunoglobulin G titer was higher for all four viruses in the patient group in comparison to the control group. This difference was significant for anti-EBV (P= 0.005), anti-CMV (P=0.0001) and anti-HSV2 (P=0.001) but not significant for anti-HSV1 (P= 0.36). Conclusion: Viruses including EBV, CMV, and HSV2 probably play a role in the pathogenesis of pemphigus in addition to the effects of genetics, toxins and other predisposing factors. In this study, no statistically significant relationship was observed between HSV1 and pemphigus vulgaris, which was probably due to the high titer of anti-HSV1 IgG in healthy individuals in the community. More studies must be done in this regard. PMID:27507994

  1. Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis.

    PubMed

    Barrs, V R; Ujvari, B; Dhand, N K; Peters, I R; Talbot, J; Johnson, L R; Billen, F; Martin, P; Beatty, J A; Belov, K

    2015-03-01

    Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1). Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

  2. Identification of gangliosides recognized by IgG anti-GalNAc-GD1a antibodies in bovine spinal motor neurons and motor nerves.

    PubMed

    Yoshino, Hiide; Ariga, Toshio; Suzuki, Akemi; Yu, Robert K; Miyatake, Tadashi

    2008-08-28

    The presence of immunoglobulin G (IgG)-type antibodies to the ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), is closely associated with the pure motor type of Guillain-Barré syndrome (GBS). In the present study, we isolated disialogangliosides from the motor neurons and motor nerves of bovine spinal cords by DEAE-Sephadex column chromatography. The disialoganglioside fraction contained GD1a, GD2, GD1b, and three gangliosides, designated X1, X2 and X3. Serum from a patient with axonal GBS with IgG anti-GalNAc-GD1a antibody yielded positive immunostaining with X1, X2, and X3. When isolated by preparative thin-layer chromatography (TLC), X1 migrated at the same position as GalNAc-GD1a from Tay-Sachs brain, suggesting that X1 is GalNAc-GD1a containing N-acetylneuraminic acid (NeuAc). TLC of isolated X2 revealed that it migrated between GD1a and GD2. On the other hand, X3 had a migratory rate on TLC between and GD1b and GT1b. Since both X2 and X3 were recognized by IgG anti-GalNAc-GD1a antibody, the results suggest that X2 is a GalNAc-GD1a species containing a mixture containing a NeuAc-and an N-glycolylneuraminic acid (NeuGc) species, and X3 is a GalNAc-GD1a species with two NeuGc. This evidence indicating the specific localization of GalNAc-GD1a and its isomers in spinal motor neurons should be useful in elucidating the pathogenic role of IgG anti-GalNAc-GD1a antibody in pure motor-type GBS.

  3. Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets.

    PubMed

    Ryan, John F; Hovde, Rachel; Glanville, Jacob; Lyu, Shu-Chen; Ji, Xuhuai; Gupta, Sheena; Tibshirani, Robert J; Jay, David C; Boyd, Scott D; Chinthrajah, R Sharon; Davis, Mark M; Galli, Stephen J; Maecker, Holden T; Nadeau, Kari C

    2016-03-01

    Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional "roadmap" of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an "anergic" Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.

  4. Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets

    PubMed Central

    Ryan, John F.; Hovde, Rachel; Glanville, Jacob; Lyu, Shu-Chen; Ji, Xuhuai; Gupta, Sheena; Tibshirani, Robert J.; Jay, David C.; Boyd, Scott D.; Chinthrajah, R. Sharon; Davis, Mark M.; Galli, Stephen J.; Maecker, Holden T.; Nadeau, Kari C.

    2016-01-01

    Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional “roadmap” of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an “anergic” Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation. PMID:26811452

  5. Total lymphoid irradiation reduces IgG autoantibody production and enhances specific antibody responses in NZB/NZW F1 mice

    SciTech Connect

    Farinas, M.C.; Strober, S.

    1989-07-01

    Thymus-independent primary antibody responses were studied in young and old (9 months) untreated and TLI-treated NZB/NZW and BALB/c mice. Untreated old NZB/NZW mice had a low primary response to Brucella abortus (BA) as compared to that of young NZB/NZW and BALB/c mice. However, TLI treatment resulted in a 130-fold increase in the IgG anti-BA primary antibody response at day 21 postimmunization, achieving similar levels to those of young NZB/NZW or nonautoimmune BALB/c mice. Anti-TNP responses to trinitrophenylated BA or Ficoll were masked by high background levels of anti-TNP antibodies. Despite the increase in the anti-BA response, spontaneous immunoglobulin secretion and autoantibody levels were markedly decreased after TLI in old NZB/NZW mice.

  6. 2D immunoblots show differential response of mouse IgG and IgM antibodies to antigens of mammary carcinoma 4 T1 cells

    PubMed Central

    2014-01-01

    Background Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer. We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells. Methods The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells. Results There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t = 0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic. Conclusions IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies

  7. Individual Restriction Of Fine Specificity Variability In Anti-GM1 IgG Antibodies Associated With Guillain-Barré Syndrome

    PubMed Central

    Lardone, Ricardo D.; Yuki, Nobuhiro; Irazoqui, Fernando J.; Nores, Gustavo A.

    2016-01-01

    Elevated titers of serum antibodies against GM1 ganglioside are associated with a variety of autoimmune neuropathies. Much evidence indicates these autoantibodies play a primary role in the disease processes, but the mechanism for their appearance is unclear. We studied the fine specificity of anti-GM1 antibodies of the IgG isotype present in sera from patients with Guillain-Barré syndrome (GBS), using thin-layer chromatogram-immunostaining of GM1, asialo-GM1 (GA1), GD1b and GM1-derivatives with small modifications on the oligosaccharide moiety. We were able to distinguish populations of antibodies with different fine specificity. Remarkably, individual patients presented only one or two of them, and different patients had different populations. This restriction in the variability of antibody populations suggests that the appearance of the anti-GM1 antibodies is a random process involving restricted populations of lymphocytes. With the origin of disease-associated anti-GM1 antibodies as a context, this finding could provide explanation for the “host susceptibility factor” observed in GBS following enteritis with GM1 oligosaccharide-carrying strains of Campylobacter jejuni. PMID:26818965

  8. Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

    PubMed Central

    Geoghegan, James C.; Fleming, Ryan; Damschroder, Melissa; Bishop, Steven M.; Sathish, Hasige A.; Esfandiary, Reza

    2016-01-01

    ABSTRACT Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies. PMID:27050875

  9. Linking the solution viscosity of an IgG2 monoclonal antibody to its structure as a function of pH and temperature.

    PubMed

    Cheng, Weiqiang; Joshi, Sangeeta B; Jain, Nishant Kumar; He, Feng; Kerwin, Bruce A; Volkin, David B; Middaugh, C Russell

    2013-12-01

    Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.

  10. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  11. Seroprevalence of Bactericidal, Specific IgG Antibodies and Incidence of Meningitis Due to Group A Neisseria meningitidis by Age in Burkina Faso 2008

    PubMed Central

    Trotter, Caroline L.; Yaro, Seydou; Njanpop-Lafourcade, Berthe-Marie; Drabo, Aly; Kroman, Sita S.; Idohou, Regina S.; Sanou, Oumarou; Bowen, Leah; Findlow, Helen; Diagbouga, Serge; Gessner, Bradford D.; Borrow, Ray; Mueller, Judith E.

    2013-01-01

    Background We investigated serological correlates of protection against Neisseria meningitidis serogroup A (NmA) in Burkina Faso before the introduction of NmA conjugate vaccine. Methodology/Principal Findings We collected blood from a representative sample (N = 1022) of Bobo-Dioulasso residents. Sera were evaluated for serum bactericidal antibody (SBA) activity against NmA strains of immunotype L11 (F8238) and L10 (3125) and NmA-specific IgG. Seroprevalence was compared to the age-specific NmA meningitis incidence in Bobo-Dioulasso during March 2007–February 2008. Meningococcal carriage was evaluated in a subset (N = 538). Geometric mean titres (GMT)/concentrations (GMC) of SBA and NmA-specific IgG increased with age, peaking around age 20 years. Overall, 70% of our sample had NmA-specific IgG ≥2 ug/mL. Meningitis incidence was highest in those aged <6 months and 5–19 years. No NmA carriers were found. Compared to the reference strain SBA, GMTs were higher against a locally isolated strain and around 40-fold lower against Dutch strain 3125. Conclusions/Significance This study provides estimates of natural immunity to NmA, according to a variety of antibody measures, which will be helpful in ascertaining antibody persistence after MenAfriVac™ introduction. Age-specific seroprevalence of reference strain SBA titres most likely reflects exposure to meningococci and consecutive reactive immunity. We could not define any serological correlate of protection. PMID:23457471

  12. A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

    PubMed Central

    Choi, Dong-Ki; Bae, Jeomil; Shin, Seung-Min; Shin, Ju-Yeon; Kim, Sunghoon; Kim, Yong-Sung

    2014-01-01

    Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents. PMID:25484049

  13. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    PubMed

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  14. Varicella-zoster virus-specific, cell-mediated immunity with interferon-gamma release assay after vaccination of college students with no or intermediate IgG antibody response.

    PubMed

    Terada, Kihei; Itoh, Yuri; Fujii, Akihide; Kitagawa, Seiko; Ogita, Satoko; Ouchi, Kazunobu

    2015-02-01

    This study measured Varicella-zoster virus (VZV) specific cell-mediated immunity (CMI) and antibodies to clarify immune response after vaccination in 68 college students with negative or intermediate IgG antibody status. The enrolled numbers of negative, intermediate, and positive VZV-IgG antibody were 27, 41, and 28 students, respectively. The positive rates of CMI were 3.7% (1/27), 41.5% (17/41), and 96.4% (27/28) before vaccination, respectively. After vaccination, the IgG antibody titers became significantly higher in the intermediate IgG group compared to those in the negative IgG group (P < 0.01), but CMI did not differ significantly between the two groups. Ninety-three percent (38/41) of the intermediate IgG antibody group and 41% (11/27) of the negative IgG antibody group became positive for the IgG antibody after vaccination (P < 0.0001). When subjects were divided into negative, intermediate, and positive CMI by interferon-gamma values before vaccination, the IgG antibody and interferon-gamma values increased significantly in the positive CMI group compared to the negative CMI group after vaccination (P < 0.01 and P < 0.01, respectively). All (17/17) of positive CMI group and 61% (27/44) of negative CMI group became positive for the IgG antibody after vaccination (P < 0.01). Ninety-four percent (16/17) of positive CMI group and 59% (28/44) of negative CMI group became positive for CMI after vaccination (P < 0.05). Ninety-six percent (22/23) of the subjects with a history of vaccination became IgG seropositive after a second dose of vaccination, but 22% (5/23) of them remained negative for CMI. CMI is valuable information to identify potential non-responders to vaccination and to predict risk of clinical VZV infection.

  15. Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

  16. Human IgE, IgG and IgA antibody responses to T101, a murine monoclonal antibody against human lymphocytes: implications for pathogenesis, risk and avoidance of adverse immunologic reactions.

    PubMed

    Dykewicz, M S; Cranberg, J A; Patterson, R; Rosen, S T; Shaughnessy, M A; Zimmer, A M

    1990-01-01

    To study immune responses that may play a role in immediate-type allergic reactions (ITAR) and serum-sickness-like reactions that have been reported with administration of monoclonal antibodies (MAb), we measured by enzyme-linked immunosorbent assay serum levels of IgE, IgG, and IgA human antimurine antibody (HAMA) to T101, a murine IgG2a antibody that has been used in the treatment of cutaneous T cell lymphoma (CTCL) and chronic lymphocytic leukemia (CLL). None of 8 patients (4 with CLL, 4 CTCL) pretreated with T101 had elevated titers of any HAMA class tested as compared to normal control sera. All CTCL patients who had elevated total serum IgE levels, but normal total serum levels of other antibody classes, had significant rises in IgE, IgG, and IgA HAMA to T101 after intravenous MAb infusion. However, the CLL patients who were hypogammaglobulinemic failed to develop significant rises in HAMA. Three patients (2 CLL, 1 CTCL) had ITAR (e.g., urticaria, angioedema, bronchospasm) associated with infusion of T101; prophylactic medication regimens based upon the control of radiographic contrast media reactions were used with apparent benefit in subsequent infusions in these patients. All 8 patients had negative immediate intradermal skin tests (1 microgram/ml) to T101 prior to its infusion. Our data confirm that (1) non-IgE-mediated mechanisms cause ITAR from this MAb, possibly by a mechanism inherent in the action of this MAb against lymphocytes, and (2) that isotypic antibody responses to MAb vary with the type of malignancy being treated.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. The effect of sugar removal on the structure of the Fc region of an IgG antibody as observed with single molecule Förster Resonance Energy Transfer.

    PubMed

    Kelliher, Michael T; Jacks, Ramiah D; Piraino, Mark S; Southern, Cathrine A

    2014-08-01

    The deglycosylation of immunoglobulin G (IgG) antibodies leads to a diminished immune response. This reduction in immune response is thought to arise from weakened binding of IgG antibodies to effector molecules as a result of a conformational change in the antibody. The nature of this structural alteration is uncertain due to the conflicting results obtained from different experimental methods. We have examined the impact of deglycosylation by the endoglycosidase PNGase F on the structure of the Fc region of a human IgG antibody using single molecule Förster Resonance Energy Transfer (FRET). The FRET efficiency histograms obtained indicate that the structure of the Fc region becomes more flexible upon deglycosylation. This is demonstrated by a change in the width of the energy transfer efficiency peak, which increases from 0.19 ± 0.02 to 0.6 ± 0.1 upon deglycosylation.

  18. Infectious gastroenteritis does not act as a triggering mechanism for the synthesis of serum IgG antibody to beta-lactoglobulin.

    PubMed

    Brüssow, H; Sidoti, J; Rahim, H; Dirren, H; Freire, W

    1991-11-01

    beta-Lactoglobulin (BLG)-specific serum IgG antibody was measured by enzyme-linked immunosorbent assay in 1,392 serum samples from newborn to 5-year-old Ecuadorian children enrolled into a representative nutrition and health survey. At a 1:100 serum dilution, 62% of the children showed specific antibody (blank-corrected optical density greater than or equal to 0.1). This prevalence did not change with increasing age. More specifically, we did not observe a prevalence or titer increase of BLG-specific antibody in age groups where the majority of these Ecuadorian children experienced infection with rotavirus (8-24-month age groups) and enterotoxigenic Escherichia coli (8-12-month age group). In addition, BLG-specific antibody did not differ between children who did or did not experience an episode of diarrhea 15 days before blood sampling. We observed a small but statistically significant difference in BLG-specific antibody between subsamples of Ecuadorian children regularly or only occasionally ingesting milk. Titers were higher in the group consuming more milk.

  19. Investigating the association between allergen-specific immunoglobulin E, cancer risk and survival

    PubMed Central

    Wulaningsih, Wahyu; Holmberg, Lars; Garmo, Hans; Karagiannis, Sophia N.; Ahlstedt, Staffan; Malmstrom, Håkan; Lambe, Mats; Hammar, Niklas; Walldius, Göran; Jungner, Ingmar; Ng, Tony; Van Hemelrijck, Mieke

    2016-01-01

    ABSTRACT Prior findings linking allergy and cancer have been inconsistent, which may be driven by diverse assessment methods. We used serum specific immunoglobulin E (IgE) against common inhalant allergens that was assessed prior to cancer diagnosis in studying this association. We selected 8,727 Swedish men and women who had measurements of serum allergen-specific IgE and total IgE between 1992 and 1996. Multivariable Cox regression using age as a timescale was performed to assess the associations of IgE sensitization, defined by any levels of serum specific IgE ≥35 kU/L, with risk of overall and specific cancers. A test for trend was performed by assigning scores derived from allergen-specific IgE levels at baseline as an ordinal scale. Kaplan–Meier curves and log-rank test were used to assess cancer survival by IgE sensitization status. During a mean follow-up of 16 year, 689 persons were diagnosed with cancer. We found an inverse association between IgE sensitization and cancer risk, with a hazard ratio (HR) of 0.83 and 95% confidence intervals (CI) of 0.70–0.99. A similar trend was seen with specific IgE scores overall (Ptrend = 0.007) and in women (Ptrend = 0.01). Although IgE sensitization was not associated with risk of common site-specific cancers, serum specific IgE scores were inversely associated with melanoma risk in men and women combined, and with risk of female breast and gynecological cancers combined. No association with survival was observed. The association between circulating IgE levels and incident cancer may point toward a role of T-helper 2 (TH2)-biased response in development of some cancers. PMID:27471625

  20. Stimulation of IgG antibody formation by sublethal irradiation during persistently repeated immunization with Brucella abortas.

    PubMed Central

    Gras, J; Morros, J M; Guix, J; Tuset, N

    1975-01-01

    Whole-body irradiation with 600 rads 8 or 10 days after the beginning of persistently repeated Brucella abortus immunization in the rabbit produces little change in the response. By contrast, irradiation with the same dose 2, 4 or 6 days after the beginning of repeated immunization stimulates the IgG response. This effect is explained by a repopulation of antigen-sensitive cells from precursor cells to a higher level than that previous to irradiation. In order to explain IgG stimulation, three main possibilities are considered. a) a T cell-dependent mechanism, consequent on a higher radioresistance of T than of B cells, or on a faster and more extensive repopulation by T cells; b) another T cell-dependent mechanism due to a greater radiosensitivity of the suppressor or inhibitory T-cell subpopulation, with the result of a predominance in the T cell population that helps IgG formation; c) a mechanism depending on antigen presence during the repopulation of antigen-sensitive cells from the precursor cells, according to previously obtained data suggesting the necessity for the persistence of a certain level of antigen for IgG formation PMID:807518

  1. Successful kidney transplant in a patient with IgG anti HLA Class-I auto-antibodies: a case report.

    PubMed

    Alhamid, Naji; Alterky, Hani; Almouslem, Amanda; Al-Rayess, Heba; Othman, Mohammad Imad

    2014-07-01

    Donor specific antibodies (DSA) play a significant role in graft rejection. Many laboratory methods, varied in sensitivity and specificity, are used to detect them. We report a case of a 38-year-old man presented with end stage renal disease considered for kidney transplantation. He had no history of blood transfusions nor transplantation procedures. Dilemma rose when he got multiple positive crossmatches with matching donors and a positive autologous crossmatch due to IgG anti HLA auto-antibodies, which are at the same time against matched donors. Since positive crossmatch is a contraindication for transplant, we couldn't perform transplant from any matched donor. Therefore, we considered a total mismatched donor then transplantation was performed. Observation after surgery showed normalization of creatinine, blood pressure and a good function of the planted allograft for two years of follow up.

  2. A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants.

    PubMed

    Jäckel, S; Eiden, M; Balkema-Buschmann, A; Ziller, M; van Vuren, P Jansen; Paweska, J T; Groschup, M H

    2013-10-01

    Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies.

  3. Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

    PubMed

    Palomares, R A; Hurley, D J; Crum, L T; Rollin, E; Collop, T; Williard, A; Felton, J; Parrish, J; Corbeil, L B

    2017-01-01

    The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of

  4. International standards for IgG and IgM anti-β2glycoprotein antibody measurement.

    PubMed

    Willis, R; Grossi, C; Orietta Borghi, M; Martos-Sevilla, G; Zegers, I; Sheldon, J; Meroni, P L

    2014-10-01

    International standards for anti-beta2 glycoprotein I (anti-β2GPI) testing are needed. We evaluated the suitability of polyclonal/monoclonal candidate reference materials (RM) for the assay. IgG/IgM anti-β2GPI were affinity-purified (AP) from high-positive antiphospholipid syndrome sera and IgG from HCAL clone supernatant. Igs were tested for purity by SDS-PAGE, pooled, concentrated, sterile-filtered and the protein concentration determined. One unit was defined as the binding activity of 1 µg/ml of AP anti-β2GPI Ig. IgG/IgM RM were each assigned a unit value using the respective AP material as a calibrator. Polyclonal/monoclonal RM and 30 samples were evaluated for linearity, unit equivalency and commutability. Polyclonal AP material was assigned a value of 100 U IgG and 15 U IgM anti-β2GPI, respectively. IgG-RM had a value of 270 IgG and the IgM-RM of 220.3 IgM anti-β2GPI U. The linearity (R (2)) of each RM curve for the various assays ranged from 0.96 to 0.99. Commutability samples fit very well within 95% prediction intervals and had excellent correlation when comparing assays. IgG and IgM polyclonal and IgG monoclonal RM displayed excellent linearity and commutability, being good candidates for better standardization of anti-β2GPI immunoassays.

  5. A rare case of Addison's disease, hepatitis, thyreoiditis, positive IgG anti-tissue transglutaminase antibodies and partial IgA deficiency

    PubMed Central

    Mihaylova, Snejina; Yankova, Petja; Atanasova, Iliana; Nikolova-Vlahova, Milena; Naumova, Elissaveta

    2016-01-01

    Introduction Selective IgA deficiency (IgAD) is the most prevalent type of primary immune deficiencies, but partial IgA deficiency is even more common. Addison's disease is a rare condition associated with primary adrenal insufficiency due to infection or autoimmune destruction of the adrenals. The association between IgA deficiency and Addison's disease is very rare. Case and laboratory data We observed a 22-year-old male patient with marked darkening of the skin, especially on the palms and areolae, jaundice on the skin and sclera, astheno-adynamia, hypotension (80/50 mm Hg), and pain in the right hypochondrium. The laboratory investigations revealed increased serum levels of total and indirect bilirubin, AST, ALT, GGT and LDH, negative HBsAg, anti-HBc IgM, anti-HCV and anti-HAV IgM, very low serum IgA levels (0.16 g/l) with normal IgG and IgM, negative ANA, ANCA, AMA, LKM-1, anti-GAD-60, anti-IA-2, anti-thyroglobulin antibodies, a mild increase in anti-TPO antibodies titer, a marked increase in IgG anti-tissue transglutaminase antibodies, with no typical changes in cellular immunity, negative T-SPOT-TB test, HLA – A*01; B*08; DRB1*03; DQB1*02, karyotype – 46, XY. Conclusions We present a rare case of partial IgA deficiency with Addison's disease, hepatitis, thyroiditis and positive anti-tissue transglutaminase antibodies. IgAD and some autoimmune disorders share several predisposing HLA genes, thus explaining the increased prevalence of IgAD in certain patient groups. PMID:27536208

  6. Application of a high-throughput screening procedure with PEG-induced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies.

    PubMed

    Gibson, Todd J; Mccarty, Katie; Mcfadyen, Iain J; Cash, Ethan; Dalmonte, Paul; Hinds, Kenneth D; Dinerman, Adam A; Alvarez, Juan C; Volkin, David B

    2011-03-01

    Protein solubility is a critical attribute in monoclonal antibody (mAb) formulation development as insolubility issues can negatively impact drug stability, activity, bioavailability, and immunogenicity. A high-throughput adaptation of an experimental method previously established in the literature to determine apparent protein solubility is described, where polyethylene glycol (PEG) is used to reduce protein solubility in a quantitatively definable manner. Utilizing an automated, high-throughput system, an immunoglobulin G (IgG)1 mAb in a variety of buffer conditions was exposed to increasing concentrations of PEG and the amount of protein remaining in solution was determined. Comparisons of PEG(midpt) values (the weight% PEG in solution required to decrease the protein concentration by 50%) to extrapolated values of apparent protein solubility (in the absence of PEG) were performed. The determination of PEG(midpt) by using sigmoidal curve fitting of the entire data set was shown to be the most precise and reproducible approach for use during high-throughput screening experiments. The high-throughput PEG methodology was then applied to the screening of different formulations to optimize relative protein solubility profiles (weight% PEG vs. protein concentration and their corresponding PEG(midpt) values) in terms of solution pH and buffer ions for both human and chimeric IgG1 mAbs. Other comparisons included evaluating relative solubility profiles of an IgG1 mAb produced from different cell lines (Chinese hamster ovary vs. murine) as well as for different IgG1 mAbs (produced from the same cell line) in a series of formulation buffers. Based on these comparisons, it was concluded that rapid, high-throughput determinations of relative protein solubility profiles can be used as a practical, experimental tool to compare mAb preparations and to rank order buffer and pH conditions during formulation development.

  7. Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization.

    PubMed Central

    Bohn, A; König, W

    1982-01-01

    Monoclonal DNP-specific IgG (lambda 2 epsilon 2), IgM (kappa 2 mu2) and IgG [kappa 2 (gamma 1)2] were isolated fom the culture supernatant of hybridomas by affinity chromatography with 2,4-dinitrophenol bovine serum albumin (DNP-BSA) sepharose and characterized by biochemical and biological methods. The molecular weights were 84,200 for the epsilon chain, 55,400 for the gamma chain and 77,500 for the mu chain as determined by sodium dodecylsulphate polyacrylamide del electrophoresis (SDS-PAGE). The association constants for [3H]-DNP-lysine determined by equilibrium dialysis were 0 . 87 X 10(7) l/mol for IgE and 1 . 91 X 10(8) 1/mol for IgG1. The isoelectric focusing of the purified monoclonal antibodies revealed for IgG1 seven bands at a pH range of 6 . 3 - 7 . 2 and for IgE sixteen bands at a pH range of 4 . 5 to 6 . 8. the binding of 125I-anti-IgE to rat basophilic leukaemia (RBL) and rat mast cells which had been preincubated with various amounts of monoclonal IgE was studied. At saturation conditions of IgE, about 2 . 14 X 10(5) molecules of anti-IgE were bound per rat mast cell. Rat mast cells coated with monoclonal anti-DNP IgE were triggered for the release of histamine in the presence of either the antigen or guinea-pig anti-mouse IgE. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against ovalbumin or rat reaginic serum directed against Nippostrongylus brasiliensis with monoclonal mouse anti-DNP IgE was demonstrated. Images Figure 2 Figure 7 Figure 8 PMID:6180975

  8. Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area

    PubMed Central

    Elanga Ndille, Emmanuel; Doucoure, Souleymane; Poinsignon, Anne; Mouchet, François; Cornelie, Sylvie; D’Ortenzio, Eric; DeHecq, Jean Sébastien; Remoue, Franck

    2016-01-01

    Background Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes. Methodology/Principal findings Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P < 0.0001), and remained low for 4 weeks post-intervention (P = 0.0002). The specific IgG decrease was associated with the decline of Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation. Conclusion/Significance Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species. PMID:27906987

  9. Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice

    PubMed Central

    Mandalasi, Msano; Dorabawila, Nelum; Smith, David F; Heimburg-Molinaro, Jamie; Cummings, Richard D; Nyame, A Kwame

    2013-01-01

    The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Lex; Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Lex, we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Lex sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Lex antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Lex epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Lex epitopes, but not to glycans with a single, terminal Lex epitope. Our results show that F8A1.1 recognizes terminal Lex epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Lex-containing glycoconjugates from schistosomes and mammalian cells. PMID:23542315

  10. Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

    PubMed

    Dada, Oluwatosin O; Jaya, Nomalie; Valliere-Douglass, John; Salas-Solano, Oscar

    2015-08-20

    Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

  11. Interdisciplinary orthodontic treatment for a patient with generalized aggressive periodontitis: Assessment of IgG antibodies to identify type of periodontitis and correct timing of treatment.

