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Sample records for antineutrophil antibodies developed

  1. Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

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    Grayson, Peter C.; Sloan, J. Mark; Niles, John L.; Monach, Paul A.; Merkel, Peter A.

    2011-01-01

    Objectives Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Methods Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 – October 2010. A PubMed database search of the medical literature was performed for papers published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. Results A 49 year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a post-recovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. Conclusions ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV. PMID:21507463

  2. Animal models of antineutrophil cytoplasm antibody-associated vasculitis.

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    Salama, Alan D

    2012-01-01

    To provide an update on the experimental models that have been developed recapitulating clinical antineutrophil cytoplasm antibody (ANCA) associated vasculitis. The application of the models in the study of pathogenesis, and the therapeutic implications of this, are covered in the article by van Timmeren and Heeringa in this issue.

  3. Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

    NARCIS (Netherlands)

    Rutgers, Abraham; Kallenberg, Cornelis

    Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received.

  4. Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis: two case reports and review of literature

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    Spoerl David

    2012-12-01

    Full Text Available Abstract Background Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis have been described in literature. Case presentation We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient. Conclusion Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis.

  5. Antineutrophil cytoplasm antibody: positivity and clinical correlation.

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    Martínez Téllez, Goitybell; Torres Rives, Bárbara; Rangel Velázquez, Suchiquil; Sánchez Rodríguez, Vicky; Ramos Ríos, María Antonia; Fuentes Smith, Lisset Evelyn

    2015-01-01

    To determine positivity and clinical correlation of anti-neutrophil cytoplasmic antibodies (ANCA), taking into account the interference of antinuclear antibodies (ANA). A prospective study was conducted in the Laboratory of Immunology of the National Cuban Center of Medical Genetic during one year. Two hounded sixty-seven patients with indication for ANCA determination were included. ANCA and ANA determinations with different cut off points and assays were determined by indirect immunofluorescense. Anti proteinase 3 and antimyeloperoxidase antibodies were determined by ELISA. Most positivity for ANCA was seen in patients with ANCA associated, primary small-vessel vasculitides, rheumatoid arthritis and systemic lupus erythematosus. Presence of ANCA without positivity for proteinase 3 and myeloperoxidase was higher in patients with ANA and little relation was observed between the perinuclear pattern confirmed in formalin and specificity by myeloperoxidase. Highest sensibility and specificity values for vasculitides diagnostic were achieved by ANCA determination using indirect immunofluorescense with a cut off 1/80 and confirming antigenic specificities with ELISA. ANCA can be present in a great number of chronic inflammatory or autoimmune disorders in the population studied. This determination using indirect immunofluorescence and following by ELISA had a great value for vasculitis diagnosis. Anti mieloperoxidasa assay has a higher utility than the formalin assay when ANA is present. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  6. Propylthiouracil-Induced Vasculitis With Antineutrophil Cytoplasmic Antibody.

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    Criado, Paulo Ricardo; Grizzo Peres Martins, Ana Claudia; Gaviolli, Camila Fatima; Alavi, Afsaneh

    2015-06-01

    Propylthiouracil (PTU)-associated vasculitis is a potentially life-threatening disease with a recent increase in the reported cases in the medical literature. This increase may suggest that some earlier cases have been unrecognized or assigned to an alternative nosology category. Although the skin can be the only organ affected by PTU-associated vasculitis, there are many reports with multiple-system involvement. Classically, the symptoms appear under a tetrad of fever, sore throat, arthralgia, and skin lesions. Cutaneous lesions in reported cases of PTU vasculitis have most commonly consisted of retiform acral, purpuric plaques, or nodules. We report a case of perinuclear antineutrophil cytoplasmic antibody-associated vasculitis developed during treatment with PTU for Grave's disease. © The Author(s) 2014.

  7. Development and validation of case-finding algorithms for the identification of patients with anti-neutrophil cytoplasmic antibody-associated vasculitis in large healthcare administrative databases.

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    Sreih, Antoine G; Annapureddy, Narender; Springer, Jason; Casey, George; Byram, Kevin; Cruz, Andy; Estephan, Maya; Frangiosa, Vince; George, Michael D; Liu, Mei; Parker, Adam; Sangani, Sapna; Sharim, Rebecca; Merkel, Peter A