    PubMed

    Ishihara, Yoshihito; Tomikawa, Kazuya; Deguchi, Toru; Honjo, Tadashi; Suzuki, Koji; Kono, Takayuki; Kuboki, Takuo; Kamioka, Hiroshi; Takashiba, Shogo; Yamashiro, Takashi

    2015-06-01

    Aggressive periodontitis is a great challenge to clinicians when providing orthodontic treatment because of the potential for progression of periodontal disease. In this article, we report the successful comprehensive orthodontic treatment of bimaxillary protrusion and severe crowding in an adult with generalized aggressive periodontitis. A woman, aged 22 years 7 months, with a chief complaint of incisal crowding was diagnosed with a skeletal Class I malocclusion associated with severe anterior crowding, possibly worsened by generalized aggressive periodontitis. In addition to a periodontal examination, a blood IgG antibody titer analysis and microbiologic examination for periodontal pathogens were used to diagnose the type of periodontal disease and determine the proper timing to initiate orthodontic treatment. The total active treatment period was 28 months, followed by periodontal prostheses and regeneration therapy. Consequently, satisfactory facial profile, occlusion, and periodontal health were maintained for at least 36 months. These results indicate that efficient screening is important for providing successful orthodontic treatment in patients with advanced periodontal disease. This report also demonstrates the diagnostic importance of blood IgG antibody titer assays and microbiologic examinations to detect periodontal pathogens.

  12. Anti-LRP/LR–Specific Antibody IgG1-iS18 Significantly Impedes Adhesion and Invasion in Early- and Late-Stage Colorectal Carcinoma Cells

    PubMed Central

    Vania, Leila; Chetty, Carryn J; Ferreira, Eloise; Weiss, Stefan FT

    2016-01-01

    Cancer is a highly complex disease that has become one of the leading causes of death globally. Metastasis, a major cause of cancer deaths, requires two crucial events, adhesion and invasion. The 37kDa/67kDa laminin receptor (laminin receptor precursor/high-affinity laminin receptor [LRP/LR]) enhances these two steps, consequently aiding in cancer progression. In this study, the role of LRP/LR in adhesion and invasion of early-stage (SW-480 and HT-29) and late-stage (DLD-1) colorectal cancer cells was investigated. Western blotting revealed that early- and late-stage colorectal cancer cells contained significantly higher total LRP/LR levels compared with poorly invasive MCF-7 breast cancer control cells. Flow cytometry revealed that both stages of colorectal cancer displayed significantly higher cell surface LRP/LR levels. Furthermore, upon treatment of colorectal cancer cells with the anti-LRP/LR–specific antibody IgG1-iS18, adhesion to laminin-1 was significantly reduced in both stages. Each stage’s invasive potential was determined using the Matrigel™ invasion assay, showing that invasion was significantly impeded in both colorectal cancer stages when the cells were incubated with IgG1-iS18. In addition, Pearson’s correlation coefficients propose that both total and cell surface LRP/LR levels are directly proportional to the adhesive and invasive potential of both stages of colorectal cancer. Hence, these findings indicate potential for use of the IgG1-iS18 antibody as a promising therapeutic tool for colorectal cancer patients at both stages. PMID:27611822

  13. Prevalence of anti-rubella, anti-measles and anti-mumps IgG antibodies in neonates and pregnant women in Catalonia (Spain) in 2013: susceptibility to measles increased from 2003 to 2013.

    PubMed

    Plans, P; de Ory, F; Campins, M; Álvarez, E; Payà, T; Guisasola, E; Compte, C; Vellbé, K; Sánchez, C; Lozano, M J; Aran, I; Bonmatí, A; Carreras, R; Jané, M; Cabero, L

    2015-06-01

    Non-immune neonates and non-immune pregnant women are at risk of developing rubella, measles and mumps infections, including congenital rubella syndrome. We describe the seroepidemiology of measles, mumps and rubella (MMR) in neonates and pregnant women in Catalonia (Spain). Anti-rubella, anti-measles and anti-mumps serum IgG titres were assessed using enzyme-linked immunosorbent assay (ELISA) tests in 353 cord blood samples from neonates of a representative sample of pregnant women obtained in 2013. The prevalence of protective antibody titres in neonates was 96 % for rubella IgG (≥8 IU/ml), 90 % for measles IgG (>300 IU/ml) and 84 % for mumps IgG (>460 EU/ml). Slightly lower prevalences of protective IgG titres, as estimated from the cord blood titres, were found in pregnant women: 95 % for rubella IgG, 89 % for measles IgG and 81 % for mumps IgG. The anti-measles and anti-mumps IgG titres and the prevalences of protective IgG titres against measles and mumps increased significantly (p < 0.001) with maternal age. The prevalence of protective anti-measles IgG titres decreased by 7 % [odds ratio (OR) = 0.15, p < 0.001), the prevalence of protective anti-rubella IgG titres increased by 3 % (OR = 1.80, p < 0.05) and the MMR vaccination coverage (during childhood) in pregnant women increased by 54 % (OR = 2.09, p < 0.001) from 2003 to 2013. We recommend to develop an MMR prevention programme in women of childbearing age based on mass MMR vaccination or MMR screening and vaccination of susceptible women to increase immunity levels against MMR.

  14. IgG Antibody Responses to Recombinant gp120 Proteins, gp70V1/V2 Scaffolds, and a CyclicV2 Peptide in Thai Phase I/II Vaccine Trials Using Different Vaccine Regimens.

    PubMed

    Karasavvas, Nicos; Karnasuta, Chitraporn; Savadsuk, Hathairat; Madnote, Sirinan; Inthawong, Dutsadee; Chantakulkij, Somsak; Rittiroongrad, Surawach; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Thongcharoen, Prasert; Siriyanon, Vinai; Andrews, Charla A; Barnett, Susan W; Tartaglia, James; Sinangil, Faruk; Francis, Donald P; Robb, Merlin L; Michael, Nelson L; Ngauy, Viseth; de Souza, Mark S; Paris, Robert M; Excler, Jean-Louis; Kim, Jerome H; O'Connell, Robert J

    2015-11-01

    RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01_AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01_AE scaffolds was higher with the AIDSVAX(®)B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.

  15. Development of antihuman IgG antibodies and hematologic deficits but not clinical abnormalities in C57BL/6 mice after repeated administration of human intravenous immunoglobulin.

    PubMed

    Loeffler, David A; Smith, Lynnae M; Klaver, Andrea C; Brzezinski, Heather A; Morrison, Essie I; Coffey, Mary P; Steficek, Barbara A; Cook, Susan S

    2012-02-01

    Intravenous immunoglobulin (IvIg) preparations consist of purified human immunoglobulins collected from large numbers of healthy persons and are used to treat autoimmune, immunodeficiency, and inflammatory disorders. Studying the effects of IvIg effects in experimental animal models might clarify its mechanisms of action in these disorders, but whether 'serum sickness' or other abnormalities occur after repeated IvIg administration to immunocompetent animals is unknown. In the current study, male C57BL/6 mice (8 to 10 wk old; n = 27) received IvIg (1 g/kg IP) weekly for 6 wk. They were observed for clinical abnormalities, and body weight, temperature, renal function, hematologic parameters, and serum antihuman IgG antibodies were measured before and during treatment. Postmortem evaluations were performed on kidney, spleen, liver, and heart. No clinical or histologic abnormalities were noted despite a transient increase in BUN. Mean antibody levels to human IgG on days 21 and 43 after IvIg administration were increased by 23-fold compared with pretreatment levels. 88% and 89% of the mice were antibody responders on those days. Unexpectedly, hemoglobin, hematocrit, and RBC, WBC, lymphocyte, and platelet counts decreased after IvIg administration. These findings suggest that although it does not produce serum sickness, repeated IvIg administration to immunocompetent mice induces a strong humoral immune response and hematologic deficits of unknown etiology. These factors could cause the effects of IvIg preparations in mouse models of human disease to differ from their effects in the human disorders.

  16. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  17. Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

    PubMed Central

    Aw-Yong, Kam Leng; Sam, I-Ching; Koh, Mia Tuang

    2016-01-01

    Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. PMID:27806091

  18. Presence of Mycoplasma fermentans in the bloodstream of Mexican patients with rheumatoid arthritis and IgM and IgG antibodies against whole microorganism

    PubMed Central

    Gil, Constantino; Rivera, Antonio; Bañuelos, David; Salinas, Salvador; García-Latorre, Ethel; Cedillo, Lilia

    2009-01-01

    Background Increasing evidence incriminates bacteria, especially Mycoplasma fermentans, as possible arthritogenic agents in humans. The purpose of this study was to investigate M. fermentans in the bloodstream of patients with rheumatoid arthritis. Methods Two hundred and nineteen blood samples from patients with rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, and healthy individuals were screened by bacterial culture and direct PCR in order to detect mycoplasmas; IgM and IgG against M. fermentans PG18 were also detected by ELISA and Immunoblotting assays in patients with rheumatoid arthritis and healthy individuals. Results Blood samples from patients with antiphospholipid syndrome and healthy individuals were negative for mycoplasma by culture or direct PCR. In blood samples from patients with systemic lupus erythematosus were detected by direct PCR M. fermentans in 2/50 (2%), M. hominis in 2/50 (2%) and U. urealyticum in 1/50 (0.5%). In patients with RA M. fermentans was detected by culture in 13/87 blood samples and in 13/87 by direct PCR, however, there was only concordance between culture and direct PCR in six samples, so M. fermentans was detected in 20/87(23%) of the blood samples from patients with RA by either culture or PCR. Antibody-specific ELISA assay to M. fermentans PG18 was done, IgM was detected in sera from 40/87 patients with RA and in sera of 7/67 control individuals, IgG was detected in sera from 48/87 RA patients and in sera from 7/67 healthy individuals. Antibody-specific immunoblotting to M. fermentans PG18 showed IgM in sera from 35/87 patients with RA and in sera from 4/67 healthy individuals, IgG was detected in sera from 34/87 patients and in sera from 5/67 healthy individuals. Conclusion Our findings show that only M. fermentans produce bacteremia in a high percentage of patients with RA. This finding is similar to those reported in the literature. IgM and IgG against M. fermentans PG18 were more frequent

  19. Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.

    PubMed

    van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia

    2014-04-01

    Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (p<0.0001) lower reactivity was demonstrated in samples from previously infected patients where acute rubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms.

  20. Interleukin-2-Dependent Allergen-Specific Tissue-Resident Memory Cells Drive Asthma.

    PubMed

    Hondowicz, Brian D; An, Dowon; Schenkel, Jason M; Kim, Karen S; Steach, Holly R; Krishnamurty, Akshay T; Keitany, Gladys J; Garza, Esteban N; Fraser, Kathryn A; Moon, James J; Altemeier, William A; Masopust, David; Pepper, Marion

    2016-01-19

    Exposure to inhaled allergens generates T helper 2 (Th2) CD4(+) T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue-resident memory cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.

  1. Antigenic proteins of Lactobacillus acidophilus that are recognised by serum IgG antibodies in children with type 1 diabetes and coeliac disease.

    PubMed

    Prangli, Anna-Liisa; Utt, Meeme; Talja, Ija; Sepp, Epp; Mikelsaar, Marika; Rajasalu, Tarvo; Uibo, Oivi; Tillmann, Vallo; Uibo, Raivo

    2010-06-01

    Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

  2. Fast, robust and high-resolution glycosylation profiling of intact monoclonal IgG antibodies using nanoLC-chip-QTOF.

    PubMed

    Jacobs, Joannes F M; Wevers, Ron A; Lefeber, Dirk J; van Scherpenzeel, Monique

    2016-10-01

    Optimal glycosylation of immunoglobulins is essential in the generation of therapeutic biologicals with respect to efficacy, pharmacokinetics and immunogenic properties. This challenge in the field of biopharmaceuticals requires technologies for fast, robust and quantitative analysis of glycosylation. Current analyses of monoclonal antibody glycosylation are proteolysis-based mass spectrometry methods, which provide detailed structural information, but suffer a number of drawbacks such as lengthy sample preparation with the possibility to introduce artifacts. Here, we describe a fast, robust and high-resolution nanoLC-chip-QTOF method for quantitative analysis of intact monoclonal IgG glycosylation profiling. The method is able to detect hypoglycosylation, i.e. the lack of whole glycans, which is an important advantage over the well-established methods for free N-glycan or glycopeptide analysis. Moreover, the method is highly amenable to automation and because no digestion steps are involved, it provides direct relative quantitative information of both glycans on each IgG attachment site. We demonstrate that the ease and robustness make this technique ideally suited for quality control of the production process of mAb biopharmaceuticals, and provides new opportunities to study the clinical impact of mAb-glycosylation in patients with monoclonal gammopathies.

  3. Evaluation of the Human IgG Antibody Response to Aedes albopictus Saliva as a New Specific Biomarker of Exposure to Vector Bites

    PubMed Central

    Doucoure, Souleymane; Mouchet, François; Cornelie, Sylvie; DeHecq, Jean Sébastien; Rutee, Abdul Hamid; Roca, Yelin; Walter, Annie; Hervé, Jean Pierre; Misse, Dorothée; Favier, François; Gasque, Philippe; Remoue, Franck

    2012-01-01

    Background The spread of Aedes albopictus, a vector for re-emergent arbovirus diseases like chikungunya and dengue, points up the need for better control strategies and new tools to evaluate transmission risk. Human antibody (Ab) responses to mosquito salivary proteins could represent a reliable biomarker for evaluating human-vector contact and the efficacy of control programs. Methodology/Principal Findings We used ELISA tests to evaluate specific immunoglobulin G (IgG) responses to salivary gland extracts (SGE) in adults exposed to Aedes albopictus in Reunion Island. The percentage of immune responders (88%) and levels of anti-SGE IgG Abs were high in exposed individuals. At an individual level, our results indicate heterogeneity of the exposure to Aedes albopictus bites. In addition, low-level immune cross-reactivity between Aedes albopictus and Aedes aegypti SGEs was observed, mainly in the highest responders. Conclusion/Significance Ab responses to saliva could be used as an immuno-epidemiological tool for evaluating exposure to Aedes albopictus bites. Combined with entomological and epidemiological methods, a “salivary” biomarker of exposure to Aedes albopictus could enhance surveillance of its spread and the risk of arbovirus transmission, and could be used as a direct tool for the evaluation of Aedes albopictus control strategies. PMID:22363823

  4. Assessing in vivo dynamics of multiple quality attributes from a therapeutic IgG4 monoclonal antibody circulating in cynomolgus monkey

    PubMed Central

    Li, Yinyin; Huang, Yu; Ferrant, Janine; Lyubarskaya, Yelena; Zhang, Yue (Emma); Li, Siyang (Peter); Wu, Shiaw-Lin (Billy)

    2016-01-01

    ABSTRACT Characterization of biopharmaceutical proteins and assessment and understanding of the critical quality attributes (CQAs) is a significant part of biopharmaceutical product development and is routinely performed in vitro. In contrast, systematic analysis of the quality attributes in vivo is not as widespread, although metabolism and clearance of multiple variants of therapeutic proteins administered to non-human primates and human subjects may have a different impact on safety, efficacy and exposure. The major hurdles of such studies are usually sample availability and technical capability. In this study, we used affinity purification coupled with liquid chromatography and mass spectrometric analysis of the digested protein for consistent and simultaneous detection of the full amino acid sequence of a therapeutic IgG4 monoclonal antibody, MAB1. This methodology allowed us to assess in vivo changes of all sequence-related modifications and quality attributes of MAB1 over the duration of a preclinical pharmacokinetic study in cynomolgus monkeys. PMID:27030286

  5. ELISA detection of specific functional antibodies in human serum to Escherichia coli, tetanus toxoid, and diphtheria-tetanus toxoids: normal values for IgG, IgA, and IgM.

    PubMed

    Moen, R C; Oemichen, S L; Kiggens, A J; Hong, R

    1986-01-01

    An inexpensive, easily performed enzyme-linked immunosorbent assay (ELISA) was developed to measure specific IgG, IgA, and IgM antibodies to the common antigens Escherichia coli, diphtheria-tetanus toxoid, and tetanus toxoid. Normal values were established. Classical antibody deficiency disease states were confirmed and delineated by these assays. Additionally, several instances were discovered when functional antibody levels were abnormal when the serum immunoglobulin levels were normal. The use of ELISA assays for antibodies to common antigens provides a useful technique to measure and monitor isotype responses of the humoral immune system.

  6. Investigation of neutron radiation effects on polyclonal antibodies (IgG) and fluorescein dye for astrobiological applications.

    PubMed

    Le Postollec, A; Coussot, G; Baqué, M; Incerti, S; Desvignes, I; Moretto, P; Dobrijevic, M; Vandenabeele-Trambouze, O

    2009-09-01

    Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Biochips have been proposed as a way to detect organic matter on extraterrestrial objects. A biochip is a miniaturized device composed of biologically sensitive systems, such as antibodies, which are immobilized on a slide. In the case of in situ measurements, the main concern is to ensure the survival of the antibodies under space radiation. Our recent computing simulation of cosmic ray interactions with the martian environment shows that neutrons are one of the dominant species at soil level. Therefore, we have chosen, in a first approach, to study antibody resistance to neutrons by performing irradiation experiments at the Applications Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine (AIFIRA) platform, a French ion beam facility at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in Bordeaux. Antibodies and fluorescent dyes, freeze-dried and in buffer solution, were irradiated with 0.6 MeV and 6 MeV neutrons. Sample analyses demonstrated that, in the conditions tested, antibody recognition capability and fluorescence dye intensity are not affected by the neutrons.

  7. Opalescence of an IgG2 monoclonal antibody solution as it relates to liquid-liquid phase separation.

    PubMed

    Mason, Bruce D; Zhang, Le; Remmele, Richard L; Zhang, Jifeng

    2011-11-01

    Opalescence for a monoclonal antibody solution was systematically studied with respect to temperature, protein concentration, ionic strength (using KCl), and pH conditions. Multiple techniques, including measurement of light scattering at 90° and transmission, Tyndall test, and microscopy, were deployed to examine the opalescence behavior. Near the vicinity of the critical point on the liquid-liquid coexistence curve in the temperature-protein concentration phase diagram, the enhanced concentration fluctuations significantly contributed to the critical opalescence evidently by formation of small liquid droplets. Furthermore, our data confirm that away from the critical point, the opalescence behavior is related to the antibody self-association (agglomeration) caused by the attractive antibody-antibody interactions. As expected, at a pH near the pI of the antibody, the solution became less opalescent as the ionic strength increased. However, at a pH below the pI, the opalescence of the solution became stronger, reached a maximum, and then began to drop as the ionic strength further increased. The change in the opalescence correlated well with the trends of protein-protein interactions revealed by the critical temperature from the liquid-liquid phase separation.

  8. Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - prediction of viscosity through protein-protein interaction measurements.

    PubMed

    Neergaard, Martin S; Kalonia, Devendra S; Parshad, Henrik; Nielsen, Anders D; Møller, Eva H; van de Weert, Marco

    2013-06-14

    The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI either at low concentration (interaction parameter (kD) obtained from dynamic light scattering, DLS) or at high concentration (solution storage modulus (G') from ultrasonic shear rheology). We also developed a novel method for the determination of PPI using the apparent radius of the protein at either low or high protein concentration determined using DLS. The PPI measurements were correlated with solution viscosity (measured by DLS using polystyrene nanospheres and ultrasonic shear rheology) as a function of pH (4-9) and ionic strength (10, 50 and 150 mM). Our measurements showed that the highest solution viscosity was observed under conditions with the most negative kD, the highest apparent radius and the lowest net charge. An increase in ionic strength resulted in a change in the nature of the PPI at low pH from repulsive to attractive. In the neutral to alkaline pH region the mAbs behaved differently with respect to increase in ionic strength. Two mAbs (A and B) showed little or no effect of increasing ionic strength, whereas mAb-C showed a remarkable decrease in attractive PPI and viscosity. Previous studies have mainly investigated mAbs of the IgG₁ and IgG₂ subclass. We show here, for the first time, that mAbs of the IgG₄ subclass behave similar as the other subclasses. By comparison of the three tested mAbs with mAbs investigated in other studies a clear linear trend emerges between the pH of strongest attractive PPI and highest solution viscosity. The determination of PPI using either kD or apparent radius is thus a useful prediction tool in the determination of solution conditions that favors low solution viscosity at high protein concentration of

  9. Molecular basis of lateral force spectroscopy nano-diagnostics: computational unbinding of autism related chemokine MCP-1 from IgG antibody.

    PubMed

    Gogolinska, Anna; Nowak, Wieslaw

    2013-11-01

    Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, is a potent chemoattractant of T cells and monocytes, involved in inflammatory and angio-proliferative brain and retinal diseases. Higher expression of MCP-1 is observed in metastatic tumors. Unusual levels of MCP-1 in the brain may be correlated with autism. Immunochemistry where atomic force microscope (AFM) tips functionalized with appropriate antibodies against MCP-1 are used could in principle support medical diagnostics. Useful signals from single molecule experiments may be generated if interaction forces are large enough. The chemokine-antibody unbinding force depends on a relative motion of the interacting fragments of the complex. In this paper the stability of the medically important MCP-1- immunoglobulin G antibody Fab fragment complex has been studied using steered molecular dynamics (SMD) computer simulations with the aim to model possible arrangements of nano-diagnostics experiments. Using SMD we confirm that molecular recognition in MCP1-IgG is based mainly on six pairs of residues: Glu39A - Arg98H, Lys56A - Asp52H, Asp65A - Arg32L, Asp68A - Arg32L, Thr32A - Glu55L, Gln61A - Tyr33H. The minimum external force required for mechanical dissociation of the complex depends on a direction of the force. The pulling of the MCP-1 antigen in the directions parallel to the antigen-antibody contact plane requires forces about 20 %-40 % lower than in the perpendicular one. Fortunately, these values are large enough that the fast lateral force spectroscopy may be used for effective nano-diagnostics purposes. We show that molecular modeling is a useful tool in planning AFM force spectroscopy experiments.

  10. Allergen-specific MHC class II tetramer+ cells are detectable in allergic, but not in nonallergic, individuals.

    PubMed

    Macaubas, Claudia; Wahlstrom, Jan; Galvão da Silva, Ana Paula; Forsthuber, Thomas G; Sønderstrup, Grete; Kwok, William W; DeKruyff, Rosemarie H; Umetsu, Dale T

    2006-04-15

    Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

  11. Aluminium in allergen-specific subcutaneous immunotherapy--a German perspective.

    PubMed

    Kramer, Matthias F; Heath, Matthew D

    2014-07-16

    We are living in an "aluminium age" with increasing bioavailability of the metal for approximately 125 years, contributing significantly to the aluminium body burden of humans. Over the course of life, aluminium accumulates and is stored predominantly in the lungs, bones, liver, kidneys and brain. The toxicity of aluminium in humans is briefly summarised, highlighting links and possible causal relationships between a high aluminium body burden and a number of neurological disorders and disease states. Aluminium salts have been used as depot-adjuvants successfully in essential prophylactic vaccinations for almost 100 years, with a convincing positive benefit-risk assessment which remains unchanged. However, allergen-specific immunotherapy commonly consists of administering a long-course programme of subcutaneous injections using preparations of relevant allergens. Regulatory authorities currently set aluminium limits for vaccines per dose, rather than per treatment course. Unlike prophylactic vaccinations, numerous injections with higher proportions of aluminium-adjuvant per injection are applied in subcutaneous immunotherapy (SCIT) and will significantly contribute to a higher cumulative life dose of aluminium. While the human body may cope robustly with a daily aluminium overload from the environment, regulatory cumulative threshold values in immunotherapy need further addressing. Based on the current literature, predisposing an individual to an unusually high level of aluminium, such as through subcutaneous immunotherapy, has the potential to form focal accumulations in the body with the propensity to exert forms of toxicity. Particularly in relation to longer-term health effects, the safety of aluminium adjuvants in immunotherapy remains unchallenged by health authorities - evoking the need for more consideration, guidance, and transparency on what is known and not known about its safety in long-course therapy and what measures can be taken to prevent or

  12. Oral glucocorticoids diminish the efficacy of allergen-specific immunotherapy in experimental feline asthma.

    PubMed

    Chang, Chee-hoon; Cohn, Leah A; Declue, Amy E; Liu, Hong; Reinero, Carol R

    2013-08-01

    Allergen-specific rush immunotherapy (RIT) shows promise in treating asthma; however, pet cats will likely require at least initial concurrent glucocorticoids (GCs) to control serious clinical signs. How the immunosuppressive effects of GCs would impact RIT in cats is unknown. The hypothesis of this study was that oral, but not inhaled GCs will diminish the efficacy of RIT in experimental feline asthma. Cats (n=6/group) were sensitized using Bermuda grass allergen (BGA) and randomized to receive BGA-specific RIT for 9 months with an oral GC (prednisolone 10mg daily), inhaled GC (fluticasone 220 μg twice daily), or placebo administered for the first 6 months. Bronchoalveolar lavage fluid (BALF) percent eosinophils and other immunological assays were performed. Eosinophilic airway inflammation was suppressed in all groups at month 6 of RIT (group mean ± SD, 5 ± 2%, 13 ± 4%, and 7 ± 2% for oral GC, inhaled GC, and placebo, respectively; P=0.291). BALF percent eosinophils significantly increased over time only in oral GC/RIT cats between months 6 and 9 (P=0.031). Placebo/RIT cats had significant decreases over time in BGA-specific serum IgE (P=0.031). Concentration of interleukin (IL)-5 in BALF significantly increased over time in inhaled GC/RIT cats (P=0.031). No significant differences were found between groups at month 6 or over time in each group for BGA-specific lymphocyte blastogenesis, percent blood T regulatory cells, or number of IL-10-producing cells. Given the significant increase of airway eosinophilia over time in RIT cats initially treated with an oral GC, inhaled GCs might be better for dampening eosinophilic inflammation until RIT normalizes the dysregulated immune system.

  13. Clinical practice recommendations for allergen-specific immunotherapy in children: the Italian consensus report.

    PubMed

    Pajno, Giovanni Battista; Bernardini, Roberto; Peroni, Diego; Arasi, Stefania; Martelli, Alberto; Landi, Massimo; Passalacqua, Giovanni; Muraro, Antonella; La Grutta, Stefania; Fiocchi, Alessandro; Indinnimeo, Luciana; Caffarelli, Carlo; Calamelli, Elisabetta; Comberiati, Pasquale; Duse, Marzia

    2017-01-23

    Allergen-specific immunotherapy (AIT) is currently recognized as a clinically effective treatment for allergic diseases, with a unique disease-modifying effect. AIT was introduced in clinical practice one century ago, and performed in the early years with allergenic extracts of poor quality and definition. After the mechanism of allergic reaction were recognized, the practice of AIT was refined, leading to remarkable improvement in the efficacy and safety profile of the treatment. Currently AIT is accepted and routinely prescribed worldwide for respiratory allergies and hymenoptera venom allergy. Both the subcutaneous (SCIT) and sublingual (SLIT) routes of administration are used in the pediatric population.AIT is recommended in allergic rhinitis/conjunctivitis with/without allergic asthma, with an evidence of specific IgE-sensitization towards clinically relevant inhalant allergens. Long-term studies provided evidence that AIT can also prevent the onset of asthma and of new sensitizations. The favorable response to AIT is strictly linked to adherence to treatment, that lasts 3-5 years. Therefore, several factors should be carefully evaluated before starting this intervention, including the severity of symptoms, pharmacotherapy requirements and children and caregivers' preference and compliance.In recent years, there have been increasing interest in the role of AIT for the treatment of IgE-associated food allergy and extrinsic atopic dermatitis. A growing body of evidence shows that oral immunotherapy represents a promising treatment option for IgE-associated food allergy. On the contrary, there are still controversies on the effectiveness of AIT for patients with atopic dermatitis.This consensus document was promoted by the Italian Society of Pediatric Allergy and Immunology (SIAIP) to provide evidence-based recommendations on AIT in order to implement and optimize current prescription practices of this treatment for allergic children.

  14. Immunosuppression in early postnatal days induces persistent and allergen-specific immune tolerance to asthma in adult mice.

    PubMed

    Chen, Yan; Zhang, Jin; Lu, Yong; Wang, Libo

    2015-01-01

    Bronchial asthma is a chronic airway inflammatory condition with high morbidity, and effective treatments for asthma are limited. Allergen-specific immunotherapy can only induce peripheral immune tolerance and is not sustainable. Exploring new therapeutic strategies is of great clinical importance. Recombinant adenovirus (rAdV) was used as a vector to make cells expressing cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4Ig) a soluble CTLA4 immunoglobulin fusion protein. Dendritic cells (DCs) were modified using the rAdVs together with allergens. Then these modified DCs were transplanted to mice before allergen sensitization. The persistence and specificity of immune tolerance were evaluated in mice challenged with asthma allergens at 3 and 7 months. DCs modified by CTLA4Ig showed increased IL-10 secretion, decreased IL-12 secretion, and T cell stimulation in vitro. Mice treated with these DCs in the early neonatal period developed tolerance against the allergens that were used to induce asthma in the adult stage. Asthma symptoms, lung damage, airway reactivity, and inflammatory response all improved. Humoral immunity indices showed that this therapeutic strategy strongly suppressed mice immune responses and was maintained for as long as 7 months. Furthermore, allergen cross-sensitization and challenge experiments demonstrated that this immune tolerance was allergen-specific. Treatment with CTLA4Ig modified DCs in the early neonatal period, inducing persistent and allergen-specific immune tolerance to asthma in adult mice. Our results suggest that it may be possible to develop a vaccine for asthma.

  15. Modelling Anti-Ov16 IgG4 Antibody Prevalence as an Indicator for Evaluation and Decision Making in Onchocerciasis Elimination Programmes

    PubMed Central

    Lont, Yvonne L.; Coffeng, Luc E.; de Vlas, Sake J.; Golden, Allison; de los Santos, Tala

    2017-01-01

    Background Onchocerciasis is targeted for elimination in Africa through annual or biannual ivermectin mass drug administration (MDA). An immunodiagnostic test, based on the detection of human IgG4 antibodies in the blood to the Onchocerca volvulus-specific antigen Ov16, is one of the recommended tools for determining whether transmission is interrupted and mass treatment can stop. For different transmission settings, the relationship between post-MDA Ov16 antibody prevalence in children (measured 1 year after the last round of MDA) and the duration and coverage of MDA, the mf prevalence in the population, and the probability that onchocerciasis is eventually eliminated is explored through mathematical modelling. Methodology The ONCHOSIM model was extended with new output on the Ov16 antibody serostatus of individuals. Seroconversion was assumed to be triggered by the first worm establishing in the host, with seroconversion occurring either before maturation, after maturation or only after the start of mf production. We are mainly interested in seroconversion rates in children, and for now ignore the possibility of seroreversion to simplify the model. Principal findings Yearly repeated MDA leads to a strong reduction in the parasite acquisition rate in humans. This reduces the seroconversion rate in newborns and young children, while those who seroconverted before the start of control remain antibody positive. Both the microfiladermia prevalence in the population aged 5 years and above and the Ov16 antibody prevalence in children under 10 declined with increasing duration of MDA. The association between either of these indicators and the model-predicted probability of elimination was not influenced much by the assumed treatment coverage levels, but was found to depend on baseline endemicity levels, assumptions regarding the trigger of seroconversion, and diagnostic test characteristics (sensitivity and specificity). Conclusions Better understanding of the dynamics

  16. Development of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG serum antibodies against measles, mumps, rubella, and varicella-zoster virus.

    PubMed

    Smits, Gaby P; van Gageldonk, Pieter G; Schouls, Leo M; van der Klis, Fiona R M; Berbers, Guy A M

    2012-03-01

    Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles, mumps, rubella, and varicella-zoster virus (MMRV). However, this method is time- and material-consuming. Therefore, a multiplex immunoassay for the simultaneous quantitative detection of antibodies against MMRV was developed. In-house as well as commercially available antigens can be used, making the assay available for all laboratories. The multiplex assay is much more sensitive than the separate ELISAs and has a high specificity, and only 5 μl of serum is needed. Heterologous inhibition did not exceed 11.5%, while homologous inhibition varied between 91.3 and 97.9%. Good correlations with the in-house ELISAs for measles (R(2) = 0.98), mumps (R(2) = 0.97), and rubella (R(2) = 0.97) virus as well as with the ELISA kit for varicella-zoster virus (R(2) = 0.95) were obtained. In conclusion, the MMRV multiplex assay is a good alternative to the conventional ELISAs and suitable for use in serosurveillance and vaccine studies.

  17. Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

    PubMed Central

    Luongo, Timothy S.; Georgiev, Ivelin S.; Matz, Julie; Schmidt, Stephen D.; Louder, Mark K.; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R.

    2013-01-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy. PMID:23843638

  18. Heavy chain-only IgG2b llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of coreceptor- and CD4-binding sites.

    PubMed

    Acharya, Priyamvada; Luongo, Timothy S; Georgiev, Ivelin S; Matz, Julie; Schmidt, Stephen D; Louder, Mark K; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R; Kwong, Peter D

    2013-09-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

  19. Emission of CdSe/ZnS and CdSeTe/ZnS quantum dots conjugated to IgG antibodies

    NASA Astrophysics Data System (ADS)

    Torchynska, T. V.; Casas Espinola, J. L.; Díaz Cano, A.; Douda, J.; Gazarian, K.

    2013-06-01

    Commercially available CdSe/ZnS QDs with emission at 605 nm (2.04 eV) and 655 nm (1.90 eV), as well as CdSeTe/ZnS QDs with emission at 705 nm (1.78 eV), have been studied by photoluminescence (PL) and Raman scattering methods in nonconjugated states and after the conjugation to immunoglobulin G (rabbit IgG) antibodies. PL spectra of nonconjugated QDs are characterized by one Gaussian shape PL band related to exciton emission in CdSe or CdSeTe cores. In PL spectra of bioconjugated QDs the PL bands shift into the high energy and become asymmetric. The PL spectrum transformation in bioconjugated QDs has been assigned to the QD interaction with the antibody electric charges (dipoles). Raman scattering spectra have been studied with the aim to reveal the CdSeTe core compositions, as well as to analyze the elastic strains in bioconjugated QDs. Optical band gaps in CdSe and CdSeTe cores of nonconjugated QDs is calculated numerically versus core radius using the effective mass approximation model with the aim to estimate the average core sizes in studied QD ensembles. It is shown that the PL energy shift increases versus QD core radius. The mechanism of PL energy shift has been discussed. The PL energy shift in bioconjugated QDs is promising for the study of bioconjugation with specific antibodies and can be a powerful technique in biology and medicine.

  20. Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

    PubMed Central

    Lian, Zhirui; Wu, Qindong; Wang, Tongtong

    2016-01-01

    ABSTRACT Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. PMID:26652198

  1. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  2. Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization.

    PubMed

    Copple, Susan S; Giles, S Rashelle; Jaskowski, Troy D; Gardiner, Anna E; Wilson, Andrew M; Hill, Harry R

    2012-05-01

    We evaluated 5 commercially available HEp-2 antinuclear antibody (ANA) indirect fluorescent antibody (IFA) assays using patient serum samples from 45 patients with rheumatoid arthritis, 50 with systemic lupus erythematosus (SLE), 35 with scleroderma, 20 with Sjögren syndrome, 10 with polymyositis, and 100 healthy control subjects. In addition, 12 defined serum samples from the Centers for Disease Control and Prevention and 100 patient serum samples sent to ARUP Laboratories (Salt Lake City, UT) for ANA IFA testing were also examined (n = 372). Standardization among the HEp-2 IFA assays occurred when they exhibited the same titer ± 1 doubling dilution. Agreement of the 5 assays was 78%. Within the specific groups of serum samples, agreement ranged from 44% in scleroderma serum samples to 93% in healthy control subjects, with 72% agreement in the SLE group. Variations in slide and substrate quality were also noted (ie, clarity, consistency of fluorescence, cell size, number and quality of mitotic cells). Along with subjectivity of interpretation, HEp-2 IFA assays are also vulnerable to standardization issues similar to other methods for ANA screening.

  3. Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii.

    PubMed

    Gondim, Luís F P; Wolf, Alexander; Vrhovec, Majda G; Pantchev, Nikola; Bauer, Christian; Langenmayer, Martin C; Bohne, Wolfgang; Teifke, Jens P; Dubey, Jitender P; Conraths, Franz J; Schares, Gereon

    2016-04-01

    Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

  4. Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

    PubMed

    Chen, Chang-Hsin; Abi-Ghanem, Daad; Waghela, Suryakant D; Chou, Wen-Ko; Farnell, Morgan B; Mwangi, Waithaka; Berghman, Luc R

    2012-04-30

    Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

  5. Blood clearance of radiolabeled antibody: enhancement by lactosamination and treatment with biotin-avidin or anti-mouse IgG antibodies

    SciTech Connect

    Klibanov, A.L.; Martynov, A.V.; Slinkin, M.A.; Sakharov, I.Yu.; Smirnov, M.D.; Muzykantov, V.R.; Danilov, S.M.; Torchilin, V.P.

    1988-12-01

    Methods of rapid blood clearance of In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.

  6. Immune Responses in Healthy and Allergic Individuals Are Characterized by a Fine Balance between Allergen-specific T Regulatory 1 and T Helper 2 Cells

    PubMed Central

    Akdis, Mübeccel; Verhagen, Johan; Taylor, Alison; Karamloo, Fariba; Karagiannidis, Christian; Crameri, Reto; Thunberg, Sarah; Deniz, Günnur; Valenta, Rudolf; Fiebig, Helmut; Kegel, Christian; Disch, Rainer; Schmidt-Weber, Carsten B.; Blaser, Kurt; Akdis, Cezmi A.

    2004-01-01

    The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-γ–, interleukin (IL)-4–, and IL-10–producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1–like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4–secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-β as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy. PMID:15173208

  7. Efficacy of IgM anti-blood type antibody monitoring by enzyme-linked immunosorbent assay after renal transplantation across the blood barrier: high-dose immunoglobulin administration blocks IgM rather than IgG anti-blood type antibodies.

    PubMed

    Ishida, H; Tanabe, K; Furusawa, M; Isizuka, T; Tokumoto, T; Shimmura, H; Shimizu, T; Miyamoto, N; Hayashi, T; Toma, H

    2004-09-01

    We used an enzyme linked immunosorbent assay (ELISA) to investigate the presence of subtypes of anti-blood-type antibodies in patients with biopsy-proven humoral rejection after ABO-incompatible renal transplantation. High agglutinin IgG and IgM anti-blood type antibodies from 12 ABO-incompatible recipients with vascular rejection were separately assessed using an ELISA. Patients who exhibited excellent renal function despite high agglutinin titers of anti-blood-type antibodies(n = 8) were also examined. All 12 rejection patients exhibited highly elevated titers of IgG and IgM, while the eight stable patients exhibited only slightly elevated IgG titers, but not IgM. IgG and IgM titers did not change after plasmapheresis and steroid pulse therapy, whereas IVIg treatment significantly blocked both IgG and IgM, with IgM being blocked to a larger extent than IgG. Blocking of IgM seems to play an important role in improving ABO-incompatible grafts.

  8. [Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents].

    PubMed

    Polat, Ceylan; Karataş, Ahmet; Sözen, Mustafa; Matur, Ferhat; Abacıoğlu, Hakan; Öktem, Mehmet Ali

    2016-04-01

    /50 and 1/100 serum dilutions and 1/5.000 and 1/10.000 conjugate dilutions were tested. The horseradish peroxidase conjugated goat anti-mouse IgG for ELISA and the alkaline phosphatase conjugated goat anti-mouse IgG for immunoblot were used. We followed the manufacturer's recommendations for the incubation parameters, substrate and the number of washes. 1/50 serum dilution and 1/10.000 conjugate dilution for ELISA and 1/100 serum dilution and 1/5.000 conjugate dilution for immunoblot were determined as optimal concentrations. By using the optimized ELISA, 26.2% (22/84) of rodents were found positive for hantavirus antibodies according the determined cut-off value (OD(450/620): 0.325). By using immunoblot as a confirmatory test, 20 out of 22 ELISA positive samples could be studied because of the insufficient amount of sera and 17 of them was found positive in terms of DOBV antibodies. Of these rodents 11 were Apodemus flavicollis, three were Apodemus agrarius, two were Microtus guentheri and one was Apodemus sylvaticus. When the results of ELISA were compared to immunoblot results, the optimized ELISA's sensitivity and specificity were found as 100% and 95%, respectively. In this study, a method that can be used in the screening of rodent sera was constituted which uses commercial antigens that can be provided easily, gives fast and reliable results. Similar serological methods optimized for different types of rodents are of great importance for the realization of active follow-up and monitoring of the studies in the field.

  9. Compatibility, physical stability, and characterization of an IgG4 monoclonal antibody after dilution into different intravenous administration bags.

    PubMed

    Kumru, Ozan S; Liu, Jun; Ji, Junyan A; Cheng, Wilson; Wang, Y John; Wang, Tingting; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B

    2012-10-01

    The physical stability of an immunoglobulin G4 monoclonal antibody (mAb) upon dilution into intravenous (i.v.) bags containing 0.9% saline was examined. Soluble aggregates and subvisible particles were observed by size-exclusion high-performance liquid chromatography (SE-HPLC) and light obscuration when formulated with suboptimal levels of polysorbate 20. The formation of soluble aggregates and particulates was further characterized by a combination of SE-HPLC, nanoparticle tracking analysis (NTA), microflow-digital imaging (MFI), and turbidity measurements. With sufficient PS20 levels, particle formation was minimized, although quantification of submicron sized particles by NTA was not possible because of the interference from PS20. Intravenous bags composed of polyvinyl chloride caused more protein particle formation than polyolefin bags. Differences between bag types were affected by removing headspace and by transferring the saline solution into glass vials. Characterization studies with Fourier transform infrared microscopy and extrinsic fluorescence spectroscopy demonstrated that isolated particles contained native-like secondary structure with partially altered tertiary structure, compared with heat-denatured and nonstressed controls. Transmission electron microscopy and MFI analysis showed particles had an amorphous morphology of varying sizes. Particles contained some non-native disulfide bond crosslinks, potentially initiated by low levels of free thiol in the native mAb. The critical role of proper formulation design to stabilize proteins against physical instability during i.v. administration is discussed.

  10. In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa.

    PubMed

    Adawi, Azmi; Bisignano, Carlo; Genovese, Tiziana; Filocamo, Angela; Khouri-Assi, Camellia; Neville, Anat; Feuerstein, Giora Z; Cuzzocrea, Salvatore; Neville, Lewis F

    2012-09-01

    The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a K(D) of 7.4x10(-10) M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD(80) of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation.

  11. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative

    PubMed Central

    Balestrino, E. A.; Daniel, T. M.; de Latini, M. D. S.; Latini, O. A.; Ma, Y.; Scocozza, J. B.

    1984-01-01

    IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA. PMID:6439426

  12. Serum pharmacokinetics and cerebrospinal fluid concentration analysis of the new IgG4 monoclonal antibody GNbAC1 to treat multiple sclerosis: A Phase 1 study

    PubMed Central

    Curtin, François; Vidal, Virginie; Bernard, Corinne; Kromminga, Arno; Lang, Alois B.; Porchet, Hervé

    2016-01-01

    ABSTRACT GNbAC1 is a humanized IgG4 monoclonal antibody antagonist of Mulitple Sclerosis Retrovirus Envelope (MSRV-Env), a protein that could play a critical role in multiple sclerosis. This randomized placebo-controlled dose-escalation study evaluated the safety and pharmacokinetics of GNbAC1 in 21 healthy volunteers after single intravenous infusion at doses of 6, 18 and 36 mg/kg. Lumbar punctures were performed at days 2, 15 or 29 to measure GNbAC1 concentrations in cerebrospinal fluid (CSF). GNbAC1 was well tolerated. Serum data show a dose-linear pharmacokinetics. A mean CSF/serum ratio of 0.12% was observed at Day 2, increasing to 0.39% at Day 15 and 0.42% at Day 29. Linear regression analysis shows a relationship between GNbAC1 CSF/serum ratio and albumin CSF/serum ratio and a relationship at the limit of statistical significance with the timing of CSF sampling. PMID:27030142

  13. Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody

    NASA Astrophysics Data System (ADS)

    Majumdar, Ranajoy; Manikwar, Prakash; Hickey, John M.; Arora, Jayant; Middaugh, C. Russell; Volkin, David B.; Weis, David D.

    2012-12-01

    Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30 % of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5 %)/ isopropanol (5 %)/ acetic acid (20 %) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.

  14. Clostridium difficile PSI polysaccharide: synthesis of pentasaccharide repeating block, conjugation to exotoxin B subunit, and detection of natural anti-PSI IgG antibodies in horse serum.

    PubMed

    Jiao, Yuening; Ma, Zuchao; Hodgins, Doug; Pequegnat, Brittany; Bertolo, Lisa; Arroyo, Luis; Monteiro, Mario A

    2013-08-30

    Clostridium difficile is the most common cause of antimicrobial-associated diarrhea in humans and may cause death. Previously, we discovered that C. difficile expresses three polysaccharides, named PSI, PSII, and PSIII. It has now been established that PSII is a conserved antigen abundantly present on the cell-surface and biofilm of C. difficile. In contrast, the expression of PSI and PSIII appears to be stochastic processes. In this work, the total chemical synthesis of the PSI pentasaccharide repeating unit carrying a linker at the reducing end, α-l-Rhap-(1→3)-β-d-Glcp-(1→4)-[α-l-Rhap-(1→3)]-α-d-Glcp-(1→2)-α-d-Glcp-(1→O(CH2)5NH2, was achieved by a linear synthesis strategy from four monosaccharide building blocks. The synthesized PSI pentasaccharide was conjugated to a subunit of C. difficile exotoxin B yielding a potential dual C. difficile vaccine. More significantly, sera from healthy horses were shown to contain natural anti-PSI IgG antibodies that detected both the synthetic non-phosphorylated PSI repeat and the native PSI polysaccharide, with a slightly higher recognition of the native PSI polysaccharide.

  15. Natural infection of baboons by Entamoeba histolytica elicits anti- gal-lectin heavy subunit IgA and IgG antibodies with shared epitope specificity to that of humans.

    PubMed

    Abd-Alla, Mohamed D; Wolf, Roman F; White, Gary L; Kosanke, Stanley D; Carey, David W; Verweij, Jaco J; El-Dessouky, Yasser M M; Zhang, Mie-Jie; Ravdin, Jonathan I

    2013-12-01

    Non-human primates, such as baboons (Papio hamadryas anubis), are natural hosts for Entamoeba species; infections can be asymptomatic or result in invasive lethal disease. It was sought to determine whether following natural infection by Entamoeba. histolytica, baboon anti-amebic antibodies recognized native Gallectin, a recombinant portion of the lectin heavy subunit (designated LC3) and specific heavy subunit epitopes; we compared the specificity of anti-amebic antibodies from baboons to that of humans following asymptomatic E. histolytica infection or cure of amebic liver abscess (ALA). Female baboons (n=54), aged one to three years of age and living in captivity were screened for infection by real time PCR. E. histolytica infection was found in 37 baboons and was associated with serum anti-LC3 IgG (73%) and anti-LC3 IgA (46%) or intestinal anti-Gal-Lectin IgA antibody responses (49%), p<0.021 for each compared to that observed with baboons having an E. dispar infection (n=10) or uninfected baboons (n=7). The ELISA OD reading for anti-LC3 or anti-lectin antibodies correlated strongly with the presence of a PCR CT value indicative of E. histolytica infection. In humans with asymptomatic E. histolytica infection or those recently cured of ALA, 63% and 57% had serum anti- LC3 IgA and 65% and 57% had serum anti-LC3 IgG antibodies respectively. Epitope- specific synthetic peptides were used as capture antigens in ELISA; for baboons that possessed anti-LC3 and anti-lectin antibodies, 74% had anti-peptide IgG or IgA antibodies, compared to 86% of asymptomatic humans and 92% of ALA subjects(P>0.05).

  16. Spectrum of allergens for Japanese cedar pollinosis and impact of component-resolved diagnosis on allergen-specific immunotherapy.

    PubMed

    Fujimura, Takashi; Kawamoto, Seiji

    2015-10-01

    The high prevalence of Japanese cedar pollinosis in Japan is associated with a negative impact on the quality of life of patients, as well as significant loss of productivity among the workforce in early spring, thus representing a serious social problem. Furthermore, the prevalence is increasing, and has risen by more than 10% in this decade. Cry j 1 and Cry j 2 were identified as the major allergens in Japanese cedar pollen (JCP), and in 2004, the existence of other major and minor allergens were revealed by a combination of two-dimensional electrophoresis and immunoblotting analysis. Allergenome analysis identified a chitinase, a lipid transfer protein, a serine protease, and an aspartic protease as novel IgE-reactive allergens in patients with JCP allergy. Thaumatin-like protein (Cry j 3) was shown to be homologous to Jun a 3, a major allergen from mountain cedar pollen. Isoflavone reductase-like protein was also characterized in a study of a JCP cDNA library. The characterization of component allergens is required to clarify the sensitizer or cross-reactive elicitor allergens for component-resolved diagnosis (CRD). Increasing evidence from numerous clinical trials indicates that CRD can be used to design effective allergen-specific immunotherapy. In this review, we summarize the eight characterized JCP allergens and discuss the impact of CRD and characterization of novel allergens on allergen-specific immunotherapy.

  17. Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies.

    PubMed

    Wang, Shujing; Zhang, Ning; Hu, Tao; Dai, Weiguo; Feng, Xiuying; Zhang, Xinyi; Qian, Feng

    2015-12-07

    Monoclonal antibodies display complicated solution properties in highly concentrated (>100 mg/mL) formulations, such as high viscosity, high aggregation propensity, and low stability, among others, originating from protein-protein interactions within the colloidal protein solution. These properties severely hinder the successful development of high-concentration mAb solution for subcutaneous injection. We hereby investigated the effects of several small-molecule excipients with diverse biophysical-chemical properties on the viscosity, aggregation propensity, and stability on two model IgG1 (JM1 and JM2) mAb formulations. These excipients include nine amino acids or their salt forms (Ala, Pro, Val, Gly, Ser, HisHCl, LysHCl, ArgHCl, and NaGlu), four representative salts (NaCl, NaAc, Na2SO4, and NH4Cl), and two chaotropic reagents (urea and GdnHCl). With only salts or amino acids in their salt-forms, significant decrease in viscosity was observed for JM1 (by up to 30-40%) and JM2 (by up to 50-80%) formulations, suggesting charge-charge interaction between the mAbs dictates the high viscosity of these mAbs formulations. Most of these viscosity-lowering excipients did not induce substantial protein aggregation or changes in the secondary structure of the mAbs, as evidenced by HPLC-SEC, DSC, and FT-IR analysis, even in the absence of common protein stabilizers such as sugars and surfactants. Therefore, amino acids in their salt-forms and several common salts, such as ArgHCl, HisHCl, LysHCl, NaCl, Na2SO4, and NaAc, could potentially serve as viscosity-lowering excipients during high-concentration mAb formulation development.

  18. Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

    PubMed Central

    Arora, Jayant; Hickey, John M; Majumdar, Ranajoy; Esfandiary, Reza; Bishop, Steven M; Samra, Hardeep S; Middaugh, C Russell; Weis, David D; Volkin, David B

    2015-01-01

    There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VH domain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA. PMID:25875351

  19. Placental transfer of IgG antibodies specific to Klebsiella and Pseudomonas LPS and to group B Streptococcus in twin pregnancies.