    2016-12-01

    The aim of this study was to develop and validate case-finding algorithms for granulomatosis with polyangiitis (Wegener's, GPA), microscopic polyangiitis (MPA), and eosinophilic GPA (Churg-Strauss, EGPA). Two hundred fifty patients per disease were randomly selected from two large healthcare systems using the International Classification of Diseases version 9 (ICD9) codes for GPA/EGPA (446.4) and MPA (446.0). Sixteen case-finding algorithms were constructed using a combination of ICD9 code, encounter type (inpatient or outpatient), physician specialty, use of immunosuppressive medications, and the anti-neutrophil cytoplasmic antibody type. Algorithms with the highest average positive predictive value (PPV) were validated in a third healthcare system. An algorithm excluding patients with eosinophilia or asthma and including the encounter type and physician specialty had the highest PPV for GPA (92.4%). An algorithm including patients with eosinophilia and asthma and the physician specialty had the highest PPV for EGPA (100%). An algorithm including patients with one of the diagnoses (alveolar hemorrhage, interstitial lung disease, glomerulonephritis, and acute or chronic kidney disease), encounter type, physician specialty, and immunosuppressive medications had the highest PPV for MPA (76.2%). When validated in a third healthcare system, these algorithms had high PPV (85.9% for GPA, 85.7% for EGPA, and 61.5% for MPA). Adding the anti-neutrophil cytoplasmic antibody type increased the PPV to 94.4%, 100%, and 81.2% for GPA, EGPA, and MPA, respectively. Case-finding algorithms accurately identify patients with GPA, EGPA, and MPA in administrative databases. These algorithms can be used to assemble population-based cohorts and facilitate future research in epidemiology, drug safety, and comparative effectiveness. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  8. Novel clinical and diagnostic aspects of antineutrophil cytoplasmic antibodies.

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    Schulte-Pelkum, Johannes; Radice, Antonella; Norman, Gary L; Lόpez Hoyos, Marcos; Lakos, Gabriella; Buchner, Carol; Musset, Lucile; Miyara, Makoto; Stinton, Laura; Mahler, Michael

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of some idiopathic systemic vasculitides. Besides the investigation of ANCA-associated vasculitis (AAV) and constant effort for a standardized nomenclature and classification of the AAV, a main focus of research during the last few years has been to constantly improve the performance of enzyme immunoassays. With the latest so called third generation ELISA, this goal seemed to be fulfilled. The International Consensus Statement on Testing and Reporting of ANCA gave recommendations for standardized strategies for the serological diagnosis of ANCA. New developments now target the system immanent drawbacks of the respective diagnostic methods, be it the need for batching and the long time to result for ELISA, or the high likelihood of error and subjectivity of indirect immunofluorescence (IIF). Random access technology and multiplexing for solid phase assays as well as digital imaging for IIF are tools which may help to expedite and simplify routine diagnostics in the lab and in emergency settings. Recent findings indicate that PR3-ANCA have clinical utility beyond the diagnosis of AAV. PR3-ANCA can also serve as an aid for the differentiation between ulcerative colitis (UC) and Crohn's disease (CrD) and the stratification of UC patients. This review provides a detailed review of what is known about ANCA and highlights the latest research and state-of-the-art developments in this area.

  9. Incidence of malignancy in patients treated for antineutrophil cytoplasm antibody-associated vasculitis : follow-up data from European Vasculitis Study Group clinical trials

    NARCIS (Netherlands)

    Heijl, C.; Harper, L.; Flossmann, O.; Stucker, I.; Scott, D. G. I.; Watts, R. A.; Hoglund, P.; Westman, K.; Mahr, A.

    Objectives Because standard immunosuppressive treatment for antineutrophil cytoplasm antibody-associated vasculitis (AAV) (granulomatosis with polyangiitis (Wegener's) (GPA) and microscopic polyangiitis (MPA)) has been associated with a significant risk of developing cancer, the cancer incidence of

  10. [Wegener's granulomatosis with anti-neutrophil cytoplasmic antibodies against anti-cathepsin G antigen].

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    Ocaña Pérez, E; Peña Casas, A M; del Campo Muñoz, T; Avila Casas, A; Luque Barona, R

    2013-12-01

    Wegener's granulomatosis belongs to the group of small vessel vasculitis associated with anti-neutrophil cytoplasmic antibodies characterized by granulomatous inflammation and necrotising vasculitis in various organs with particular involvement of the upper and lower respiratory tracts and kidneys. Wegener's granulomatosis is a rare disorder in childhood and early diagnosis of this disease is critical to the long-term prognosis of the disease. The presence of positive cytoplasmic antineutrophil cytoplasmic antibody staining or a high titre of proteinase 3 antibodies were added as new criteria of vasculitis in childhood. This article presents a case of Wegener's granulomatosis, with the presence of anti-neutrophil cytoplasm antibodies with cytoplasmic pattern with absence of anti-proteinase 3 antibodies and presence of high levels of anti-cathepsin G antibodies, rarely described in Wegener's granulomatosis. Copyright © 2012 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  11. Central Diabetes Insipidus in Refractory Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

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    Ohashi, Keiji; Morishita, Michiko; Watanabe, Haruki; Sada, Ken-Ei; Katsuyama, Takayuki; Miyawaki, Yoshia; Katsuyama, Eri; Narazaki, Mariko; Tatebe, Noriko; Watanabe, Katsue; Kawabata, Tomoko; Wada, Jun

    2017-11-01

    We herein describe two cases of refractory antineutrophil cytoplasmic antibody-associated vasculitis (AAV) complicated with diabetes insipidus (DI) possibly related to hypertrophic pachymeningitis (HP). One patient had microscopic polyangiitis and HP, which were refractory to cyclophosphamide, azathioprine, rituximab, mycophenolate mofetil (MMF), and mizoribine. Remission was finally achieved with the use of etanercept, but DI occurred 5 years later. The other patient had granulomatosis with polyangiitis, which that was refractory to cyclophosphamide, methotrexate, MMF, and rituximab. DI subsequently developed, but was successfully treated with etanercept. Dura mater hypertrophy was macroscopically observed in the latter case.