    PubMed

    Stach, S C L; Brizot, M L; Liao, A W; Palmeira, P; Francisco, R P V; Carneiro-Sampaio, M M S; Zugaib, M

    2015-02-01

    Group B Streptococcus (GBS), Klebsiella spp. and Pseudomonas spp. are important aetiological agents of neonatal infections in Brazil. There is a lack of data in the literature regarding the specific transport of immunoglobulin G (IgG) against these pathogens in multiple pregnancies. Maternal (n = 55) and umbilical cord (n = 110) blood samples were prospectively collected at birth from 55 twin pregnancies. The factors associated with cord levels and transfer ratios of IgG against GBS, Klebsiella and Pseudomonas were examined. The IgG umbilical cord serum levels specific to GBS, Klebsiella LPS and Pseudomonas LPS were significantly associated with maternal-specific IgG concentrations and the presence of diabetes. The anti-Klebsiella IgG cord serum concentrations were also related to birthweight and the presence of hypertension. The transfer ratios against GBS and Pseudomonas LPS were associated with maternal-specific IgG concentrations. The transfer ratios for GBS and Pseudomonas LPS were associated with gestational age at delivery and the presence of diabetes, respectively. None of the examined parameters were related to Klebsiella LPS transfer ratios. We conclude that in twin pregnancies, specific maternal IgG serum concentrations and diabetes were the parameters associated with umbilical cord serum IgG concentrations reactive with the three pathogens investigated. All the other parameters investigated showed different associations with neonatal-specific IgG levels according to the antigen studied. There was no uniformity of the investigated parameters regarding association with placental IgG transfer ratios against the GBS, Pseudomonas LPS and Klebsiella LPS.

  20. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model.

    PubMed

    Li, Qiang; Ujiie, Hideyuki; Shibaki, Akihiko; Wang, Gang; Moriuchi, Reine; Qiao, Hong-jiang; Morioka, Hiroshi; Shinkuma, Satoru; Natsuga, Ken; Long, Heather A; Nishie, Wataru; Shimizu, Hiroshi

    2010-12-15

    Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

  1. Effects of maternal diet during late pregnancy and lactation on the development of IgE and egg- and milk-specific IgE and IgG antibodies in infants.

    PubMed

    Lilja, G; Dannaeus, A; Foucard, T; Graff-Lonnevig, V; Johansson, S G; Oman, H

    1991-03-01

    The IgE levels and food-allergen-specific IgE- and IgG-antibodies (Ab) to ovalbumin (OA), ovomucoid (OVO) and beta-lactoglobulin (BLG) were determined up to 18 months of age in 163 infants born to women who were atopic. A high (HIGH group) or a low (REDUCED group) intake of hen's egg and cow's milk by the mother during the third trimester gave no significant differences in the concentrations of IgE or in IgE-Ab (OVO, BLG) and IgG-Ab (OA, OVO, BLG). Similarly, a prolongation of the abstention diet to the early lactation period did not influence the immune response. The IgG-Ab levels to all three food allergens decreased significantly (P less than 0.001) in both study groups between birth and 2 months of age, but then increased significantly (P less than 0.001) between 6 and 18 months of age. The presence in serum of IgE-Ab to OVO (greater than or equal to 0.15 PRU/ml) was associated with significantly higher IgG-Ab levels to OVO at 6 months (P less than 0.001) and at 18 months (P less than 0.05). Infants with positive skin-prick tests (SPT) to OA and OVO showed higher IgG-Ab levels at 6 and 18 months of age than did infants with negative SPT reactions to the two egg allergens. This indicates a relation between the IgE- and IgG-Ab response and it also suggests that some individuals are 'high responders' to both types of immunoglobulin isotypes while others are 'low responders'.

  2. IgG RF and anti-CCP2 antibody can be positive in undifferentiated arthritis due to streptococcal infection, hepatitis B virus, tuberculosis, trauma and hypothyroidism: a preliminary study.

    PubMed

    Singh, Usha; Verma, Pamod Kumar; Bhagat, Priyanka; Singh, Sangeeta; Singh, Suman; Singh, Nand Kumar

    2012-09-01

    Anti-CCP2 antibody and rheumatoid (RF) tests are used for the diagnosis of rheumatoid arthritis (RA). Out of these two, anti-CCP2 antibody is supposed to be more specific for RA. Aim of the study was to present 33 cases of undifferentiated arthritis (UA) in which features of RA were not present, but anti-CCP2 antibody was positive. Out of the 33 cases of UA, 19 had well-known disease like hyperthyroidism, hypothyroidism, tubercular arthritis, traumatic arthritis, pneumonia with arthritis, varicose vein with pain in legs, cervical spondylitis and SSA. The duration of disease was more than one year in 67.86% cases. Majority of the patients were females (63.64%). Knee joint involvement was seen in maximum number (i.e. 20 cases). All 33 cases were positive for anti-CCP2 Ab. Maximum number of cases (78.78%) had involvement of one or two joints. CRP positivity was seen in 23.07% cases. Morning stiffness was present in (36.36%) cases, while swelling of the joint was present in 33.33% cases. In 16 cases, only serum sample was available for further analysis. About 62.5% cases showed IgG RF positivity. Antitubercular IgM and IgG were detected in 18.75% cases; ASO was elevated in 12.5% cases, and HBs Ag was positive in 6.25% cases. None of the controls (30 cases) were positive for these infections, anti-CCP2 antibody or RF. Thus, our study concludes that chronic infections like streptococcus, hepatitis B, tuberculosis and autoimmune thyroid diseases can produce raised levels of anti-CCP2 antibody and IgG RF.

  3. The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    PubMed Central

    Park, Sung-Hyun; Chen, Wen-Chi; Durmus, Nedim; Bleck, Bertram; Reibman, Joan; Riemekasten, Gabriela; Grunig, Gabriele

    2015-01-01

    Air pollution is known to exacerbate chronic inflammatory conditions of the lungs including pulmonary hypertension, cardiovascular diseases and autoimmune diseases. Directly pathogenic antibodies bind pro-inflammatory cell receptors and cause or exacerbate inflammation. In contrast, anti-inflammatory antibody isotypes (e.g. mouse immunoglobulin G1, IgG1) bind inhibitory cell receptors and can inhibit inflammation. Our previous studies showed that co-exposure to antigen and urban ambient particulate matter (PM2.5) induced severe pulmonary arterial thickening and increased right ventricular systolic pressures in mice via T-cell produced cytokines, Interleukin (IL)-13 and IL-17A. The aim of the current study was to understand how B cell and antibody responses integrate into this T cell cytokine network for the pulmonary hypertension phenotype. Special focus was on antigen-specific IgG1 that is the predominant antibody in the experimental response to antigen and urban ambient PM2.5. Wild type and B cell-deficient mice were primed with antigen and then challenged with antigen and urban particulate matter and injected with antibodies as appropriate. Our data surprisingly showed that B cells were necessary for the development of increased right ventricular pressures and molecular changes in the right heart in response to sensitization and intranasal challenge with antigen and PM2.5. Further, our studies showed that both, the increase in right ventricular systolic pressure and right ventricular molecular changes were restored by reconstituting the B cell KO mice with antigen specific IgG1. In addition, our studies identified a critical, non-redundant role of B cells for the IL-17A-directed inflammation in response to exposure with antigen and PM2.5, which was not corrected with antigen-specific IgG1. In contrast, IL-13-directed inflammatory markers, as well as severe pulmonary arterial remodeling induced by challenge with antigen and PM2.5 were similar in B cell

  4. [Investigation of mold fungi in air samples of elementary schools and evaluation of allergen-specific IgE levels in students' sera].

    PubMed

    Ovet, Habibe; Ergin, Cağrı; Kaleli, Ilknur

    2012-04-01

    Atmospheric fungal spores play important role in allergic reactions in atopic individuals. Monitorization of those spores found in the environment of atopic cases is crucial for the choice of the antigens that will be included in allergen screening procedures and precautions to be taken against mold-originated health problems. Since most of the people spend plenty of time indoors in recent years, the effects of exposure to indoor air fungi on human health have gained importance. This study was aimed to investigate the indoor air mold distribution of elementary schools in Denizli province (located in west Anatolia, Turkey) and to compare the allergen-specific IgE levels of children against the most frequently detected mold genus. A questionnaire (MM080) was distributed to the 4967 students (6-8 year-old) attending first and second degrees of 16 different elementary schools with scattered locations in city center. This questionnaire form included the questions related to the general information about the child, school environment, allergic complaints since last year, home environment and nutrition. Response rate to the questionnaire was 51.6% (2565/4967). Air samples were collected from 18 classrooms in March 2009, during which high rates of allergic symptoms were observed according to the questionnaire results. Mold fungi belonging to 10 different genera (Penicillium spp. 46%; Aspergillus spp. 18%; Cladosporium spp. 17%; Alternaria spp. 15%; Drechslera spp. 1%; Chrysosporium, Fusarium, Conidiobolus and Cladothecium species 0.5%; unidentified 1%) were isolated from indoor air of classrooms. Since the most frequently detected mold was Penicillium spp. (46%), the 48 children with atopic symptoms were called to the hospital for the determination of total IgE and Penicillium specific IgE in their sera. Twenty two students accepted the invitation and serum total IgE (Immulite 2000; Diagnostic Product Corporation, USA) and allergen-specific IgE (Penicillium brevicompactum

  5. The FcγR of humans and non-human primates and their interaction with IgG: implications for induction of inflammation, resistance to infection and the use of therapeutic monoclonal antibodies.

    PubMed

    Hogarth, P Mark; Anania, Jessica C; Wines, Bruce D

    2014-01-01

    Considerable effort has focused on the roles of the individual members of the FcγR receptor (FcγR) family in inflammatory diseases and humoral immunity. Recent work has revealed major roles in infection and in particular HIV pathogenesis and immunity. In addition, FcγR functions underpin the action of many of the successful therapeutic monoclonal antibodies. This emphasises the need for a greater understanding of FcγR function in humans and in the NHP which provides a key model for human immunity and preclinical testing of antibodies. We discuss recent key aspects of the human FcγR receptor biology and structure to define differences and similarities in activity between the human and macaque Fc receptors. These differences and similarities nuance the interpretation of infection and vaccine studies in the macaque. Indeed passive IgG antibody protection in lentivirus infection models in the macaque provided early evidence for the role of Fc receptors in anti-HIV immunity that have subsequently gained support from human vaccine trials. None-the-less the diverse functions and cellular contexts of FcγR receptor expression ensure there is much still to understand of the protective and deleterious effects of FcγRs in HIV infection. Careful comparative studies of human and non-human primate FcγRs will facilitate our appreciation of what attributes of HIV specific IgG antibodies, either acquired naturally or via vaccination, are most important for protection.

  6. Agreement between allergen-specific IgE assays and ensuing immunotherapy recommendations from four commercial laboratories in the USA

    PubMed Central

    Plant, Jon D; Neradelik, Moni B; Polissar, Nayak L; Fadok, Valerie A; Scott, Brian A

    2014-01-01

    Background Canine allergen-specific IgE assays in the USA are not subjected to an independent laboratory reliability monitoring programme. Hypothesis/Objectives The aim of this study was to evaluate the agreement of diagnostic results and treatment recommendations of four serum IgE assays commercially available in the USA. Methods Replicate serum samples from 10 atopic dogs were submitted to each of four laboratories for allergen-specific IgE assays (ACTT®, VARL Liquid Gold, ALLERCEPT® and Greer® Aller-g-complete®). The interlaboratory agreement of standard, regional panels and ensuing treatment recommendations were analysed with the kappa statistic (κ) to account for agreement that might occur merely by chance. Six comparisons of pairs of laboratories and overall agreement among laboratories were analysed for ungrouped allergens (as tested) and also with allergens grouped according to reported cross-reactivity and taxonomy. Results The overall chance-corrected agreement of the positive/negative test results for ungrouped and grouped allergens was slight (κ = 0.14 and 0.13, respectively). Subset analysis of the laboratory pair with the highest level of diagnostic agreement (κ = 0.36) found slight agreement (κ = 0.13) for ungrouped plants and fungi, but substantial agreement (κ = 0.71) for ungrouped mites. The overall agreement of the treatment recommendations was slight (κ = 0.11). Altogether, 85.1% of ungrouped allergen treatment recommendations were unique to one laboratory or another. Conclusions and clinical importance Our study indicated that the choice of IgE assay may have a major influence on the positive/negative results and ensuing treatment recommendations. PMID:24461034

  7. Exposure to allergen and diesel exhaust particles potentiates secondary allergen-specific memory responses promoting asthma susceptibility

    PubMed Central

    Brandt, Eric B.; Biagini Myers, Jocelyn M.; Acciani, Thomas H.; Ryan, Patrick H.; Sivaprasad, Umasundari; Ruff, Brandy; LeMasters, Grace K.; Bernstein, David I.; Lockey, James; LeCras, Timothy D.; Khurana Hershey, Gurjit K.

    2015-01-01

    Background Exposure to traffic pollution particulate matter, predominantly diesel exhaust particles (DEP), increases risk for asthma and asthma exacerbation, however the underlying mechanisms remain poorly understood. Objective To examine the impact of DEP exposure on the generation and persistence of allergen-specific memory T-cells in asthma and translate these findings by determining the impact of early DEP exposure on the prevalence of allergic asthma in children. Methods The impact of DEP on HDM-specific memory responses was determined using an asthma model. Data from children enrolled in the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) birth cohort were analyzed to determine the impact of the DEP exposure on asthma outcomes. Results DEP co-exposure with HDM resulted in persistent Th2/Th17 CD127+ effector/memory cells in the lungs, spleen and lymph nodes of adult and neonatal mice. After 7 weeks of rest, a single exposure to HDM resulted in airway hyperresponsiveness and increased levels of Th2 cytokines in only mice that had been previously exposed to both HDM and DEP versus HDM alone. Based on these data, we examined whether DEP exposure was similarly associated increased asthma prevalence in children in the presence or absence of allergen exposure/sensitization in the CCAAPS birth cohort. Early life exposure to high DEP was associated with significantly increased asthma prevalence among allergic children, but not among non-allergic children. Conclusion These findings suggest that DEP exposure results in accumulation of allergen specific Th2/Th17 cells in the lungs, potentiating secondary allergen recall responses and promoting the development of allergic asthma. PMID:25748065

  8. Development of an indirect ELISA method for the parallel measurement of IgG and IgM antibodies against Crimean-Congo haemorrhagic fever (CCHF) virus using recombinant nucleoprotein as antigen.

    PubMed

    Dowall, S D; Richards, K S; Graham, V A; Chamberlain, J; Hewson, R

    2012-02-01

    Recombinant nucleoprotein from Crimean-Congo Haemorrhagic Fever (CCHF) virus was successfully derived from a baculovirus expression system and purified for use in a novel enzyme-linked immunosorbent assay (ELISA) diagnostic test. Comparable tests were used for detection of IgG and IgM antibodies, thus allowing efficient detection of both antibodies in parallel. The major benefits of the assay also included removing any requirement for polyclonal sera, thus eliminating variation in preparations and allowing standardisation between laboratories. The assay was successfully tested using a panel of positive sera supplied from samples identified as being positive in Turkey, Tajikistan and Kosovo and shown to be sensitive and specific. It is envisaged that this simple diagnostic ELISA for CCHF virus infection which removes the reliance on polyclonal antibody preparations, will be accessible to a wider range of laboratories enabling them to carry out routine diagnosis. This will improve the efficiency of diagnosis and subsequent management of infected patients.

  9. The effect of maternal avoidance of eggs, cow's milk, and fish during lactation on the development of IgE, IgG, and IgA antibodies in infants.

    PubMed

    Hattevig, G; Kjellman, B; Sigurs, N; Grodzinsky, E; Hed, J; Björkstén, B

    1990-01-01

    Serum levels of IgE, IgE antibodies to egg white (EW) and cow's milk (CM), IgG, and IgA antibodies to ovalbumin (OA) and beta-lactoglobulin (BLG) were measured in a group of 115 infants with a family history of atopy/allergy at birth and at 3, 6, 9, 12, and 18 months of age. The mothers of 65 infants avoided eggs, CM, and fish during the first 3 months of lactation (maternal antigen avoidance diet, D group), whereas the remaining 50 mothers had no diet restrictions (no maternal antigen avoidance diet, ND group). CM was introduced after 6 months of age and EW after 9 months. The only statistically significant difference between the D and ND group infants was a lower rate of specimens with IgE antibodies to EW and/or CM in the infants at 3 months of age (p = 0.008). IgE antibodies to EW and/or CM appeared in 62 infants during the study period and often during complete breast-feeding. In 40 of the infants, IgE antibodies appeared before the introduction of EW and CM into the diet. The IgE concentrations of the D and the ND group infants were similar. Cord-blood IgE was a poor predictor of atopy/allergy; for example, only seven of 103 infants with double heredity for atopy/allergy had values above the 90th percentile of our normal reference. The concentrations of IgG antibodies to OA and BLG were similar in the two groups. The levels decreased significantly (p less than 0.001) from birth to 6 months of age, indicating a passive placental transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA], IgG, and IgM) after natural and experimental infections.

    PubMed Central

    Peeters, J E; Villacorta, I; Vanopdenbosch, E; Vandergheynst, D; Naciri, M; Ares-Mazás, E; Yvoré, P

    1992-01-01

    Fecal and serum anti-Cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme-linked immunosorbent assay after experimental and natural infection of calves with C. parvum. Although all experimentally infected calves showed high levels of colostral antibodies in the feces, they acquired C. parvum infection. Three of five animals died. Calves which acquired natural infection showed only diarrhea. Levels of colostral coproantibodies dropped quickly. Experimental infection was followed by a rise in local anti-C. parvum IgM levels from day 5 postinfection (p.i.). IgM peaked at day 14 p.i. and then disappeared quickly. Anti-C. parvum IgA levels rose between days 7 and 14 p.i. and decreased slowly. Rising levels of coproantibodies coincided with falling oocyst output. Fecal anti-C. parvum IgG levels rose slightly during oocyst output, and IgG disappeared 3 weeks p.i. Similar kinetics were established in naturally infected calves. Although fecal anti-C. parvum IgA levels declined slowly, reinfections were established 5, 7, and 14 weeks after the primary contact. Serum anti-C. parvum IgG levels rose during maximal oocyst excretion, whereas serum anti-C. parvum IgA levels peaked later than did local IgA levels. Challenge reinfection of naturally infected calves at day 112 was not followed by clinical signs or oocyst output or by a secondary antibody response. Sequential Western immunoblotting with fecal extracts revealed up to 32 different parasite antigens. Convalescent-phase sera recognized up to 23 antigens. Fecal IgA reacted intensely with antigens with relative molecular weights (M(r)) of approximately 11,000 and 15,000. These antigens were not recognized by convalescent-phase serum IgG. Both local IgA and serum IgG also showed strong reactions with 23,000- and 44,000-M(r) antigens and with several antigens of between 66,200 and 200,000 M(r). Most bands remained detectable for at least 16 weeks p.i. Images PMID:1587597

  11. Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG), IgM, and IgA with broad specificity for enteroviruses.

    PubMed Central

    Swanink, C M; Veenstra, L; Poort, Y A; Kaan, J A; Galama, J M

    1993-01-01

    An antibody-capture enzyme-linked immunosorbent assay (ELISA) with coxsackievirus B1 as the antigen was evaluated for detection of immunoglobulin G (IgG), IgM, and IgA antibodies and showed broad specificity for enteroviruses. In total, 116 serum or cerebrospinal fluid samples from 62 patients were tested by ELISA and the complement fixation test (CFT). Additionally, 15 serum samples that contained poliovirus-specific IgM antibody were tested. Serum samples from 200 healthy blood donors were used for standardization of the assays. The sensitivity of the ELISA varied with time of serum sampling, with a relatively low sensitivity when serum was collected within 3 days after the onset of symptoms (23%; 5 of 22) but good sensitivity when serum was collected later (83%; 20 of 24). The sensitivity was better than that of the CFT. The ELISAs were broadly reactive as concluded from typing of virus isolates that were simultaneously obtained. The assay did, furthermore, detect antibody against poliovirus type 3. Sera that contained rheumatoid factor, antinuclear antibody, or cardiolipin antibody (by the Venereal Disease Research Laboratory test) did not react in this ELISA. Nonspecific reactivity did occur, however, in cases of infectious mononucleosis and in Mycoplasma pneumoniae infection. The enterovirus-specific ELISA is found to be simple to perform, more sensitive than the CFT, and far less laborious than the neutralization test. PMID:8308117

  12. Development and Evaluation of a Multiplex Microsphere Assay for Quantitation of IgG and IgA Antibodies against Neisseria meningitidis Serogroup A, C, W, and Y Polysaccharides

    PubMed Central

    Bårnes, Guro K.; Kristiansen, Paul A.; Caugant, Dominique A.

    2015-01-01

    We developed and evaluated a rapid and simple multiplex microsphere assay for the quantification of specific IgG and IgA antibodies against meningococcal serogroup A, C, W, and Y capsular polysaccharides in serum and saliva. Meningococcal polysaccharides were conjugated to distinct magnetic carboxylated microspheres, and the performance of the assay was assessed using the CDC1992 standard meningococcal reference serum and a panel of serum and saliva samples. The standard curve was linear over an eight 3-fold dilution range in the IgG assay and a seven 3-fold dilution range in the IgA assay. No cross-reactivity was discovered, and the assay showed high specificity with ≥91% homologous inhibition and ≤11% heterologous inhibition for all serogroups and immunoglobulin classes. Lower limits of detections were ≤280 pg/ml for IgG and ≤920 pg/ml for IgA antibodies. The assay was reproducible, with a mean coefficient of variation of ≤5% for intra-assay duplicates, a mean coefficient of variation of ≤20% for interassay repeated analysis with different conjugations of microspheres, and a mean coefficient of variation within 25.8% for interoperator variation. The assay showed good correlation to the standard meningococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies. This multiplex assay is robust and reliable and requires less sample volume, and less time and workload are needed than for ELISA, making this method highly relevant for serological and salivary investigations on the effect of meningococcal vaccines and for immunosurveillance studies. PMID:25924767

  13. Immunoblot analysis of IgE and IgG antibodies to honey bee venom: cross sectional and sequential studies in bee sensitive subjects.

    PubMed

    Roberts-Thomson, P J; Koh, S; Shepherd, K; Kupa, A; Heddle, R J

    1991-12-01

    To investigate the specific IgE and IgG immune response to honey bee venom (bv), we performed immunoblot analysis of sera from 47 bee sensitive subjects and followed the response during and after venom immunotherapy in 15 of these subjects. Fifteen venom proteins varying in molecular size from 20 to 105 kDa were identified as being antigenic and consisted of a high molecular weight (HMW) group (5 to 105 kDa, containing the previously identified allergens B and C) and a low molecular weight group (LMW) containing hyaluronidase and phospholipase A. In general for a given individual the anti-venom IgE and IgG response was qualitatively similar although some variation between individuals was apparent. Reactivity with hyaluronidase and phospholipase A appeared only in those subjects showing reactivity with HMW components. During immunotherapy specific anti-venom IgG and IgE responses tended to be linked. Increased responses being seen against all components in 4 of 12 subjects, reductions in 3 and unchanged responses in the remainder. Following immunotherapy (mean 4.0 years), spontaneous reduction of IgE and IgG was seen in 5 of 5 subjects. Loss of reactivity with the LMW components was prominent in these sera.

  14. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

    PubMed

    Foo, D G W; Ang, R X; Alonso, S; Chow, V T K; Quak, S H; Poh, C L

    2008-03-01

    A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.

  15. Mutations in Toll-Like Receptor 3 Are Associated with Elevated Levels of Rotavirus-Specific IgG Antibodies in IgA-Deficient but Not IgA-Sufficient Individuals

    PubMed Central

    Günaydın, Gökçe; Nordgren, Johan; Svensson, Lennart

    2014-01-01

    Double-stranded RNA (dsRNA) triggers immune-mediated responses through toll-like receptor 3 (TLR3), which is involved in innate antiviral defense. Low expression of TLR3 was recently suggested to contribute to susceptibility to rotavirus infection. Thus, we investigated the role of two TLR3 polymorphisms (rs3775291 and rs5743305), both of which resulted in reduced protein function or expression, in healthy blood donors and IgA-deficient (IgAD) individuals. These polymorphisms were associated with elevated rotavirus-specific IgG titers in IgAD individuals but not in healthy individuals. Thus, we propose that TLR3 signaling does not contribute to the rotavirus-specific antibody response in IgA-sufficient individuals, whereas it is associated with elevated antibody titers in IgAD individuals. PMID:24371259

  16. Effects of MF59 Adjuvant on Induction of Isotype-Switched IgG Antibodies and Protection after Immunization with T-Dependent Influenza Virus Vaccine in the Absence of CD4+ T Cells

    PubMed Central

    Ko, Eun-Ju; Lee, Young-Tae; Kim, Ki-Hye; Jung, Yu-Jin; Lee, Youri; Denning, Timothy L.

    2016-01-01

    ABSTRACT CD4+ T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4+ T cells in the effects of adjuvants, we investigated the efficacy of a T-dependent influenza virus split vaccine with MF59 or alum in CD4 knockout (CD4KO) and wild-type (WT) mice. CD4+ T cells were required for the induction of IgG antibody responses to the split vaccine and the effects of alum adjuvant. In contrast, MF59 was found to be highly effective in raising isotype-switched IgG antibodies to a T-dependent influenza virus split vaccine in CD4KO mice or CD4-depleted WT mice equivalent to those in intact WT mice, thus overcoming the deficiency of CD4+ T cells in helping B cells and inducing immunity against influenza virus. Vaccination with the MF59-adjuvanted influenza virus vaccine was able to induce protective CD8+ T cells and long-lived antibody-secreting cells in CD4KO mice. The effects of MF59 adjuvant in CD4KO mice might be associated with uric acid, inflammatory cytokines, and the recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, the elderly, immunocompromised patients, as well as healthy adults. IMPORTANCE MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4+ T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to a T-dependent influenza vaccine independent of CD4+ T cells. These findings are highly significant for the following reasons: (i) MF59 can overcome a defect of CD4+ T cells in inducing protective immunity to vaccination with a T-dependent influenza

  17. Beta 2-microglobulin-deficient mice are protected from hypergammaglobulinemia and have defective antibody responses because of increased IgG catabolism.

    PubMed

    Christianson, G J; Brooks, W; Vekasi, S; Manolfi, E A; Niles, J; Roopenian, S L; Roths, J B; Rothlein, R; Roopenian, D C

    1997-11-15

    The goal of this study was to determine whether class I proteins play an important role in the regulation of Ig and to elucidate the mechanism(s) involved. We analyzed the phenotype imposed by a null allele of beta 2-microglobulin (beta 2m). Serum Ig levels of several mouse strains showed a beta 2m dependence that was most evident in mice genetically predisposed to develop chronic systemic lupus erythematosus, was preferential to IgG isotypes, and was greatly exaggerated in aging mice that normally develop hypergammaglobulinemia. Beta 2m-deficient mice, regardless of genetic background, also displayed a substantial reduction of specific Ab in response to a prototypic T cell-dependent Ag and a prototypic T cell-independent 2 Ag. This reduction could be accounted for by a selective diminution of Abs of the IgG class. Therefore, class I proteins play a considerable role in the regulation of Ig. The beta 2m dependence could not be explained by class I-dependent immunoregulatory cells (CD8+ cells, NK1.1+ T cells, or conventional NK+ cells) or by the transfer of maternal IgG into the prenatal/neonatal mouse made possible by the beta 2m-dependent Fc receptor (FcRn). However, a beta 2m-dependent increase in the half-lives of IgG, presumably conferred by lifelong FcRn expression, was observed in all mice regardless of genetic background and age. We conclude that FcRn-mediated protection of IgG from catabolism is a generic mechanism that best explains the lifelong beta 2m dependence of Ig in both normal and pathologic situations.

  18. Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo.

    PubMed

    Zhong, W; Wiesmüller, K H; Kramer, M D; Wallich, R; Simon, M M

    1996-11-01

    Plasmid DNA-based vaccination is an efficient way to evoke various forms of protective immunity in laboratory animals. Our previous experiments have shown that mice immunized with either plasmid DNA encoding the outer surface lipoprotein A (pOspA) of Borrelia burgdorferi or the respective lipoprotein (Lip-OspA) produce protective antibodies against subsequent challenge with virulent spirochetes. In the present study, we compared the specificity and function of T cells generated in AKR/N mice previously immunized to either pOspA or Lip-OspA. T cell populations derived by either of the two protocols consistently responded by proliferation in vitro to one (residues 186-203; B4) out of a panel of 27 overlapping 20-mer peptides spanning the entire OspA molecule of strain ZS7. B4 was shown to express allele-specific ligand motifs for I-Ek. Most of the other peptides produced variable and much less pronounced or marginal proliferative T cell responses. T cells reactive to B4 as well as to some minor epitopes were CD4+CD8- T cells which produced IFN-gamma but no detectable IL-4 upon antigen stimulation in vitro. Priming of AKR/N mice with B4 but not with inactive peptides of OspA led to an enhanced production of IgG antibodies, mainly of the IgG1 isotype, including those to a prominent protective epitope (LA-2) upon subsequent challenge with Lip-OspA or intact spirochetes. The data demonstrate that both plasmid DNA and protein immunization with OspA results in T cell responses with specificity for a dominant OspA epitope and suggest that priming of mice with immunodominant peptides accelerates the appearance of protective antibodies in vivo. The identification of T helper cell epitopes relevant for the induction of protective antibodies will also facilitate the design of more potent vaccines against Lyme disease.

  19. Measurement of IgG, IgA and IgE antibodies to Dermatophagoides pteronyssinus by antigen-binding assay, using a partially purified fraction of mite extract (F4P1).

    PubMed Central

    Chapman, M D; Platts-Mills, T A

    1978-01-01

    An extract of Dermatophagoides pteronyssinus culture has been fractionated by chromatography on Sephadex G-100 and Pevikon block electrophoresis to obtain a partially purified allergen (F4P1). This preparation has a molecular weight of between 15--25,000 Dalton, migrates slowly on electrophoresis, and is colourless in solution. The skin-test reactivity of F1P1 was comparable to that of crude D. pteronyssinus extract. F4P1 was radio-labelled with 125I and used in an antigen-binding radioimmunoassay to measure IgG, IgA and IgE antibody (ab) to D. pteronyssinus. IgG, ab was detected in serum from 32/34 (94%) mite-allergic persons, and from 10/31 (30%) nonallergic persons. IgA ab and IgE ab were found in sera from 22/34 (65%) and 37/34 (79%) allergic persons respectively. Neither IgA nor IgE ab could be detected in sera from non-allergic persons. An excellent correlation was found between radioallergo-sorbent technique (RAST), using crude D. pteronyssinus extract and IgE-binding activity (BA) for F4P1, (r=0.94, P less than 0.001). The antigen-binding assay for IgE BA was as sensitive as RAST, but less sensitive than PK testing. There was a very good quantitative correlation between IgG BA and IgE BA (r = 0.84, P less than 0.001). IgG BA was shown to rise in the serum of three patients treated with injections of D. pteronyssinus extract. PMID:750116

  20. FoxP3 Tregs Response to Sublingual Allergen Specific Immunotherapy in Children Depends on the Manifestation of Allergy.

    PubMed

    Stelmaszczyk-Emmel, Anna; Zawadzka-Krajewska, Anna; Głodkowska-Mrówka, Eliza; Demkow, Urszula

    2015-01-01

    Over the last decades allergic diseases has become a major health problem worldwide. The only specific treatment to date is allergen specific immunotherapy (ASIT). Although it was shown that ASIT generates allergen-tolerant T cells, detailed mechanism underlying its activity is still unclear and there is no reliable method to monitor its effectiveness. The aim of our study was to evaluate ASIT influence on the frequency of forkhead box P3 (FoxP3) Tregs in allergic children with various clinical manifestations. The relative number of FoxP3 Tregs in 32 blood samples from allergic children at baseline and/or after 1 year of ASIT was assessed by flow cytometry. In the entire studied group, the percentage of FoxP3 Tregs did not increase 1 year after ASIT. Nevertheless, the percentage of FoxP3 Tregs after ASIT significantly increased in children with respiratory allergy (conjunctivitis, asthma, and rhinitis) coexisting with nonrespiratory manifestations (food allergy and/or atopic dermatitis), whereas, in patients with respiratory allergy only, the percentage of FoxP3 Tregs decreased. To the best of our knowledge, this is the first report showing various differential FoxP3 Tregs response to ASIT in allergic children. FoxP3 Tregs number could be useful in treatment monitoring. Further studies are warranted to confirm these observations.

  1. Induction of T regulatory cells by the superagonistic anti-CD28 antibody D665 leads to decreased pathogenic IgG autoantibodies against desmoglein 3 in a HLA-transgenic mouse model of pemphigus vulgaris.

    PubMed

    Schmidt, Thomas; Willenborg, Sebastian; Hünig, Thomas; Deeg, Cornelia A; Sonderstrup, Grete; Hertl, Michael; Eming, Rüdiger

    2016-04-01

    Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune disease of the skin and mucous membranes. Its pathogenesis is based on IgG autoantibodies that target the desmosomal cadherins, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1) and induce intra-epidermal loss of adhesion. Although the PV pathogenesis is well-understood, therapeutic options are still limited to immunosuppressive drugs, particularly corticosteroids, which are associated with significant side effects. Dsg3-reactive T regulatory cells (Treg) have been previously identified in PV and healthy carriers of PV-associated HLA class II alleles. Ex vivo, Dsg3-specific Treg cells down-regulated the activation of pathogenic Dsg3-specific T-helper (Th) 2 cells. In this study, in a HLA-DRB1*04:02 transgenic mouse model of PV, peripheral Treg cells were modulated by the use of Treg-depleting or expanding monoclonal antibodies, respectively. Our findings show that, in vivo, although not statistically significant, Treg cells exert a clear down-regulatory effect on the Dsg3-driven T-cell response and, accordingly, the formation of Dsg3-specific IgG antibodies. These observations confirm the powerful immune regulatory functions of Treg cells and identify Treg cells as potential therapeutic modulators in PV.

  2. Development of a fluorescent microbead-based immunoassay for the detection of hepatitis E virus IgG antibodies in pigs and comparison to an enzyme-linked immunoassay.

    PubMed

    Owolodun, Olajide A; Giménez-Lirola, Luis G; Gerber, Priscilla F; Sanford, Brenton J; Feagins, Alicia R; Meng, Xiang-Jin; Halbur, Patrick G; Opriessnig, Tanja

    2013-11-01

    Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in-house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n=72), (B) samples from known HEV-negative pigs (negative controls, n=62) and (C) samples from pigs of unknown HEV infection status (n=182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa=0.92).

  3. Two years' performance of an in-house ELISA for diagnosis of Legionnaires' disease: detection of specific IgM and IgG antibodies against Legionella pneumophila serogroup 1, 3 and 6 in human serum.

    PubMed

    Elverdal, P L; Jørgensen, C S; Krogfelt, K A; Uldum, S A

    2013-08-01

    The aim of this study was to evaluate the performance of an in-house ELISA for the diagnosis of Legionnaires' disease (LD) by detection of IgM and IgG antibodies against Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6. The evaluation was done throughout a two-year period in a diagnostic routine laboratory. Furthermore, the sensitivity of four different methods, the in-house L. pneumophila antibody test (ELISA), the urinary antigen test (Binax® EIA), an in-house PCR and culture, both alone and in combination was evaluated. From 2008 to 2010, 12,158 serum samples from 10,503 patients were analysed. During the same period, 361 cases of laboratory-confirmed LD cases were recorded in Denmark, but of these only 113 had a serum sample examined. The positive predictive value of the in-house ELISA was calculated to be 12.8 and the negative predictive value was 99.6, using only the confirmed LD cases as true positives. The sensitivity of the in-house ELISA for the detection of IgM and IgG antibodies in the confirmed LD cases was 61% and 36%, respectively. By combining the two ELISA assays the sensitivity increased to 66%. The sensitivity of the Legionella urinary antigen test (Binax® EIA) was 63%, of the in-house PCR 87% and of culture 69%. When all the different methods were combined, a higher sensitivity was calculated--for in-house ELISA (IgM+IgG) and Binax® EIA 91%, in-house ELISA (IgM+IgG) and in-house PCR 93%, in-house ELISA (IgM+IgG) and culture 93%, Binax® EIA and in-house PCR 79%, Binax® EIA and culture 68% and in-house PCR and culture 94%. This study confirms that the detection of IgG and IgM antibodies by ELISA is an important diagnostic tool, also during the initial phase of the disease. Furthermore, we showed that LD in Denmark with or without serum samples collected exhibits the same age and sex distribution and epidemiology, as in the rest of Europe, i.e., mostly men are infected, infections are mostly community acquired, followed by infection from

  4. A Vaccine of L2 Epitope Repeats Fused with a Modified IgG1 Fc Induced Cross-Neutralizing Antibodies and Protective Immunity against Divergent Human Papillomavirus Types

    PubMed Central

    Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

    2014-01-01

    Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17–36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development. PMID:24802101

  5. Chimeric Anti-CD14 IGG2/4 Hybrid Antibodies for Therapeutic Intervention in Pig and Human Models of Inflammation

    PubMed Central

    Lau, Corinna; Gunnarsen, Kristin S.; Høydahl, Lene S.; Andersen, Jan Terje; Berntzen, Gøril; Pharo, Anne; Lindstad, Julie K.; Ludviksen, Judith K.; Brekke, Ole-Lars; Barratt-Due, Andreas; Nielsen, Erik Waage; Stokes, Christopher R.; Espevik, Terje; Sandlie, Inger

    2013-01-01

    CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases. PMID:24062486

  6. Opsonizing antibodies (IgG1) up-regulate monocyte proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and IL-6 but not anti-inflammatory cytokine IL-10 in mycobacterial antigen-stimulated monocytes-implications for pathogenesis.

    PubMed

    Hussain, R; Shiratsuchi, H; Phillips, M; Ellner, J; Wallis, R S

    2001-02-01

    Cachexia is one of the prominent features of advanced tuberculosis (TB) seen in association with increased expression of the monokine TNF-alpha. Several mycobacterial proteins, including PPD, stimulate TNF-alpha secretion from monocytes. Host factors that may play a role in cytokine expression from monocytes remain largely unknown. One such factor is the opsonizing antibodies. Monocytes have high-affinity receptors (FcgammaI and FcgammaIII) for IgG1 and IgG3 antibodies that mediate antigen uptake. We have reported selective up-regulation of IgG1 (which bind to Fcgamma receptors) in advanced TB and have recently shown the ability of PPD-specific IgG1 antibodies to augment TNF-alpha expression in PPD-stimulated monocytes. These observations have now been extended to other cytokines with semipurified fractions from secreted antigens of Mycobacterium tuberculosis (containing 30 kD and 58 kD) that were devoid of lipids, glycolipids and carbohydrates. In the presence of heat-inactivated TB plasma containing known amounts of antigen-specific IgG1 antibodies, these fractions induced significantly increased TNF-alpha, IL-6 and IL-10 secretion. Absorption of IgG1 with Protein 'A' removed the augmenting activity for TNF-alpha and IL-6 secretion from the TB plasma samples. In the case of IL-10, removal of IgG1 resulted in increased rather than decreased IL-10 secretion. These results suggest a possible pathogenic role for antibodies in TB by enhancing proinflammatory and blocking down-regulatory cytokines such as IL-10 cytokines during the chronic phase of TB.

  7. Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects, patients with coronary heart disease and inflammatory bowel disease.

    PubMed

    Füst, George; Uray, Katalin; Bene, László; Hudecz, Ferenc; Karádi, István; Prohászka, Zoltán

    2012-03-01

    Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.

  8. Pathogenic IgG antibodies against desmoglein 3 in pemphigus vulgaris are regulated by HLA-DRB1*04:02-restricted T cells.

    PubMed

    Eming, Rüdiger; Hennerici, Tina; Bäcklund, Johan; Feliciani, Claudio; Visconti, Kevin C; Willenborg, Sebastian; Wohde, Jessica; Holmdahl, Rikard; Sønderstrup, Grete; Hertl, Michael

    2014-11-01

    Pemphigus vulgaris (PV) is considered as a model for an autoantibody-mediated organ-specific autoimmune disorder. IgG autoantibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), the major autoantigen in PV, cause loss of epidermal keratinocyte adhesion, resulting in blisters and erosions of the skin and mucous membranes. The association of human autoimmune diseases with distinct HLA alleles is a well-known phenomenon, such as the association with HLA-DRB1*04:02 in PV. However, direct evidence that HLA-DRB1*04:02-restricted autoreactive CD4(+) T cells recognizing immunodominant epitopes of Dsg3 initiate the production of Dsg3-reactive IgG autoantibodies is still missing. In this study, we show in a humanized HLA-DRB1*04:02-transgenic mouse model that HLA-DRB1*04:02-restricted T cell recognition of human Dsg3 epitopes leads to the induction of pathogenic IgG Abs that induce loss of epidermal adhesion, a hallmark in the immune pathogenesis of PV. Activation of Dsg3-reactive CD4(+) T cells by distinct human Dsg3 peptides that bind to HLA-DRβ1*04:02 is tightly regulated by the HLA-DRB1*04:02 allele and leads, via CD40-CD40L-dependent T cell-B cell interaction, to the production of IgG Abs that recognize both N- and COOH-terminal epitopes of the human Dsg3 ectodomain. These findings demonstrate key cellular and humoral immune events in the autoimmune cascade of PV in a humanized HLA-transgenic mouse model. We show that CD4(+) T cells recognizing immunodominant Dsg3 epitopes in the context of the PV-associated HLA-DRB1*04:02 induce the secretion of Dsg3-specific IgG in vivo. Finally, these results identify Dsg3-reactive CD4(+) T cells as potential therapeutic targets in the future.

  9. A histoplasma capsulatum-specific IgG1 isotype monoclonal antibody, H1C, to a 70-kilodalton cell surface protein is not protective in murine histoplasmosis.

    PubMed

    Lopes, Livia Cristina Liporagi; Guimarães, Allan J; de Cerqueira, Mariana Duarte; Gómez, Beatriz L; Nosanchuk, Joshua D

    2010-07-01

    Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

  10. Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies

    PubMed Central

    Palafox Sánchez, Claudia Azucena; Satoh, Minoru; Chan, Edward KL; Carcamo, Wendy C; Muñoz Valle, José Francisco; Orozco Barocio, Gerardo; Oregon Romero, Edith; Navarro Hernández, Rosa Elena; Salazar Páramo, Mario; Cabral Castañeda, Antonio; Vázquez del Mercado, Mónica

    2009-01-01

    Introduction Systemic lupus erythematosus is characterized by production of autoantibodies to RNA or DNA–protein complexes such as small nuclear ribonucleoproteins (snRNPs). A role of Epstein–Barr virus in the pathogenesis has been suggested. Similar to Epstein–Barr virus, cytomegalovirus (CMV) infects the majority of individuals at a young age and establishes latency with a potential for reactivation. Homology of CMV glycoprotein B (UL55) with the U1snRNP-70 kDa protein (U1–70 k) has been described; however, the role of CMV infection in production of anti-snRNPs is controversial. We investigated the association of CMV serology and autoantibodies in systemic lupus erythematosus. Methods Sixty-one Mexican patients with systemic lupus erythematosus were tested for CMV and Epstein–Barr virus serology (viral capsid antigen, IgG, IgM) and autoantibodies by immunoprecipitation and ELISA (IgG and IgM class, U1RNP/Sm, U1–70 k, P peptide, rheumatoid factor, dsDNA, β2-glycoprotein I). Results IgG anti-CMV and IgM anti-CMV were positive in 95% (58/61) and 33% (20/61), respectively, and two cases were negative for both. Clinical manifestation and autoantibodies in the IgM anti-CMV(+) group (n = 20) versus the IgM anti-CMV(-)IgG (+) (n = 39) group were compared. Most (19/20) of the IgM anti-CMV(+) cases were IgG anti-CMV(+), consistent with reactivation or reinfection. IgM anti-CMV was unrelated to rheumatoid factor or IgM class autoantibodies and none was positive for IgM anti-Epstein–Barr virus–viral capsid antigen, indicating that this is not simply due to false positive results caused by rheumatoid factor or nonspecific binding by certain IgM. The IgM anti-CMV(+) group has significantly lower levels of IgG anti-U1RNP/Sm and IgG anti-U1–70 k (P = 0.0004 and P = 0.0046, respectively). This finding was also confirmed by immunoprecipitation. Among the IgM anti-CMV(-) subset, anti-Su was associated with anti-U1RNP and anti-Ro (P < 0.05). High levels of IgG

  11. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  12. Detection of anti-U3-RNP/fibrillarin IgG antibodies by line immunoblot assay has comparable clinical significance to immunoprecipitation testing in systemic sclerosis.

    PubMed

    Peterson, Lisa K; Jaskowski, Troy D; Mayes, Maureen D; Tebo, Anne E

    2016-04-01

    The aim of this study was to evaluate the performance and clinical relevance of a commercially available line immunoblot assay (LIA) for detecting anti-U3-RNP/fibrillarin (anti-U3-RNP), against immunoprecipitation (gold standard). This study involved a multi-ethnic cohort of 1000 American systemic sclerosis (SSc) patients and 50 healthy controls. Antinuclear antibodies and centromere antibodies were detected by indirect immunofluorescent antibody test, anti-topo I by immunodiffusion and anti-RNAP III by ELISA. The presence of anti-U3-RNP in select serum samples was detected by immunoprecipitation (IP) and LIA. By IP, U3-RNP antibody was detected in 75 (7.5 %) patients with SSc. Overall agreement between LIA and IP was very good (κ = 0.966). Analytic sensitivity and specificity of the U3-RNP LIA was 100 and 94.7 %, respectively. Clinical features associated with positivity for the anti-U3-RNP antibody include diffuse cutaneous SSc and increased prevalence of renal crisis, consistent with previous studies that used IP. Testing for U3-RNP antibodies is only performed by a small number of laboratories due to the complexity of both performance and interpretation of the IP. LIA is faster and less complex than IP. Excellent agreement between IP and LIA demonstrates that LIA is an acceptable and attractive alternative to IP for anti-U3-RNP detection.

  13. Prevalence of IgG Antibodies against Human Papillomavirus (HPV) type 6, 11, 16, and 18 Virus-Like Particles in Women of Childbearing Age in Port Harcourt, Nigeria.

    PubMed

    Okonko, I O; Ofoedu, V

    2015-01-01

    Most HPV prevalence studies have been carried out in high-resource countries with few studies focused on low-resource regions where highest HPV prevalence in the world occurs. This study reports on prevalence of IgG antibodies against HPVs among women of childbearing age in Port Harcourt, Nigeria. One hundred and eighty-two consented women (age-range 19-45 years) were consecutively recruited. Demographic/behavioral data and 5 mL blood samples were collected from each woman. Plasma of each sample was assayed for HPV-6/11/16/18 virus-like particles using a HPV IgG ELISA kit. The overall anti-HPV prevalence was 4.9% while 7.7% with itching/wound in the private part tested positive. Most (88.9%) of the seropositive women were sexually active. Group-specific seropositivity was low (0.0-10.0%). It also showed that all the 9(100.0%) who tested positive to the HPV responded "yes" to no information on the source of HPV information. Being younger, married, high educational level, religion, and lack of information on HPV were the main correlates of HPV positivity among these women. None was vaccinated and would have been naturally exposed to at least one of HPV-6/11/16/18. With 4.9% seropositivity and lack of information regarding HPV among these women, this study recommends a statewide enlightenment campaign and vaccination.

  14. Allergen-specific IgE levels against crude mould and storage mite extracts and recombinant mould allergens in sera from horses affected with chronic bronchitis.

    PubMed

    Eder, C; Crameri, R; Mayer, C; Eicher, R; Straub, R; Gerber, H; Lazary, S; Marti, E

    2000-03-15

    Immunoglobulin E antibody (IgE) levels against four recombinant (r) mould allergens (r-Aspergillus fumigatus [rAsp f] 7, 8 and 9; r-Alternaria alternata 1 [rAlta1]) and crude mould (Aspergillus fumigatus, Alternaria alternata, Penicillium notatum) and storage mite extracts were determined by ELISA in sera from 24 pulmonary sound control horses and 26 horses suffering from chronic bronchitis/bronchiolitis (CB), also called chronic obstructive pulmonary disease (COPD). Serum IgG and IgA titres were also determined against Aspergillus fumigatus extract and rAsp f 8.IgE against the crude extracts could be measured in all sera, but there was no significant difference between CB-affected and control horses. In contrast, only 8-30% of the horses, depending on the r-allergen tested, had detectable IgE levels in serum against the r-allergens. Horses with CB had significantly more often detectable IgE levels than controls against rAlt a 1 (10/26 and 3/24, respectively, p=0. 054), rAsp f 7 (13/26 and 2/24, respectively, p<0.01) and rAsp f 8 (11/26 and 1/24, respectively, p<0.01). Only four horses (three CB-affected and one healthy, p0.05) had detectable IgE levels against rAsp f 9. Furthermore, CB-affected horses were often sensitised against two or more r-allergens (13/26 of the CB-affected horses) while only one of the 24 healthy horses had positive IgE levels against more than one r-allergens. Similarly to IgE levels, no significant differences between CB-affected and healthy horses were found for IgG titres against the Aspergillus fumigatus extract. However, horses with CB had significantly higher serum IgG titres against rAsp f 8 than healthy controls (median=28 versus 10 relative ELISA units [REU], p<0.01). Additionally, horses with detectable IgE titres against rAsp f 8 had significantly higher IgG titres against this r-allergen than horses with undetectable IgE titres (median IgG titres=46 and 13 REU, respectively; p<0.01). For serum IgA titres, neither differences

  15. Human IgG4: a structural perspective

    PubMed Central

    Davies, Anna M; Sutton, Brian J

    2015-01-01

    IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs. PMID:26497518

  16. Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera.

    PubMed

    Carvalho, Fernando R; Silva, Deise A O; Cunha-Júnior, Jair P; Souza, Maria A; Oliveira, Taísa C; Béla, Samantha R; Faria, Gabriele G; Lopes, Carolina S; Mineo, José R

    2008-08-01

    The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

  17. Novel Hybrid Compound of a Plinabulin Prodrug with an IgG Binding Peptide for Generating a Tumor Selective Noncovalent-Type Antibody-Drug Conjugate.

    PubMed

    Muguruma, Kyohei; Yakushiji, Fumika; Kawamata, Ryosuke; Akiyama, Daichi; Arima, Risako; Shirasaka, Takuya; Kikkawa, Yamato; Taguchi, Akihiro; Takayama, Kentaro; Fukuhara, Takeshi; Watabe, Tetsuro; Ito, Yuji; Hayashi, Yoshio

    2016-07-20

    Although several approaches for making antibody-drug conjugates (ADC) have been developed, it has yet to be reported that an antibody binding peptide such as Z33 from protein A is utilized as the pivotal unit to generate the noncovalent-type ADC (NC-ADC). Herein we aim to establish a novel probe for NC-ADC by synthesizing the Z33-conjugated antitumor agent, plinabulin. Due to the different solubility of two components, including hydrophobic plinabulin and hydrophilic Z33, an innovative method with a solid-supported disulfide coupling reagent is required for the synthesis of the target compounds with prominent efficiency (29% isolated yield). We demonstrate that the synthesized hybrid exhibits a binding affinity against the anti-HER2 antibody (Herceptin) and the anti-CD71 antibody (6E1) (Kd = 46.6 ± 0.5 nM and 4.5 ± 0.56 μM, respectively) in the surface plasmon resonance (SPR) assay. In the cell-based assays, the hybrid provides a significant cytotoxicity in the presence of Herceptin against HER2 overexpressing SKBR-3 cells, but not against HER2 low-expressing MCF-7 cells. Further, it is noteworthy that the hybrid in combination with Herceptin induces cytotoxicity against Herceptin-resistant SKBR-3 (SKBR-3HR) cells. Similar results are obtained with the 6E1 antibody, suggesting that the synthesized hybrid can be widely applicable for NC-ADC using the antibody of interest. In summary, a series of evidence presented here strongly indicate that NC-ADCs have high potential for the next generation of antitumor agents.

  18. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens

    PubMed Central

    Santos, Alexandra F.; James, Louisa K.; Bahnson, Henry T.; Shamji, Mohammed H.; Couto-Francisco, Natália C.; Islam, Sabita; Houghton, Sally; Clark, Andrew T.; Stephens, Alick; Turcanu, Victor; Durham, Stephen R.; Gould, Hannah J.; Lack, Gideon

    2015-01-01

    Background Most children with detectable peanut-specific IgE (P-sIgE) are not allergic to peanut. We addressed 2 non–mutually exclusive hypotheses for the discrepancy between allergy and sensitization: (1) differences in P-sIgE levels between children with peanut allergy (PA) and peanut-sensitized but tolerant (PS) children and (2) the presence of an IgE inhibitor, such as peanut-specific IgG4 (P-sIgG4), in PS patients. Methods Two hundred twenty-eight children (108 patients with PA, 77 PS patients, and 43 nonsensitized nonallergic subjects) were studied. Levels of specific IgE and IgG4 to peanut and its components were determined. IgE-stripped basophils or a mast cell line were used in passive sensitization activation and inhibition assays. Plasma of PS subjects and patients submitted to peanut oral immunotherapy (POIT) were depleted of IgG4 and retested in inhibition assays. Results Basophils and mast cells sensitized with plasma from patients with PA but not PS patients showed dose-dependent activation in response to peanut. Levels of sIgE to peanut and its components could only partially explain differences in clinical reactivity between patients with PA and PS patients. P-sIgG4 levels (P = .023) and P-sIgG4/P-sIgE (P < .001), Ara h 1–sIgG4/Ara h 1–sIgE (P = .050), Ara h 2–sIgG4/Ara h 2–sIgE (P = .004), and Ara h 3–sIgG4/Ara h 3–sIgE (P = .016) ratios were greater in PS children compared with those in children with PA. Peanut-induced activation was inhibited in the presence of plasma from PS children with detectable P-sIgG4 levels and POIT but not from nonsensitized nonallergic children. Depletion of IgG4 from plasma of children with PS (and POIT) sensitized to Ara h 1 to Ara h 3 partially restored peanut-induced mast cell activation (P = .007). Conclusions Differences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children. Blocking IgG4 antibodies provide an additional

  19. Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota).

    PubMed

    Foetisch, Kay; Dahl, Lotte; Jansen, Baerbel; Becker, Wolf-Meinhard; Lidholm, Jonas; van Ree, Ronald; Broll, Hermann; Kaul, Susanne; Vieths, Stefan; Holzhauser, Thomas

    2011-01-01

    Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

  20. Mumps virus infection in vaccinated patients can be detected by an increase in specific IgG antibodies to high titres: a retrospective study.

    PubMed

    Borgmann, S; Schwab, F; Santibanez, S; Mankertz, A

    2014-11-01

    Mumps outbreaks in highly vaccinated populations with genotype G have been reported repeatedly. Detection of these outbreaks can be difficult in a setting with relatively high vaccination coverage when acute cases of mumps are routinely diagnosed by IgM serology since this marker is not reliable for diagnosis of mumps re-infection. To learn whether diagnostic tests performed in a large private laboratory may be useful to detect mumps outbreaks retrospectively, we reviewed the results of almost 7000 mumps tests. Two groups were compared: group 1 comprised of 3438 samples from patients submitted by physicians and clinicians (it was assumed that these patients visited their doctor due to acute disease). Group 2 comprised of 3398 samples submitted from company medical officers and occupational physicians. Since these patients usually attend for routine check-ups and certification of immunity to vaccine-preventable diseases, these samples comprised a control group. From July 2010 to May 2011, a mumps virus outbreak with more than 300 cases occurred in Bavaria, Southeast Germany. Our study includes samples received for serological mumps tests from January 2009 until December 2011 (36 months). The two groups were analysed with regard to the number of IgM-positive cases per month and the level of IgG titre. We found a marked increase for both parameters in group 1 during the time of the outbreak, while the samples submitted by the occupational medical physicians did not display significant alterations. These parameters reflect the outbreak with high accuracy, indicating that a retrospective analysis of IgG titres may be a useful tool for detection of mumps outbreaks when, as was the case in Germany, (i) a nationwide notification system has not been implemented and (ii) a highly vaccinated population is affected.

  1. Reformatting Rituximab into Human IgG2 and IgG4 Isotypes Dramatically Improves Apoptosis Induction In Vitro

    PubMed Central

    Könitzer, Jennifer D.; Sieron, Annette; Wacker, Angelika; Enenkel, Barbara

    2015-01-01

    The direct induction of cell death, or apoptosis, in target cells is one of the effector mechanisms for the anti CD20 antibody Rituximab. Here we provide evidence that Rituximab’s apoptotic ability is linked to the antibody IgG isotype. Reformatting Rituximab from the standard human IgG1 heavy chain into IgG2 or IgG4 boosted in vitro apoptosis induction in the Burkitt’s lymphoma B cell line Ramos five and four-fold respectively. The determinants for this behavior are located in the hinge region and CH1 domain of the heavy chain. By transplanting individual IgG2 or IgG4 specific amino acid residues onto otherwise IgG1 like backbones, thereby creating hybrid antibodies, the same enhancement of apoptosis induction could be achieved. The cysteines at position 131 of the CH1 domain and 219 in the hinge region, involved in IgG2 and IgG4 disulfide formation, were found to be of particular structural importance. Our data indicates that the hybrid antibodies possess a different CD20 binding mode than standard Rituximab, which appears to be key in enhancing apoptotic ability. The presented work opens up an interesting engineering route for enhancing the direct cytotoxic ability of therapeutic antibodies. PMID:26713448

  2. Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking

    PubMed Central

    Bergström, Joakim J. E.; Xu, Hui; Heyman, Birgitta

    2017-01-01

    Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG–antigen complexes and/or that IgG “hides” the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking. PMID:28321225

  3. Analysis of negative result in serum anti-H. pylori IgG antibody test in cases with gastric mucosal atrophy

    PubMed Central

    Adachi, Kyoichi; Mishiro, Tomoko; Tanaka, Shino; Kinoshita, Yoshikazu

    2016-01-01

    The purpose is to elucidate factors related to negative results of anti-H. pylori antibody test in cases with gastric mucosal atrophy. A total of 859 individuals without past history of eradication therapy for H. pylori (545 males, 314 females; mean age 52.4 years) who underwent an upper GI endoscopy examination and serological test were enrolled as subjects. Serological testing was performed using SphereLight H. pylori antibody J®, and endoscopic findings of gastric mucosal atrophy by the classification of Kimura and Takemoto and post-eradication findings were analyzed. The positive rates for the anti-H. pylori antibody test in subjects with and without gastric mucosal atrophy were 85.6% and 0.9%, respectively. In analysis of subjects with gastric mucosal atrophy, a low positive rate and serum titer was observed in subjects with C1, C2 and O3 atrophy. When the analysis was performed separately in male and female subjects, low positive rate was observed in males with O3 atrophy and females with C2 atrophy. Suspected post-eradication endoscopic findings were more frequently observed in cases with C2 atrophy. In conclusion, negative result of anti-H. pylori antibody test was frequently observed in middle-aged subjects with C1, C2 and O3 gastric mucosal atrophy. PMID:27698543

  4. Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to an IgG4κ Inhibitory Antibody

    PubMed Central

    Lin, Jasper C.; Ettinger, Ruth A.; Schuman, Jason T.; Zhang, Ai-Hong; Wamiq-Adhami, Muhammad; Nguyen, Phuong-Cac T.; Nakaya-Fletcher, Shelley M.; Puranik, Komal; Thompson, Arthur R.; Pratt, Kathleen P.

    2015-01-01

    The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies. PMID:25615825

  5. Evaluation of a novel hexavalent humanized anti-IGF-1R antibody and its bivalent parental IgG in diverse cancer cell lines.

    PubMed

    Chang, Chien-Hsing; Wang, Yang; Trisal, Preeti; Li, Rongxiu; Rossi, Diane L; Nair, Anju; Gupta, Pankaj; Losman, Michele; Cardillo, Thomas M; Rossi, Edmund A; Goldenberg, David M

    2012-01-01

    A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.

  6. Anti-LRP/LR specific antibody IgG1-iS18 and knock-down of LRP/LR by shRNAs rescue cells from Aβ42 induced cytotoxicity

    PubMed Central

    Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle; Moodley, Kiashanee; Reusch, Uwe; Knackmuss, Stefan; Penny, Clement; Weinberg, Marc S.; Little, Melvyn; Weiss, Stefan F. T.

    2013-01-01

    Alzheimer's disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Amyloid beta (Aβ) is proposed to elicit neuronal loss through cell surface receptors. As Aβ shares common binding partners with the 37 kDa/67 kDa laminin receptor (LRP/LR), we investigated whether these proteins interact and the pathological significance of this association. An LRP/LR-Αβ42 interaction was assessed by immunofluorescence microscopy and pull down assays. The cell biological effects were investigated by 3-(4,5-Dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide and Bromodeoxyuridine assays. LRP/LR and Αβ42 co-localised on the cell surface and formed immobilized complexes suggesting an interaction. Antibody blockade by IgG1-iS18 and shRNA mediated down regulation of LRP/LR significantly enhanced cell viability and proliferation in cells co-treated with Αβ42 when compared to cells incubated with Αβ42 only. Results suggest that LRP/LR is implicated in Αβ42 mediated cytotoxicity and that anti-LRP/LR specific antibodies and shRNAs may serve as potential therapeutic tools for AD. PMID:24048171

  7. IgG (Gm) allotypes and multiple sclerosis in a French population: phenotype distribution and quantitative abnormalities in CSF with respect to sex, disease severity, and presence of intrathecal antibodies.

    PubMed

    Sesboüé, R; Daveau, M; Degos, J D; Martin-Mondiere, C; Goust, J M; Schuller, E; Rivat-Peran, L; Coquerel, A; Dujardin, M; Salier, J P

    1985-11-01

    The association of a given Gm allotype or phenotype with MS susceptibility, as previously described in some Caucasian populations, was not observed in a large French MS group, whether or not considering the possible influence of sex or disease severity. This result could be related to variations in geographical distribution of Gm alleles and MS susceptibility gene(s) or suggests the simultaneous involvement of Gm and other genetic system(s). In contrast, the corresponding CSFs exhibited already known MS-associated abnormalities of IgG1 (G1m) allotype contents, which therefore did not merely result from a Gm-associated MS susceptibility. These quantitative abnormalities were not sex dependent, but may fluctuate with MS severity. The G1m allotype levels in each CSF were not correlated with titers of various intrathecal antibodies but with the number of antibody specificities detected, a picture arguing for a polyclonal, non-antigen-specific activation of G1m allotype-producing B cells present in MS brain.

  8. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs.

    PubMed

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H; Gutiérrez, Andrés H; Rueda, Luis D; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H; Sheen, Patricia

    2012-01-15

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.

  9. Serum allergen-specific immunoglobulin E in atopic and healthy cats: comparison of a rapid screening immunoassay and complete-panel analysis.

    PubMed

    Diesel, Alison; DeBoer, Douglas J

    2011-02-01

    Feline and canine atopic dermatitis are thought to have a similar immunopathogenesis. As with dogs, detection of allergen-specific IgE in cat serum merely supports a diagnosis of feline atopy based on compatible history, clinical signs and elimination of other pruritic dermatoses. In this study, a rapid screening immunoassay (Allercept(®) E-Screen 2nd Generation; Heska AG, Fribourg, Switzerland; ES2G) was compared with a complete-panel serum allergen-specific IgE assay (Allercept(®); Heska AG; CP) in healthy cats with no history of skin disease and in atopic cats. The latter had no diagnosis of external parasitism, infection, food hypersensitivity or other skin disease explaining their pruritus, and expressed cutaneous reaction patterns typically associated with feline allergic skin disease (head, neck or pinnal pruritus, miliary dermatitis, self-induced alopecia, eosinophilic granuloma complex). The proportion of cats positive on either the ES2G or the CP assays was not significantly different between the atopic and healthy cat groups. There was, however, strong agreement between the results of the ES2G and CP assay; overall, the two tests were in agreement for 43 of 49 (88%) serum samples. There was also strong agreement when individual allergen groups were evaluated (agreement noted: indoor, 41 of 49 samples; grasses/weeds, 37 of 49 samples; and trees, 41 of 49 samples). These results indicate that although neither test is diagnostic for feline atopic dermatitis, the screening assay is beneficial for predicting the results of a complete-panel serum allergen-specific IgE assay in cats.

  10. Tolerogenic Dendritic Cells Derived from Donors with Natural Rubber Latex Allergy Modulate Allergen-Specific T-Cell Responses and IgE Production

    PubMed Central

    Escobar, Alejandro; Aguirre, Adam; Guzmán, María Antonieta; González, Rodrigo; Catalán, Diego; Acuña-Castillo, Claudio; Larrondo, Milton; López, Mercedes; Pesce, Barbara; Rolland, Jennifer; O’Hehir, Robyn; Aguillón, Juan Carlos

    2014-01-01

    Natural rubber latex (NRL; Hevea brasiliensis) allergy is an IgE-mediated reaction to latex proteins. When latex glove exposure is the main sensitizing agent, Hev b 5 is one of the major allergens. Dendritic cells (DC), the main antigen presenting cells, modulated with pharmacological agents can restore tolerance in several experimental models, including allergy. In the current study, we aimed to generate DC with tolerogenic properties from NRL-allergic patients and evaluate their ability to modulate allergen-specific T and B cell responses. Here we show that dexamethasone-treated DC (dxDC) differentiated into a subset of DC, characterized by low expression of MHC class II, CD40, CD80, CD86 and CD83 molecules. Compared with LPS-matured DC, dxDC secreted lower IL-12 and higher IL-10 after CD40L activation, and induced lower alloantigenic T cell proliferation. We also show that dxDC pulsed with the dominant Hev b 5 T-cell epitope peptide, Hev b 546–65, inhibited both proliferation of Hev b 5-specific T-cell lines and the production of Hev b 5-specific IgE. Additionally, dxDC induced a subpopulation of IL-10-producing regulatory T cells that suppressed proliferation of Hev b 5-primed T cells. In conclusion, dxDC generated from NRL-allergic patients can modulate allergen-specific T-cell responses and IgE production, supporting their potential use in allergen-specific immunotherapy. PMID:24465795

  11. Comparison of intradermal skin testing (IDST) and serum allergen-specific IgE determination in an experimental model of feline asthma.

    PubMed

    Lee-Fowler, Tekla M; Cohn, Leah A; DeClue, Amy E; Spinka, Christine M; Ellebracht, Ryan D; Reinero, Carol R

    2009-11-15

    Intradermal skin testing (IDST) and allergen-specific IgE determination are used to determine allergen sensitization. In cats, studies have found poor correlation between the two tests. However, these studies were mainly conducted in pet cats sensitized to unknown allergens with unknown dose and duration of exposure. We hypothesized that in an experimental model of allergic sensitization where these variables are controlled, IDST would demonstrate greater sensitivity and specificity than would serum allergen-specific IgE determination. A model of feline asthma employing Bermuda grass allergen (BGA) or house dust mite allergen (HDMA) was used to test the hypothesis. Thirteen cats were assigned to undergo sensitization to BGA, HDMA or saline (placebo). Bronchoalveolar lavage fluid confirmed development of an asthmatic phenotype. Serum collection and IDST were performed on D0, D28 and D50. A portion of serum was pooled, and an aliquot heat inactivated (HI) to destroy IgE. Individual, pooled, and pooled HI samples were used for allergen-specific IgE determination using an Fc epsilon R1 alpha-based ELISA; pooled samples were also analyzed using an enzymoimmunometric assay. Sensitivity (SE), specificity (SP), and positive and negative predictive values (PPV and NPV) were calculated for IDST and for BGA- and HDMA-specific IgE. Combined results for IDST found SE=90.9%, SP=86.7%, PPV=83.3%, and NPV=92.9%. For ELISA-based serum IgE testing, the SE=22.7%, SP=100%, PPV=100% and NPV=63.8%. The enzymoimmunometric assay did not detect sensitizing IgE, but did detect IgE reactivity to a variety of irrelevant allergens (even in HI samples). Sensitivity of IDST was greater than sensitivity of serum IgE measurement supporting use as a screening test for aeroallergens. Both IDST and allergen-specific IgE determination via ELISA were specific; either test can be used to guide selection of allergens for immunotherapy. The enzymoimmunometric assay was unreliable and cannot be recommended.

  12. A study of the human immune response to Lolium perenne (rye) pollen and its components, Lol p I and Lol p II (rye I and rye II). I. Prevalence of reactivity to the allergens and correlations among skin test, IgE antibody, and IgG antibody data.

    PubMed

    Freidhoff, L R; Ehrlich-Kautzky, E; Grant, J H; Meyers, D A; Marsh, D G

    1986-12-01

    In a stratified random sample of 320 white adults, the prevalence of puncture skin test positivity (ST +) to Lolium perenne (rye grass)-pollen extract (LPE) was 16%. Fifteen percent of all subjects (or 84% of subjects classified LPE IgE antibody positive [Ab +]) was classified IgE Ab + to highly purified Lol p I (Rye I), and 4% of all subjects (or 26% of subjects classified LPE IgE Ab +) was classified IgE Ab + to highly purified Lol p II (Rye II). These data and similar results obtained in an allergy-enriched group of 361 subjects are consistent with previous studies that Lol I is a major allergen and Lol II is a minor allergen of LPE. Whether we studied LPE, Lol I, or Lol II, responder subjects were younger than nonresponder subjects and more male than female subjects were responders. We then investigated the quantitative interrelationships among ST, IgE, and IgG Ab responsiveness to LPE, Lol I, and Lol II in the allergy-enriched group. For each allergen, log-log correlations were strong and significant for ST versus IgE Ab and for IgE Ab versus IgG Ab. All subjects IgE Ab + to Lol I or Lol II were IgG Ab + to that allergen, supporting other evidence for a commonality in the genetic control influencing the production of IgE and IgG Abs to a given allergen. Log-log correlations among ST end points, IgE Ab levels, or IgG Ab levels were strong for LPE versus either Lol I or Lol II but weak between Lol I and Lol II, consistent with the reported lack of cross-reactivity between Lol I and Lol II. Despite these findings, almost all Lol II + subjects were Lol I + by ST (98%), IgE Ab (91%), and IgG Ab (83%), suggesting that the Ia-restricted immune recognition of both these molecules is at least in part under a common genetic control.

  13. [Antinuclear antibodies, patterns and characteristics obtained by immunofluorescence. The importance of the IgA, IgM and IgG isotypes].

    PubMed

    Arcavi, Miriam; Dadone, Jorge

    2009-01-01

    The indirect immunofluorescence with epitelial cell line from human laryngeal carcinoma as substrate (IIF-HEp2) and anti-IgG or anti-total Ig as antisera, is the technique currently used for the detection of antinuclear antibodies. The most important antibodies for the diagnosis and follow-up of connective tissue diseases (CTD) are the IgG-ANA, while the IgM-ANA have no clinical relevance. However the IgA-ANA have not been thoroughly investigated so far. The aim of this work was to study the prevalence of different ANA isotypes of Ig antibodies in CTD patients and to evaluate the convenience of the use of monovalent or polyvalent conjugate. We examined the sera of 100 patients with different CTD by IIF-HEp2 and detected a prevalence of 38% IgA-ANA (titles > or = 1:80) and 12% IgM-ANA (titles < or = 1:160). In twenty nine cases we detected IgA-ANA in absence of IgM-ANA, and in 3 cases IgM-ANA in absence of IgA-ANA. In all the cases IgG-ANA were present. In 6 sera a change in the immunofluorescence pattern was observed while using anti-IgA conjugate, whereas in 3 the change was observed with the use of anti-IgM conjugate. Because of the high prevalence of ANA-IgA detected by IIF-HEp2, we emphasize the convenience of employing anti-total Ig in spite of anti-IgG conjugated until the role of ANA-IgA is dilucidated in CTD patients, in order to establish its relevance in the diagnosis, prognosis and follow-up of systemic rheumatic diseases.

  14. Mannosamine-biotin as a novel masking agent for coating IgG for immune response silencing and augmentation of antibody-antigen interaction.

    PubMed

    Vaya, Jacob; Aizenshtein, Elina; Khatib, Soliman; Gefen, Tal; Fassler, Michael; Musa, Ramadan; Krispel, Simi; Pitcovski, Jacob

    2009-11-16

    A variety of protein-coating procedures are used to modify proteins' properties. The principle coating agent used is PEGylation, in which proteins are coated by conjunction to polyethylene glycol (PEG). In the present study, we describe a novel approach that makes use of small molecules with multifunctional groups as the protein-coating agent. The new coating molecule was produced by reacting two endogenous molecules, mannosamine and biotin, to form mannose-biotin adducts (MBA). hIgG was coated with MBA at various MBA/protein ratios. The immunogenicity of MBA-coated hIgG was tested in chickens. A dose-responsive effect of MBA/hIgG ratio on immune response suppression was detected, with an optimal masking effect at a 12:1 ratio. The immune response to MBA-coated hIgG was about eightfold lower than that to PEG-coated hIgG. MBA also increased antibody-antigen-binding affinity, and decreased recognition of the Fc domain of MBA-coated hIgG by Fc receptor and secondary antibodies. While the PEG molecule consists of inert repeating units of ethylene oxide with no additional functional group to allow for potentially desirable modifications, the MBA has several functional groups, including vicinal hydroxyls, which can easily be converted to active residues such as aldehydes or carboxyls. This may be of importance for developing passive immunizations or for achieving tolerance of the immune response to an immunogenic molecule or virus. In summary, we developed a new protein-coating molecule with the ability to mask foreign antigens and in the case of antibodies, to enhance activity.

  15. Assessment of HPV 16 and HPV 18 antibody responses by pseudovirus neutralization, Merck cLIA and Merck total IgG LIA immunoassays in a reduced dosage quadrivalent HPV vaccine trial.

    PubMed

    Krajden, Mel; Cook, Darrel; Yu, Amanda; Chow, Ron; Su, Qiang; Mei, Wendy; McNeil, Shelly; Money, Deborah; Dionne, Marc; Palefsky, Joel; Karunakaran, Karuna; Kollmann, Tobias; Ogilvie, Gina; Petric, Martin; Dobson, Simon

    2014-01-23

    We assessed HPV 16 and 18 antibody responses of female subjects enrolled in a 2- vs. 3-dose quadrivalent HPV (Q-HPV) vaccine trial (ClinicalTrials.gov NCT00501137) using the Merck competitive Luminex (cLIA) and total IgG Luminex (TIgG) immunoassays, and a pseudovirus neutralizing antibody (PsV NAb) assay. Subjects were enrolled in one of three groups: (1) 9-13yr, 2 doses of Q-HPV at 0, 6 months (n=259); (2) 9-13yr, 3 doses at 0, 2, 6 months (n=260); and (3) 16-26yr, 3 doses at 0, 2, 6 months (n=305). Sera were collected from all subjects at baseline, months 7 and 24, and from half the subjects at months 18 and 36. High correlation was observed between all three assays. At month 36, HPV 16 antibodies remained detectable in all subjects by all assays, whereas 86.4%, 99.6% and 100% of subjects respectively were HPV 18 cLIA, TIgG and PsV NAb (partial neutralization endpoint) seropositive. The proportion seropositive for HPV 18 by cLIA at 36 months was not significantly different for 2-dose girls vs. 3-dose adults (85.9% vs. 79.4%; p=0.51), whereas the proportion for 3-dose girls was significantly higher than for 3-dose adults (95.3% vs. 79.4%; p<0.01). The HPV 18 seropositive proportions by the TIgG and PsV NAb (partial neutralization endpoint) assays were the same for all subjects. High baseline HPV 16 and HPV 18 seropositivity was observed for the TIgG assay and it is unclear if all the detected TIgG antibodies are type-specific and/or neutralizing. For the PsV NAb assay, 90% and partial neutralization geometric mean titres were consistently 2-8-fold higher than for 100% neutralization, which enabled detection of HPV 18 NAb in subjects who lost detectable cLIA antibodies over time. We conclude that the PsV NAb assay is more sensitive than the cLIA, and likely more specific than the TIgG assay.

  16. A Naturally Occurring Hypoallergenic Variant of Vespid Antigen 5 from Polybia scutellaris Venom as a Candidate for Allergen-Specific Immunotherapy

    PubMed Central

    Vinzón, Sabrina E.; Marino-Buslje, Cristina; Rivera, Elena; Biscoglio de Jiménez Bonino, Mirtha

    2012-01-01

    Stings by insects from the Hymenoptera order are known to cause life-threatening allergic reactions and impair life quality. Despite the effectiveness of conventional vespid venom immunotherapy, more standardized and safer allergy vaccines are required and recombinant hypoallergenic variants are important clinical tools. Antigen 5 is a major allergen of vespid venoms and it was previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) could be a hypoallergenic variant. In this work we assess the immunological behavior and allergenic activity of Poly s 5 in order to explore its suitability for specific immunotherapy. With this aim, recombinant Poly s 5 was expressed in Pichia pastoris and the presence of cross-reactive epitopes with Pol a 5, a known allergenic Antigen 5, was investigated both at the IgG and IgE levels, by ELISA assays and a basophil-mediator release assay respectively. A molecular model was also built to better understand the relationship between immunological and structural aspects. In mice, Poly s 5 induced IgG antibodies which cross-reacted with Pol a 5. However, Poly s 5 induced only minimal amounts of IgE and was a poor inducer of basophil-mediator release, even when the cells were sensitized with Pol a 5-specific IgE. Moreover, Poly s 5-specific serum showed a specific protective activity and was able to inhibit the Pol a 5-induced basophil degranulation. Structural analysis from the molecular model revealed that a few amino acid substitutions in the N-terminal region of Poly s 5 should lead to an alteration of the surface topography and electrostatic potential of the epitopes which could be responsible for its hypoallergenic behavior. These findings, taken as a whole, show that Poly s 5 is likely a naturally occurring hypoallergenic Antigen 5 variant. PMID:22844463

  17. Biochemical characterization and three-dimensional structures of the Fab and Fc portions of a human IgG1 antibody

    NASA Astrophysics Data System (ADS)

    Bourne, Christina R.

    2003-07-01

    A monoclonal antibody was isolated from the serum of a patient (Nav) with multiple myeloma for investigation of mechanisms of antibody-mediated damage. It was believed that the over-expressed molecule possessed intrinsic properties contributing unfavorably to disease progression. The monoclonal component was present in large quantities prior to treatment, thus creating a window for uncontrolled and potentially damaging events. Amino acid sequences were determined for the Fab VH and V L domains. This information suggested probable positive selective pressure from antigen. Crystals were obtained in the P21 space group, and X-ray data were collected to 2.5A (99.4% complete). This structure was refined to an Rwork of 22.5% and an Rfree of 28.0%. Unusual structural properties were located in the antigen-binding site. The LCDR3 loop contained a double proline sequence that directed its extrusion into the bulk solvent and partitioned the binding site, which has a groove-type structure. Ligands conforming to this site were identified with a library of phage-displayed peptides. Crystal packing interactions of the Nav Fab were partially mediated by the antigen-binding site, with prevalent involvement of the LCDR3 loop. The crystallographic asymmetric unit was an atypical trimer. Two molecules interacted by edge beta-strand pairing; the third was accommodated through steric complementarity. Edge beta-strand pairing has become a dominant crystal packing motif in antibodies. The frequency of occurrence of this motif revealed sequence preferences for kappa-type light chains. These interactions may play a role in lethal aggregation and amyloid fibrillogenesis. Similar aggregation behavior of the Nav kappa-type Bence-Jones protein probably resulted in impaired renal function. Crystals of the Fc portion suitable for X-ray analysis could be obtained only in gelled media. The space group was P212 121. X-ray data collected at room temperature to 2.5A were 76.0% complete; the final

  18. A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

    PubMed Central

    Ichiyoshi, Yuji; Zhou, Min; Casali, Paolo

    2015-01-01

    We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and Vκ segments and used them to generate the full (unmutated) “germ-line revertant” of the “wild-type” (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full “germ-line revertant” bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Avrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 × 10−8 vs 4.91 × 10−9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and Vκ segments, that the increased Avrel for insulin of mAb13 when compared with its full “germ-line revertant” counterpart was entirely dependent on the mutations in the VH not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 × 10−8 to 5.14 × 10−9 g/μl) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 × 10−3 to 1.54 × 10−3 s−1)but not an increase in association rate constant (Kon, from 2.60 × 104 to 2.61 × 104 M−1 s−1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor

  19. Immunological effects of sublingual immunotherapy: clinical efficacy is associated with modulation of programmed cell death ligand 1, IL-10, and IgG4.

    PubMed

    Piconi, Stefania; Trabattoni, Daria; Rainone, Veronica; Borgonovo, Linda; Passerini, Simone; Rizzardini, Giuliano; Frati, Franco; Iemoli, Enrico; Clerici, Mario

    2010-12-15

    Sublingual immunotherapy (SLIT) is an alternate route of administration of allergen-specific immunotherapy with an improved safety profile; to clarify the immune mechanisms elicited by this therapy, we analyzed the clinical and immunologic effects of SLIT in patients with a clinical history of ragweed sensitization. To analyze possible difference among immunotherapeutic protocols, we also compared patients receiving preseasonal, seasonal, or prolonged sublingual therapy (≥ 3 y); patients receiving symptomatic therapy alone were enrolled as well in the study. Clinical and immunological parameters were measured twice in and out of the pollination period. Clinical benefits, as measured by the visual analog scale for symptoms and for use of drugs, were evident in all three groups of individuals receiving immunotherapy, but were significantly better in patients undergoing prolonged SLIT. Immunologically, SLIT resulted in increased IL-10 production, programmed cell death ligand 1 expression, and concentration of allergen-specific IgG4, as well as in the reduction of CD80 and CD86 expression and IL-4 production. SLIT, thus, is associated with modulation of programmed cell death ligand 1 expression and IL-10 synthesis and favors the production of allergen-specific IgG4. These effects are evident from the first pollen season, independently from therapeutic regimen (preseasonal or seasonal) even if a prolonged treatment is necessary to obtain full clinical efficacy. A more detailed understanding of the interaction of allergen and APCs within the oral mucosa will allow improved targeting of allergy vaccine.

  20. Chronic cat-allergen exposure induces a TH2 cell-dependent IgG4 response related to low-sensitization

    PubMed Central

    Renand, Amedee; Archila, Luis D.; McGinty, John; Wambre, Erik; Robinson, David; Hales, Belinda J.; Thomas, Wayne R.; Kwok, William W.

    2015-01-01

    Background In human subjects, allergen-tolerance has been observed after high dose allergen exposure or after completed allergen immunotherapy, which is related to the accumulation of anti-inflammatory IgG4. However, the specific T cell response that leads to the induction of IgG4 during chronic allergen exposure remains poorly understood. Objective To evaluate the relationship between cat allergen-specific T cell frequency, cat allergen-specific IgE and IgG4 titers and clinical status in cat allergic adults with and without cat ownership and the cellular mechanism by which IgG4 is produced. Methods Fel d 1, Fel d 4, Fel d 7 and Fel d 8- specific T cell responses were characterized by CD154 expression after antigen stimulation. Results In allergic subjects without cat ownership, the frequency of cat allergen (Fel d 1 and Fel d 4) specific TH2 cells (sTH2 cells) correlates with IgE level and is linked to asthma. Paradoxically, we observed that cat allergic subjects with chronic cat exposure maintain high frequency of sTH2 cells, which correlates with IgG4 level and low-sensitization. B cells from allergic, but not from non-allergic subjects, are able to produce IgG4 after cognate interactions with sTH2 clones, and Fel d 1 peptide or the Fel d 1 recombinant protein. Conclusion These experiments suggest that 1) allergen-experienced B cells with capacity to produce IgG4 are present in allergic subjects; and 2) cat-allergen exposure induces an IgG4 response in a TH2 cell-dependent manner. Thus, IgG4 accumulation could be mediated by chronic activation of the TH2 response, which in turn drives desensitization. PMID:26371841

  1. Pathogenicity and Epitope Characteristics Do Not Differ in IgG Subclass-Switched Anti-Desmoglein 3 IgG1 and IgG4 Autoantibodies in Pemphigus Vulgaris.

    PubMed

    Lo, Agnes S; Mao, Xuming; Mukherjee, Eric M; Ellebrecht, Christoph T; Yu, Xiaocong; Posner, Marshall R; Payne, Aimee S; Cavacini, Lisa A

    2016-01-01

    Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties.

  2. Pathogenicity and Epitope Characteristics Do Not Differ in IgG Subclass-Switched Anti-Desmoglein 3 IgG1 and IgG4 Autoantibodies in Pemphigus Vulgaris

    PubMed Central

    Ellebrecht, Christoph T.; Yu, Xiaocong; Posner, Marshall R.; Payne, Aimee S.

    2016-01-01

    Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties. PMID:27304671

  3. Detection of Lassa virus antinucleoprotein immunoglobulin G (IgG) and IgM antibodies by a simple recombinant immunoblot assay for field use.

    PubMed

    Ter Meulen, J; Koulemou, K; Wittekindt, T; Windisch, K; Strigl, S; Conde, S; Schmitz, H

    1998-11-01

    The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 microgram of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 microgram. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative

  4. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12.

    PubMed

    Nie, Jianhui; Zhao, Juan; Chen, Qingqing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The CD4 binding site (CD4bs) of envelope glycoprotein (Env) is an important conserved target for anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12 (b12) could recognize conformational epitopes that overlap the CD4bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4bs epitopes.

  5. Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure.

    PubMed

    Giménez-Lirola, L G; Xiao, C T; Halbur, P G; Opriessnig, T

    2012-10-01

    A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.

  6. Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody.

    PubMed

    Viski, Kornél; Gengeliczki, Zsolt; Lenkey, Krisztián; Baranyáné Ganzler, Katalin

    2016-10-01

    Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.

  7. IgG subclass co-expression brings harmony to the quartet model of murine IgG function.

    PubMed

    Collins, Andrew M

    2016-11-01

    A model of murine IgG function is presented in which the co-expression of the IgG subclasses is a central feature, class switching occurs before the commencement of somatic hypermutation, and there is little switching between subclasses. It is named the quartet model to emphasize the harmony that comes from the simultaneous presence of the four subclasses. In this model, IgG3 and IgG2b antibodies are particularly important early in the response, when T-cell help may be limiting. IgG3 initiates inflammation through complement fixation, whereas IgG2b provides early FcγR-mediated effector functions. As T-cell help strengthens, IgG2a antibodies increase the power of the response, whereas IgG1 production helps limit the inflammatory drive and limits immunopathology. The model highlights the fact that murine IgG subclasses function quite differently to human IgG subclasses. This allows them to serve the special immunological needs of a species that is vulnerable because of its small size.

  8. Fluorescent IgG fusion proteins made in E. coli.

    PubMed

    Luria, Yael; Raichlin, Dina; Benhar, Itai

    2012-01-01

    Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called "Inclonals." By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the "Inclonals" technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.

  9. Relationship between Antibody Levels, IgG Binding to Plasmodium falciparum-Infected Erythrocytes, and Disease Outcome in Hospitalized Urban Malaria Patients from Dakar, Sénégal

    PubMed Central

    Mbengue, Babacar; Fall, Mouhamadou Mansour; Sylla Niang, Maguette; Niang, Birahim; Varela, Marie Louise; Diatta, Antoine Marie; Mbow, Moustapha; Ndiaye, Kantome; Ndiaye Diallo, Rokhaya; Dieye, Alioune; Perraut, Ronald

    2016-01-01

    Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease. Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated. Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p = 0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia. Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis. PMID:27563669

  10. Phase transitions in human IgG solutions

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F.; Laubach, Jacob P.; Hideshima, Teru; Richardson, Paul G.; Munshi, Nikhil C.; Anderson, Kenneth C.; Benedek, George B.

    2013-09-01

    Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ˜100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to

  11. Phase transitions in human IgG solutions.

    PubMed

    Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F; Laubach, Jacob P; Hideshima, Teru; Richardson, Paul G; Munshi, Nikhil C; Anderson, Kenneth C; Benedek, George B

    2013-09-28

    Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ~100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to

  12. IgG subclasses compared in maternal and cord serum and breast milk.

    PubMed Central

    Gasparoni, A; Avanzini, A; Ravagni Probizer, F; Chirico, G; Rondini, G; Severi, F

    1992-01-01

    Total and specific IgG subclass antibodies against 14 pneumococcal capsular polysaccharide antigens on the cord serum from 11 healthy term infants at birth and on serum from their mothers at delivery were evaluated. The same evaluation was performed five days after delivery on the serum and the milk obtained from the six mothers who were breast feeding their infants. Mean neonatal: maternal serum ratio of total IgG1 was significantly higher than the ratios of total IgG2, IgG3, and IgG4 and higher than the ratios of pneumococcal IgG subclass antibodies. Total IgG3 and IgG4 ratios were higher than the specific antibody ratios of the same IgG subclass. Type 1 and type 14 IgG1 antibodies were the highest antipneumococcal ratios. Although the maternal milk:serum ratios of total IgG subclasses were very low, significant amounts of specific antibodies were found in the milk, at about half the concentration observed in mother's serum. PMID:1536584

  13. IgGs containing light chains of the lambda and kappa type and of all subclasses (IgG1-IgG4) from sera of patients with multiple sclerosis hydrolyze DNA.

    PubMed

    Parkhomenko, Taisiya A; Legostaeva, Galina A; Doronin, Boris M; Buneva, Valentina N; Nevinsky, Georgy A

    2010-01-01

    We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1-IgG4) are catalytically active in the hydrolysis of DNA and on average their relative activity (nM supercoiled DNA/1mg IgG/1 h) increases in the order: IgG1 (0.58) < IgG2 (0.94) < IgG3 (1.4) < IgG4 (4.1), while their approximate relative contribution to the total activity of abzymes increases in the order: IgG1 (6.9%) < IgG3 (9.3%) < IgG2 (18.2%) < IgG4 (65.6%). On average IgGs containing light chains of the lambda-type are severalfold more active in the hydrolysis of DNA than IgGs with light chains of the kappa-type. Using different physicochemical methods of antibody analysis we have shown that the immune system of multiple sclerosis patients generates a variety of anti-DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis.

  14. Anti-fixed Leishmania chagasi promastigotes IgG antibodies detected by flow cytometry (FC-AFPA-IgG) as a tool for serodiagnosis and for post-therapeutic cure assessment in American visceral leishmaniasis.

    PubMed

    Garcia, Lúcia Maria; Coelho-Dos-Reis, Jordana Grazziela Alves; Peruhype-Magalhães, Vanessa; Teixeira-Carvalho, Andréa; Rocha, Roberta Dias Rodrigues; Araújo, Márcio Sobreira Silva; Gomes, Izabelle Teixeira; Carvalho, Sílvio Fernando Guimarães; Dietze, Reynaldo; Lemos, Elenice Moreira; Andrade, Mariléia Chaves; Martins-Filho, Olindo Assis

    2009-10-31

    Visceral leishmaniasis (VL) is a systemic infection, caused by an intracellular protozoan parasite belonging to the Leishmania donovani complex. The diagnosis of VL is complex because most clinical features are shared with other commonly occurring febrile hepatosplenic diseases that can be endemic along with VL. A number of serological devices are available but still require improvement mainly due to residual post-therapeutic serology and the cross-reactivity with other Trypanosomatidae protozooses. This study intended to describe and evaluate the performance of an indirect immunofluorescence assay referred as flow cytometry anti-fixed Leishmania chagasi promastigote IgG antibodies (FC-AFPA-IgG) for serodiagnosis of VL and assessment of post-therapeutic cure. The sera reactivity is reported as the percentage of positive fluorescent parasite (PPFP) along the titration curve. The analysis of sera titration curve indicated the sera dilution 1/32,000 and the PPFP=25% as the cut-off to segregate positive and negative results. Using these parameters, the FC-AFPA-IgG displayed outstanding sensitivity and specificity for diagnosis and post-therapeutic cure assessment purposes. The inter-test reproducibility of FC-AFPA-IgG was also verified, considering two independent Analysts and validated the results obtained by FC-AFPA-IgG. Moreover, the comparison between FC-AFPA-IgG and the conventional serologic test (ELISA) showed that besides the statistically analogous results with strong positive correlation the FC-AFPA-IgG displayed higher performance indexes. Further analysis demonstrated that while cross-reactivity was observed in 8% of samples tested by ELISA, no cross-reactivity was detected by FC-AFPA-IgG. Together, the findings presented in this study showed the potential of FC-AFPA-IgG in both diagnosis and post-therapeutic cure assessment of VL.

  15. Potential Mechanisms for IgG4 Inhibition of Immediate Hypersensitivity Reactions.

    PubMed

    James, Louisa K; Till, Stephen J

    2016-03-01

    IgG4 is the least abundant IgG subclass in human serum, representing less than 5% of all IgG. Increases in IgG4 occur following chronic exposure to antigen and are generally associated with states of immune tolerance. In line with this, IgG4 is regarded as an anti-inflammatory antibody with a limited ability to elicit effective immune responses. Furthermore, IgG4 attenuates allergic responses by inhibiting the activity of IgE. The mechanism by which IgG4 inhibits IgE-mediated hypersensitivity has been investigated using a variety of model systems leading to two proposed mechanisms. First by sequestering antigen, IgG4 can function as a blocking antibody, preventing cross-linking of receptor bound IgE. Second IgG4 has been proposed to co-stimulate the inhibitory IgG receptor FcγRIIb, which can negatively regulate FcεRI signaling and in turn inhibit effector cell activation. Recent advances in our understanding of the structural features of human IgG4 have shed light on the unique functional and immunologic properties of IgG4. The aim of this review is to evaluate our current understanding of IgG4 biology and reassess the mechanisms by which IgG4 functions to inhibit IgE-mediated allergic responses.

  16. Association of HLA-DRB3*0202 and serum IgG antibodies to Chlamydia pneumoniae with essential hypertension in a highly homogeneous population from Majorca (Balearic Islands, Spain).

    PubMed

    Zabay, J M; Marco, J; Soler, J; Contu, L; Cappai, L; Carcassi, C; Gómez, G; Mulet, J M; Munar, M A; Viader, C

    2005-08-01

    Separate studies investigating the relationship of essential hypertension (EH) with the HLA system and with Chlamydia pneumoniae (C. pneumoniae) infection have given conflicting results. Our aim was to clarify these relationships and determine whether the HLA system and C. pneumoniae infection interact with respect to the risk for EH. An association study (110 essential hypertensives and 107 controls) was conducted in a highly homogeneous population in the Balearic Island of Majorca (Spain). Molecular typing of HLA-B and HLA-DRB and quantification of serum levels of IgG antibodies to C. pneumoniae (sIgGa-Cp) were determined. Student's t-test, chi(2)-statistics, logistic regression analysis, and general linear model ANOVA were used for statistical analysis. The results showed that EH was related with HLA-DRB3*0202 in the whole study population, and with levels of sIgGa-Cp>63.5 BU/ml in the group of individuals with sIgGa-Cp>30 BU/ml (OR (95% CI) adjusted for obesity, familial history of EH and diabetes=2.06 (1.07-3.97), P=0.03, and =4.60 (1.06-19.90), P=0.04, respectively). The association between EH and sIgGa-Cp was observed in the DRB3*0202(+) individuals, but not in the DRB3*0202(-) subgroup (OR (95% CI)=11.14 (1.92-64.54), P=0.004, and =0.98 (0.22-4.43), P=0.64, respectively (P of the Mantel-Haenszel test for homogeneity of OR=0.06)). In our population, EH was positively associated with HLA-DRB3*0202 and with high levels of sIgGa-Cp. Moreover, a significant interaction of DRB3*0202 on the effect of sIgGa-Cp was observed, as the association of EH with these antibodies depended on the presence of DRB*0202.

  17. Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

    PubMed

    Garmatiuk, Tetiana; Swoboda, Ines; Twardosz-Kropfmüller, Anna; Dall'antonia, Fabio; Keller, Walter; Singh, Mohan B; Bhalla, Prem L; Okada, Takashi; Toriyama, Kinya; Weber, Milena; Ghannadan, Minoo; Sperr, Wolfgang R; Blatt, Katharina; Valent, Peter; Klein, Brigitte; Niederberger, Verena; Curin, Mirela; Balic, Nadja; Spitzauer, Susanne; Valenta, Rudolf

    2013-09-01

    The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

  18. CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells

    PubMed Central

    Selb, Regina; Eckl-Dorna, Julia; Neunkirchner, Alina; Schmetterer, Klaus; Marth, Katharina; Gamper, Jutta; Jahn-Schmid, Beatrice; Pickl, Winfried F.; Valenta, Rudolf; Niederberger, Verena

    2017-01-01

    Background Increasing evidence suggests that the low-affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen-specific T cells through IgE-facilitated allergen presentation. Objective We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen-presenting cells is associated with IgE levels and influences allergen uptake and allergen-specific T-cell activation. Methods Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen-specific IgE levels, as well as with allergen-induced immediate skin reactivity. Allergen uptake and allergen-specific T-cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T-cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE-allergen immune complexes were formed with human monoclonal allergen-specific IgE and Bet v 1. Results In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen-induced skin reactions (RS = 0.63, P = .008). Uptake of allergen-IgE complexes into B cells and activation of allergen-specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. Conclusion CD23 surface density on B cells of allergic patients is correlated with allergen-specific IgE levels and determines allergen uptake and subsequent activation of T cells. PMID:27372566

  19. IKKβ in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites.

    PubMed

    Bonnegarde-Bernard, A; Jee, J; Fial, M J; Aeffner, F; Cormet-Boyaka, E; Davis, I C; Lin, M; Tomé, D; Karin, M; Sun, Y; Boyaka, P N

    2014-03-01

    Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKβ) in IECs (IKKβ(ΔIEC)), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKβ(ΔIEC) mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKβ(ΔIEC) mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKβ(ΔIEC) mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKβ(ΔIEC) mice, but did not affect IgA Ab responses. In summary, we show that IKKβ in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.

  20. Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

    PubMed

    Carrasco-Yepez, Maricela; Campos-Rodriguez, Rafael; Lopez-Reyes, Israel; Bonilla-Lemus, Patricia; Rodriguez-Cortes, Antonio Yahve; Contis-Montes de Oca, Arturo; Jarillo-Luna, Adriana; Miliar-Garcia, Angel; Rojas-Hernandez, Saul

    2014-11-01

    The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1β by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1β). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.

  1. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    PubMed

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

  2. IgGs containing λ- and κ-type light chains and of all subclasses (IgG1-IgG4) from the sera of patients with autoimmune diseases and viral and bacterial infections hydrolyze DNA.

    PubMed

    Parkhomenko, Taisiya A; Buneva, Valentina N; Doronin, Boris M; Volkova, Margarita V; Senkovich, Sergey A; Generalov, Igor I; Nevinsky, Georgy A

    2012-07-01

    We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1-IgG4) from patients with viral (tick-borne encephalitis), bacterial infections (streptococcal infection or erysipelas), and suppurative surgical infections caused by epidermal staphylococci as well as from patients with autoimmune diseases (systemic lupus erythematosus and multiple sclerosis) are catalytically active in the hydrolysis of supercoiled DNA. The hydrolysis of DNA was analyzed by agarose gel electrophoresis. The catalytic activities of nonfractionated IgGs increased in the following order: tick-borne encephalitis < suppurative surgical infection < streptococcal infection < multiple sclerosis < systemic lupus erythematosus, whereas IgGs of healthy donors were inactive. However, the pools of antibodies corresponding to any particular disease were characterized by a specific ratio of IgGs of all four subclasses (IgG1-IgG4) and IgGs containing λ- and κ-type light chains, and each of these subfractions of immunoglobulins demonstrated characteristic relative DNase activity. The relative activities of IgGs containing λ-type light chains may on average be higher, lower, or comparable with those for IgGs with κ-type light chains. The relative contributions of IgGs of different subclasses to the total activity of IgGs also varied widely in the case of various diseases: IgG1 (7%-45%), IgG2 (0.4%-73%), IgG3 (0%-12%), and IgG4 (9%-66%). Thus, immune systems of patients with different diseases can generate a variety of anti-DNA abzymes of different types and with different catalytic properties, which can play an important role in the pathogenesis or protection from the development of these diseases.

  3. A review of clinical efficacy, safety, new developments and adherence to allergen-specific immunotherapy in patients with allergic rhinitis caused by allergy to ragweed pollen (Ambrosia artemisiifolia).

    PubMed

    Turkalj, Mirjana; Banic, Ivana; Anzic, Srdjan Ante

    2017-01-01

    Allergic rhinitis is a common health problem in both children and adults. The number of patients allergic to ragweed (Ambrosia artemisiifolia) is on the rise throughout Europe, having a significant negative impact on the patients' and their family's quality of life. Allergen-specific immunotherapy (AIT) has disease-modifying effects and can induce immune tolerance to allergens. Both subcutaneous immunotherapy and sublingual immunotherapy with ragweed extracts/preparations have clear positive clinical efficacy, especially over pharmacological treatment, even years after the treatment has ended. AIT also has very good safety profiles with extremely rare side effects, and the extracts/preparations used in AIT are commonly well tolerated by patients. However, patient adherence to treatment with AIT seems to be quite low, mostly due to the fact that treatment with AIT is relatively time-demanding and, moreover, due to patients not receiving adequate information and education about the treatment before it starts. AIT is undergoing innovations and improvements in clinical efficacy, safety and patient adherence, especially with new approaches using new adjuvants, recombinant or modified allergens, synthetic peptides, novel routes of administration (epidermal or intralymphatic), and new protocols, which might make AIT more acceptable for a wider range of patients and novel indications. Patient education and support (eg, recall systems) is one of the most important goals for AIT in the future, to further enhance treatment success.

  4. A review of clinical efficacy, safety, new developments and adherence to allergen-specific immunotherapy in patients with allergic rhinitis caused by allergy to ragweed pollen (Ambrosia artemisiifolia)

    PubMed Central

    Turkalj, Mirjana; Banic, Ivana; Anzic, Srdjan Ante

    2017-01-01

    Allergic rhinitis is a common health problem in both children and adults. The number of patients allergic to ragweed (Ambrosia artemisiifolia) is on the rise throughout Europe, having a significant negative impact on the patients’ and their family’s quality of life. Allergen-specific immunotherapy (AIT) has disease-modifying effects and can induce immune tolerance to allergens. Both subcutaneous immunotherapy and sublingual immunotherapy with ragweed extracts/preparations have clear positive clinical efficacy, especially over pharmacological treatment, even years after the treatment has ended. AIT also has very good safety profiles with extremely rare side effects, and the extracts/preparations used in AIT are commonly well tolerated by patients. However, patient adherence to treatment with AIT seems to be quite low, mostly due to the fact that treatment with AIT is relatively time-demanding and, moreover, due to patients not receiving adequate information and education about the treatment before it starts. AIT is undergoing innovations and improvements in clinical efficacy, safety and patient adherence, especially with new approaches using new adjuvants, recombinant or modified allergens, synthetic peptides, novel routes of administration (epidermal or intralymphatic), and new protocols, which might make AIT more acceptable for a wider range of patients and novel indications. Patient education and support (eg, recall systems) is one of the most important goals for AIT in the future, to further enhance treatment success. PMID:28243068

  5. Beneficial cross-protection of allergen-specific immunotherapy on airway eosinophilia using unrelated or a partial repertoire of allergen(s) implicated in experimental feline asthma.

    PubMed

    Reinero, Carol; Lee-Fowler, Tekla; Chang, Chee-Hoon; Cohn, Leah; Declue, Amy

    2012-06-01

    The study hypothesis was that in experimentally asthmatic cats rush immunotherapy (RIT) using allergens not completely matched with sensitizing allergen(s) would at least partially attenuate the asthmatic phenotype and modulate the aberrant immune response. In phase I, cats sensitized to Bermuda grass allergen (BGA), house dust mite allergen (HDMA) or placebo received BGA RIT. In phase II, cats dually sensitized to BGA and HDMA received RIT using BGA, HDMA or placebo. Efficacy of RIT was assessed using percentage bronchoalveolar lavage fluid (BALF) eosinophils. Additionally, a variety of immunologic assays were performed. Eosinophilic airway inflammation significantly decreased over time in asthmatic cats given RIT using sensitizing allergen or unrelated allergen (P<0.001). In dually sensitized cats, single allergen RIT but not placebo reduced airway eosinophilia (P=0.038). Differences in allergen-specific lymphocyte proliferation, in the number of IL-10 producing cells and in the percentage T regulatory cells were detected between asthmatic cats getting RIT and controls. Cross-protection manifested by reduced airway eosinophilia was noted in cats treated with RIT allergens which did not completely match allergen used in asthma induction. However, the mechanism of immunologic tolerance may differ when improperly matched allergens to the sensitizing allergens are used in RIT.

  6. Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: Lessons from the past and novel mechanisms of action for the future.

    PubMed

    Valenta, Rudolf; Campana, Raffaela; Focke-Tejkl, Margit; Niederberger, Verena

    2016-02-01

    In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination.

  7. IgGs containing light chains of the λ- and κ- type and of all subclasses (IgG1-IgG4) from the sera of patients with systemic lupus erythematosus hydrolyze myelin basic protein.

    PubMed

    Bezuglova, Anna M; Konenkova, Ludmila P; Buneva, Valentina N; Nevinsky, Georgy A

    2012-12-01

    Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies from systemic lupus erythematosus (SLE) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from SLE patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hMBP at different optimal pH values (5.3-9.5); the pH profiles of IgG1, IgG2, IgG3 and IgG4 were unique. IgGs containing the λ-type of light chains demonstrated higher relative activities (RAs) in the hydrolysis of hMBP and its oligopeptides (OPs) than κ-IgGs. IgGs of all four subclasses were catalytically active; their RAs in the hydrolysis of hMBP increased in the following order: IgG4 < IgG2 < IgG3 < IgG1. Metal-dependent proteolytic activity of λ-IgG, IgG1, IgG2 and IgG3 was higher than their serine protease-like activity, while these activities of κ-IgG were comparable. Phenylmethylsulfonylfluoride had almost no effect on the activity of IgG4, while EDTA significantly suppressed its activity. The RAs of λ-IgG in the hydrolysis of four OPs corresponding to different cleavage sites of hMBP were remarkably higher than those for κ-IgGs. IgG1-IgG4 demonstrated different RAs and patterns of hydrolysis of these four OPs. Although combination of Ca²⁺ plus Mg²⁺ was the best in the activation of IgG1 and IgG2, IgG3 and IgG4 demonstrated the highest activity in the presence of Ca²⁺ plus Co²⁺. The ratio of the RAs of λ-IgG, κ-IgG and IgG1-IgG4 preparations in all analyzed cases was individual for each preparation.

  8. IgG Placental Transfer in Healthy and Pathological Pregnancies

    PubMed Central

    Palmeira, Patricia; Quinello, Camila; Silveira-Lessa, Ana Lúcia; Zago, Cláudia Augusta; Carneiro-Sampaio, Magda

    2012-01-01

    Placental transfer of maternal IgG antibodies to the fetus is an important mechanism that provides protection to the infant while his/her humoral response is inefficient. IgG is the only antibody class that significantly crosses the human placenta. This crossing is mediated by FcRn expressed on syncytiotrophoblast cells. There is evidence that IgG transfer depends on the following: (i) maternal levels of total IgG and specific antibodies, (ii) gestational age, (iii) placental integrity, (iv) IgG subclass, and (v) nature of antigen, being more intense for thymus-dependent ones. These features represent the basis for maternal immunization strategies aimed at protecting newborns against neonatal and infantile infectious diseases. In some situations, such as mothers with primary immunodeficiencies, exogenous IgG acquired by intravenous immunoglobulin therapy crosses the placenta in similar patterns to endogenous immunoglobulins and may also protect the offspring from infections in early life. Inversely, harmful autoantibodies may cross the placenta and cause transitory autoimmune disease in the neonate. PMID:22235228

  9. Monoclonal Antibodies against Epidermal Growth Factor Receptor Acquire an Ability To Kill Tumor Cells through Complement Activation by Mutations That Selectively Facilitate the Hexamerization of IgG on Opsonized Cells.

    PubMed

    Tammen, Annalina; Derer, Stefanie; Schwanbeck, Ralf; Rösner, Thies; Kretschmer, Anna; Beurskens, Frank J; Schuurman, Janine; Parren, Paul W H I; Valerius, Thomas

    2017-02-15

    Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets.

  10. Allergen-specific immune response suppresses interleukin 10 expression in B cells via increasing micro-RNA-17-92 cluster.

    PubMed

    Geng, Xiao-Rui; Qiu, Shu-Qi; Yang, Li-Tao; Liu, Zhi-Qiang; Yang, Gui; Liu, Jiang-Qi; Zeng, Lu; Li, Xiao-Xi; Mo, Li-Hua; Liu, Zhi-Gang; Yang, Ping-Chang

    2016-08-01

    Interleukin (IL)-10-expressing B cells play a critical role in the immune homeostasis in the body; its regulation has not been fully understood. Micro-RNA (miR)-17-92 cluster has strong regulation in the immunity. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells. In this study, peripheral B cells were collected from patients with allergic rhinitis (AR). The B cells were treated with specific allergens, dust mite extracts, in the culture. The expressions of miR-17-92 cluster and IL-10 in the culture were assessed by real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that the levels of miR-19a, but not the rest of the 5 members (miR-17, miR-18a, miR-19b, miR-20a, and miR-92a), were significantly higher in peripheral B cells from AR patients as than in B cells from healthy participants. Exposure of B cells from AR patients to specific allergen, dust mite extracts, significantly increased the levels if miR-19a and suppressed the expression of IL-10 in B cells. The levels of histone deacetylase 11 and acetylated H3K9 were higher, and the RNA polymerase II and c-Maf (the IL-10 transcription factor) were lower, at the IL-10 promoter locus. In conclusion, miR-19a mediates the allergen-specific immune response-decreased IL-10 expression in B cells.

  11. Investigation on the effects of ciclosporin (Atopica) on intradermal test reactivity and allergen-specific immunoglobulin (IgE) serology in atopic dogs.

    PubMed

    Goldman, Candice; Rosser, Edmund; Petersen, Annette; Hauptman, Joe

    2010-08-01

    The ability to use ciclosporin (Atopica®: Novartis Animal Health, Greensboro, NC, USA) prior to intradermal testing (IDT) would help avoid exacerbation of clinical disease that can be associated with drug withdrawal. This study evaluated the effects of 30 days of administration of ciclosporin at a dose of 5 mg/kg once daily on IDT reactivity (immediate phase reactions) in a group of dogs with atopic dermatitis (AD) with initial positive IDT reactions. 16 dogs diagnosed with AD were included in the study. Eight dogs (group A) were treated with ciclosporin orally at 5 mg/kg once daily for 30 days. Eight dogs (group P) were treated with a placebo orally once daily for 30 days. IDT was performed at day 0 and day 30 on all dogs enrolled using a standardized panel of 45 aqueous allergens (Greer Laboratories, Lenoir, NC, USA) appropriate to our geographical region. IDT reactivity was assessed by both subjective and objective methods at 15 min post-intradermal injection. Serum for allergen-specific immunoglobulin (IgE) serology was obtained at day 0 and day 30. The study was designed as a double-blinded, placebo-controlled, cross-over study. Data were analysed using a split-plot analysis of variance with the grouping factor of treatment and the repeat factor of time (SAS System for Windows). At week 4, ciclosporin did not have a statistically significant effect on IDT reactivity or serology results. It therefore appears that, no withdrawal is recommended to evaluate immediate phase reactions.

  12. IgG4-related sialadenitis and Sjögren's syndrome.

    PubMed

    Fragoulis, G E; Zampeli, E; Moutsopoulos, H M

    2017-03-01

    IgG4-related disease (IgG4-RD) has emerged as a new entity in the last decade. It comprises numerous conditions previously thought to be unrelated. Macroscopically, these diseases cause diffuse organ swelling and formation of pseudotumorous masses. Histopathologically, they are characterized by a lymphoplasmacytic infiltrate with increased IgG4+ plasma cells and storiform fibrosis. Despite rapid progress within the last years, our knowledge on these conditions is still fragmented. To date, more than forty organs have been reported to be included in IgG4-RD, and salivary gland involvement is amongst the most common organs affected [IgG4-related sialadenitis (IgG4-RS)]. Interestingly, IgG4-RS shares commonalities with Sjögren's syndrome (SS), like glandular enlargement, sicca symptoms, arthralgias, hypergammaglobulinemia, hypocomplementemia, and circulating antinuclear antibodies. Nonetheless, they differ in that the incidence of anti-Ro and anti-La reactivity is not frequently found in patients with IgG4-RS, their salivary glands are infiltrated by a large number of IgG4+ plasma cells and IgG4-RS symptoms respond promptly to steroids. The aim of this review was to describe the clinical, serological, histopathological and pathophysiological aspects of IgG4-RS in the context of IgG4-RD and highlight the differences between IgG4-RS and SS.

  13. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  14. IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome

    PubMed Central

    Guhsl, E E; Hofstetter, G; Lengger, N; Hemmer, W; Ebner, C; Fröschl, R; Bublin, M; Lupinek, C; Breiteneder, H; Radauer, C

    2015-01-01

    Background Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. Methods Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. Results Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. Conclusion Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac. PMID:25327982

  15. Subclass specificity of the Fc receptor for human IgG on K562.

    PubMed

    Chiofalo, M S; Teti, G; Goust, J M; Trifiletti, R; La Via, M F

    1988-07-01

    The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).

  16. Selective IgG subclass deficiency: quantification and clinical relevance.

    PubMed Central

    Jefferis, R; Kumararatne, D S

    1990-01-01

    Each of the four human IgG subclasses exhibits a unique profile of effector functions relevant to the clearance and elimination of infecting microorganisms. The quantitative response within each IgG subclass varies with the nature of the antigen, its route of entry and, presumably, the form in which it is presented to the immune system. This results in antibody responses to certain antigens being predominantly or exclusively of a single IgG subclass. An inability to produce antibody of the optimally protective isotype can result in a selective immunodeficiency state. This is particularly apparent for responses to certain bacterial carbohydrate antigens that are normally of IgG2 isotype. A failure to produce the appropriate specific antibody response may result in recurrent upper and/or lower respiratory tract infection. Careful patient investigation can identify such deficiencies and suggest appropriate clinical management. In this review we outline the biology and clinical relevance of the IgG subclasses and summarize current rational treatment approaches. PMID:2204502

  17. The action of NIR (808nm) laser radiation and gold nanorods labeled with IgA and IgG human antibodies on methicillin-resistant and methicillin sensitive strains of Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Tuchina, Elena S.; Petrov, Pavel O.; Ratto, Fulvio; Centi, Sonia; Pini, Roberto; Tuchin, Valery V.

    2015-03-01

    The effect of NIR laser radiation (808 nm) on methicillin-sensitive and methicillin resistant strains of Staphylococcus aureus incubated with gold nanorods is studied. Nanorods having length of 44 (± 4) nm and diameter of 10 (± 3) nm with the absorption maximum in the NIR (800 nm), functionalized with human immunoglobulins IgA and IgG, were synthesized and used in the studies. The killing ability up to 97% of the microorganism populations by using this nanotechnology was shown.

  18. IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

    PubMed

    Koneczny, Inga; Stevens, Jo A A; De Rosa, Anna; Huda, Saif; Huijbers, Maartje G; Saxena, Abhishek; Maestri, Michelangelo; Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Tzartos, Socrates; Verschuuren, Jan; van der Maarel, Silvère M; van Damme, Philip; De Baets, Marc H; Molenaar, Peter C; Vincent, Angela; Ricciardi, Roberta; Martinez-Martinez, Pilar; Losen, Mario

    2017-02-01

    Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies

  19. The sequential appearance of IgG subclasses and IgE during the course of Trichinella spiralis infection.

    PubMed Central

    Ljungström, I; Hammarström, L; Kociecka, W; Smith, C I

    1988-01-01

    Earlier studies have shown that IgG1 and IgG4 are the dominant IgG subclasses in the specific response during a chronic helminthic infection. It has also been suggested that IgG4 production results from chronic or repetitive antigenic stimulation and a correlation between IgG4 and IgE levels exists. An outbreak of Trichinella spiralis infection in Poland provided the opportunity to follow the sequential appearance of the IgG subclass and IgE responses in 15 patients during the early stage of Trichinella infection and to compare these observations in sera obtained one year later from the same patients. The results show that the sequential appearance of the IgG subclasses were IgG1 before IgG3 and IgG3 before IgG4. IgG1 antibodies dominated the immune response in all patients. A statistically significant increase in the number of IgG4 positive sera was observed in patients during the chronic stage compared to the findings during the early stage of infection (13% vs 73%; p less than 0.001), supporting the view that IgG4 results from a chronic antigenic stimulation. A correlation between the appearance of IgG4 and IgE was not found. The highest levels of IgE were seen in the first serum samples obtained, with a decrease during the course of infection. PMID:3224442

  20. Myocarditis in different experimental models infected by Trypanosoma cruzi is correlated with the production of IgG1 isotype.

    PubMed

    Caldas, Ivo Santana; Diniz, Livia de Figueiredo; Guedes, Paulo Marcos da Matta; Nascimento, Álvaro Fernando da Silva do; Galvão, Lúcia Maria da Cunha; Lima, Wanderson Geraldo de; Caldas, Sérgio; Bahia, Maria Terezinha

    2017-03-01

    This study was designed to verify the relationship between IgG antibodies isotypes and myocarditis in Trypanosoma cruzi infection using mice and dogs infected with different T. cruzi strains. The animals were infected with benznidazole-susceptible Berenice-78 and benznidazole-resistant AAS and VL-10 strains. The IgG subtypes were measured in serum samples from dogs (IgG, IgG1, and IgG2) and mice (IgG, IgG1, IgG2a, and IgG2b). The infection of dogs with VL-10 strain induced the highest levels of heart inflammation while intermediate and lower levels were detected with Berenice-78 and AAS strains, respectively. Similar results were found in mice infected with VL-10, but not in those infected with AAS or Berenice-78 strains. The AAS strain induced higher levels of heart inflammation in mice, while Berenice-78 strain was not able to induce it. Correlation analysis between myocarditis and antibody reactivity index revealed very interesting results, mainly for IgG and IgG1, the latter being the most exciting. High IgG1 showed a significant correlation with myocarditis in both experimental models, being more significant in dogs (r=0.94, p<0.0001) than in mice (r=0.58, p=0.047). Overall, our data suggest that IgG1 could be a good marker to demonstrate myocarditis intensity in Chagas disease.

  1. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  2. IgG1 deficiency exacerbates experimental autoimmune myasthenia gravis in BALB/c mice.

    PubMed

    Huda, Ruksana; Strait, Richard T; Tüzün, Erdem; Finkelman, Fred D; Christadoss, Premkumar

    2015-04-15

    Myasthenia gravis is an autoimmune disease characterized by muscle weakness due to neuromuscular junction (NMJ) damage by anti-acetylcholine receptor (AChR) auto-antibodies and complement. In experimental autoimmune myasthenia gravis (EAMG), which is induced by immunization with Torpedo AChR in CFA, anti-AChR IgG2b and IgG1 are the predominant isotypes in the circulation. Complement activation by isotypes such as IgG2b plays a crucial role in EAMG pathogenesis; this suggested the possibility that IgG1, which does not activate complement through the classical pathway, may suppress EAMG. In this study, we show that AChR-immunized BALB/c mice genetically deficient for IgG1 produce higher levels of complement-activating isotypes of anti-AChR, especially IgG3 and IgG2a, and develop increased IgG3/IgG2a deposits at the NMJ, as compared to wild type (WT) BALB/c mice. Consistent with this, AChR-immunized IgG1(-/-) BALB/c mice lose muscle strength and muscle AChR to a greater extent than AChR-immunized WT mice. These observations demonstrate that IgG1 deficiency leads to increased severity of EAMG associated with an increase in complement activating IgG isotypes. Further studies are needed to dissect the specific role or mechanism of IgG1 in limiting EAMG and that of EAMG exacerbating role of complement activating IgG3 and IgG2a in IgG1 deficiency.

  3. Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients

    PubMed Central

    Westra, J; van Assen, S; Wilting, K R; Land, J; Horst, G; de Haan, A; Bijl, M

    2014-01-01

    Rituximab (RTX) treatment in rheumatoid arthritis (RA) patients severely hampers humoral response after influenza vaccination as determined by haemagglutination inhibition assay (HI). It is not known whether HI reflects both immunoglobulin (Ig)M and IgG (subclass) influenza response, and whether IgM antibodies contribute to the low rate of influenza infection seen in RA patients. Twenty RA patients on methotrexate (MTX), 23 on RTX and 28 healthy controls (HC) received trivalent influenza subunit vaccination. Before and 28 days after vaccination, H1N1- and H3N2-specific antibodies were measured by HI and by IgM and IgG (subclass) enzyme-linked immunosorbent assay (ELISA). B cell activating factor (BAFF) levels were determined in serum samples before vaccination. Vaccination induced a significant increase of IgM and IgG (IgG1 and IgG3) antibodies against both strains in the HC and MTX groups (all P < 0·01), but not in the RTX group. HI correlated significantly in all cases with IgG (IgG1) but not with IgM. In RTX late patients (RTX treatment 6–10 months before vaccination), IgG (IgG1 and IgG3) response to vaccination was restored, but not IgM response. BAFF levels were significantly increased in RA-RTX patients and correlated with total IgG levels. Haemagglutination inhibition assay, used as gold standard, detects primarily IgG (IgG1) responses. IgM- and IgG influenza-specific antibodies increase after vaccination in HC and RA patients except in patients on RTX treatment. BAFF levels are increased in both early and late RTX-treated patients, but do not correlate with an influenza-specific antibody response. PMID:24889761

  4. Epstein-Barr Virus Antibodies Test

    MedlinePlus

    ... and services. Advertising & Sponsorship: Policy | Opportunities Epstein-Barr Virus (EBV) Antibody Tests Share this page: Was this ... EA-D IgG Ab Formal name: Epstein-Barr Virus Antibody to Viral Capsid Antigen, IgM, IgG; Epstein- ...

  5. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  6. Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

    PubMed Central

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-01-01

    The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease. PMID:26137589

  7. Glomerular lesions induced in the rabbit by physicochemically altered homologous IgG.

    PubMed Central

    Cavalot, F.; Miyata, M.; Vladutiu, A.; Terranova, V.; Dubiski, S.; Burlingame, R.; Tan, E.; Brentjens, J.; Milgrom, F.; Andres, G.

    1992-01-01

    Immunization of rabbits with physicochemically altered homologous or even autologous IgG induces formation of antibodies combining with IgG of rabbit and of foreign species. Cardiac but not renal lesions were reported in such animals. This study examined the nephritogenic potential of the immune response to cationized or heat-aggregated homologous IgG of b9 or b4 allotype in rabbits of the b4 allotype. Rabbits injected with either b9 or b4 cationized IgG produced antibodies reactive with rabbit and human IgG and with histones; they also developed abnormal glomerular deposits of IgG b4 and C3 corresponding to alterations of the glomerular basement membranes (GBM). Rabbits injected with either b9 or b4 aggregated IgG developed antibodies reactive with rabbit and human IgG and abnormal glomerular deposits of IgG b4 and C3 in the GBM and in the mesangium with subendothelial and mesangial electron-dense deposits. Some rabbits in both groups had proliferative and exudative glomerulonephritis and proteinuria. The results showed that immunization of rabbits with physicochemically altered homologous IgG induces an immune response to rabbit and human IgG and to histones as well as glomerular deposits of autologous IgG and C3 and other glomerular lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 PMID:1546743

  8. Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease.

    PubMed

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-05-01

    The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease.

  9. The birthday of a new syndrome: IgG4-related diseases constitute a clinical entity.

    PubMed

    Takahashi, Hiroki; Yamamoto, Motohisa; Suzuki, Chisako; Naishiro, Yasuyoshi; Shinomura, Yasuhisa; Imai, Kohzoh

    2010-07-01

    IgG4-related disease is a distinct clinical entity, whose characteristic features are the following; Serum IgG4 is prominently elevated, IgG4-positive plasma cells infiltrate in involved tissues, various mass-forming lesions with fibrosis develop in a timely and spatial manner and the response to corticosteroids is prompt and good. IgG4-related diseases mainly target two organs. One is the pancreas (autoimmune pancreatitis; AIP), and the other comprises the lacrimal and salivary glands, the clinical phenotype is Mikulicz's disease (MD). MD has long been considered a manifestation of Sjögren's syndrome (SS). However, we noticed several clinical differences in case of MD from SS; no deflection of female sex differences, mild sicca syndrome, good response to corticosteroids, no positivity of anti-SS-A/SS-B antibodies. In addition, elevated level of serum IgG4 and abundant infiltration of plasma cells expressing IgG4 were reported in MD patients. Those are common features of IgG4-related diseases. MD often coexisted with IgG4-related diseases such as AIP, retroperitoneal fibrosis, and IgG4-associated nephropathy. Based on those findings, it has been considered to recognize IgG4-related diseases including MD as a new clinical entity. The etiology of IgG4-related systemic diseases remains to be elucidated. It is necessary to accumulate and analyze larger data from patients worldwide.

  10. Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

    PubMed Central

    Boesch, Austin W.; Osei-Owusu, Nana Yaw; Crowley, Andrew R.; Chu, Thach H.; Chan, Ying N.; Weiner, Joshua A.; Bharadwaj, Pranay; Hards, Rufus; Adamo, Mark E.; Gerber, Scott A.; Cocklin, Sarah L.; Schmitz, Joern E.; Miles, Adam R.; Eckman, Joshua W.; Belli, Aaron J.; Reimann, Keith A.; Ackerman, Margaret E.

    2016-01-01

    Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies. PMID:28018355

  11. In vitro functional characterization of feline IgGs.

    PubMed

    Strietzel, Catherine J; Bergeron, Lisa M; Oliphant, Theodore; Mutchler, Veronica T; Choromanski, Leszek J; Bainbridge, Graeme

    2014-04-15

    Very little is known about the functional properties of feline IgGs. Here we report the in vitro characterization of cloned feline IgGs. Rapid amplification of cDNA ends (RACE) and full-length PCR of cat splenic cDNA were used to identify feline sequences encoding IgG heavy chain constant regions (IGHC). Two of the sequences are possibly allelic and have been previously reported in the literature as the only feline IgG, IgG1. Although we confirmed these alleles to be highly abundant (∼98%), analysis of numerous amplification products revealed an additional sequence (∼2%). We cloned and characterized chimeric monoclonal antibodies with each of these heavy chains. Using RACE we revealed the sequences for feline Fc gamma receptor I (FcγRI) and feline Fc neonatal receptor (FcRn). We constructed these recombinant receptors as well as fFcγRIII and determined their binding affinities to the chimeras. All of the chimeras bound to Protein A but not to Protein G, and bound tightly to fFcRn (KD=2-5 nM). Both IgG1 alleles have a high affinity for fFcγRI (KD=10-20 nM), they bind to the low-affinity fFcγRIII receptor (2-4 μM), and also bind to human complement C1q. Thus, feline IgG1a and 1b are expected to induce strong effector function in vivo. The additional IgG detected does not bind to recombinant fFcγRI or fFcγRIII and has negligible binding to hC1q. Consequently, although this putative subclass is projected to have a similar serum half-life as the IgG1 alleles based on comparable in vitro affinity to FcRn, it may not elicit the effector responses mediated by fFcγRI or fFcγRIII. Further testing with native receptors and functional cell-based assays would confirm effector function capabilities of feline IgG subclasses; however this is the first report characterizing affinities of feline IgGs to their Fc receptors and helps pave the way for construction of feline-specific IgGs for therapeutic use.

  12. Hypogammaglobulinaemia in nephrotic rats is attributable to hypercatabolism of IgG.

    PubMed Central

    Beaman, M; Oldfield, S; MacLennan, I C; Michael, J; Adu, D

    1988-01-01

    The effect of the nephrotic syndrome induced by puromycin aminonucleoside (PA) in rats on specific antibody responses to 2,4 dinitrophenyl (DNP) conjugated to either spider crab haemocyanin (MSH), a T cell-dependent antigen, or hydroxyethyl starch (HES), a T cell-independent type 2 antigen were studied. The serum IgG anti-DNP levels following immunization with both antigens were reduced in nephrotic animals compared with controls while IgM anti-DNP antibody titres were higher. The half-life of IgG anti-DNP antibodies passively transferred into non-immunized nephrotic rats was markedly reduced while the half-life of anti-DNP antibodies of the IgM class was comparable to that in controls. Low serum IgG and elevated IgM levels were seen in nephrotic animals compared to controls. Antibody-forming cells specific for DNP were demonstrated by immunohistology on rat spleens and the numbers of both IgG and IgM-producing cells were found to be significantly increased (P less than 0.05) in nephrotic animals in response to both DNP-HES and DNP-MSH. These data indicate that in nephrotic rats the alteration seen in the serum immunoglobulin levels is not attributable to reduced antibody production but increased catabolism of serum IgG antibodies. PMID:3233791

  13. The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc.

    PubMed

    Leo, Jack C; Goldman, Adrian

    2009-05-01

    The immunoglobulin-binding proteins from Escherichia coli (Eibs) comprise a family of six proteins homologous to the Yersinia adhesin YadA. These proteins are postulated to bind to the Fc portion of immunoglobulin G (IgG) in a non-immune manner. However, a recent study [Ghumra, A., Pleass, R.J., 2007. Escherichia coli do not express Fc-receptors for human immunoglobulin G (IgG). Mol. Immunol. 44, 2144-2146] appeared to show that these proteins do not bind Fc and suggested that the binding seen in earlier studies is due to the polyclonal preparations used in the assays containing antibodies specific to epitopes in the Eib proteins. To resolve this matter, we produced purified, recombinant Eibs for the first time and investigated their binding to intact antibodies and Fc fragments by immunoblot and ELISA techniques. We were able to purify four members of the family, EibA, -C, -D and -F, and show conclusively that these bind IgG Fc. We were also able to block the binding of full-length antibody with IgG Fc, but not with IgG Fab. Binding to IgG Fab was not detectable by surface plasmon resonance, whereas the affinities of Eibs to IgG and IgG Fc were in the range of 50-200 nM. We further demonstrate that deglycosylating IgG Fc does not affect Eib binding. Our results show that the Eib proteins do indeed bind human IgG Fc and that IgG Fc receptors are present in E. coli.

  14. Highly parallel characterization of IgG Fc binding interactions