  12. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

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    Hong, Ying

    2012-01-01

    The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

  13. Urinary Biomarkers in Relapsing Antineutrophil Cytoplasmic Antibody-associated Vasculitis

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    Lieberthal, Jason G.; Cuthbertson, David; Carette, Simon; Hoffman, Gary S.; Khalidi, Nader A.; Koening, Curry L.; Langford, Carol A.; Maksimowicz-McKinnon, Kathleen; Seo, Philip; Specks, Ulrich; Ytterberg, Steven R.; Merkel, Peter A.; Monach, Paul A.

    2015-01-01

    Objective Glomerulonephritis (GN) is common in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but tools for early detection of renal involvement are imperfect. We investigated 4 urinary proteins as markers of active renal AAV: alpha-1 acid glycoprotein (AGP), kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL). Methods Patients with active renal AAV (n = 20), active nonrenal AAV (n = 16), and AAV in longterm remission (n = 14) were identified within a longitudinal cohort. Urinary biomarker concentrations (by ELISA) were normalized for urine creatinine. Marker levels during active AAV were compared to baseline remission levels (from 1–4 visits) for each patient. Areas under receiver-operating characteristic curves (AUC), sensitivities, specificities, and likelihood ratios (LR) comparing disease states were calculated. Results Baseline biomarker levels varied among patients. All 4 markers increased during renal flares (p < 0.05). MCP-1 discriminated best between active renal disease and remission: a 1.3-fold increase in MCP-1 had 94% sensitivity and 89% specificity for active renal disease (AUC = 0.93, positive LR 8.5, negative LR 0.07). Increased MCP-1 also characterized 50% of apparently nonrenal flares. Change in AGP, KIM-1, or NGAL showed more modest ability to distinguish active renal disease from remission (AUC 0.71–0.75). Hematuria was noted in 83% of active renal episodes, but also 43% of nonrenal flares and 25% of remission samples. Conclusion Either urinary MCP-1 is not specific for GN in AAV, or it identifies early GN not detected by standard assessment and thus has potential to improve care. A followup study with kidney biopsy as the gold standard is needed. PMID:23547217

  14. Anticorpos contra o citoplasma de neutrófilos Antineutrophil cytoplasmic antibodies

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    Ari Stiel Radu

    2005-07-01

    Full Text Available A descoberta do marcador sorológico denominado anticorpo anticitoplasma de neutrófilos revolucionou o diagnóstico e o seguimento das vasculites pulmonares, especialmente da granulomatose de Wegener. Seu padrão pode ser citoplasmático e perinuclear. Sua titulação auxilia no diagnóstico e no seguimento das vasculites pulmonares.The discovery of the serological markers known as antineutrophil cytoplasmic antibodies revolutionized the diagnosis and follow-up treatment of the various forms of pulmonary vasculitis, especially that of Wegener's granulomatosis. The antineutrophil cytoplasmic antibodies pattern can be cytoplasmic or perinuclear. Determination of antineutrophil cytoplasmic antibodies titers aids the diagnosis and follow-up treatment of pulmonary vasculitis.

  15. A case of propylthiouracil-induced antineutrophilic cytoplasmic antibody-positive vasculitis successfully treated with radioactive iodine

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    C. Bes

    2013-07-01

    Full Text Available Antineutrophilic cytoplasmic antibody (ANCA associated vasculitis is one of the rare complications of propylthiouracil treatment. Having a variable clinical spectrum, it may be presented with both skin limited vasculitis and life-threatening systemic vasculitis. In this study, we present a case that developed ANCA-positive vasculitis with skin and kidney involvement (hematuria and proteinuria six months after propylthiouracil treatment was initiated for toxic nodular goiter. Proteinuria recovered dramatically subsequent to radioactive iodine treatment following ceasing the drug.

  16. Clinical significance of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune liver diseases

    NARCIS (Netherlands)

    Roozendaal, C; de Jong, MA; van den Berg, AP; Limburg, PC; Kallenberg, CGM; van Wijk, R.

    Background/Aims: The clinical relevance of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune liver disease is unclear. Defining the antigenic specificities of ANCA in these diseases may improve their clinical significance. Methods: We studied the target antigens of ANCA in 88 patients with

  17. Plasma exchange in antineutrophil cytoplasmic antibody-associated vasculitis--a 25-year perspective

    DEFF Research Database (Denmark)

    Szpirt, Wladimir M

    2015-01-01

    Demonstration of a pathogenic role for antineutrophil cytoplasmic antibodies (ANCA) underlies the scientific rationale for plasma exchange (PLEX) in the treatment of ANCA-associated vasculitis (AAV). Most clinical evidence of efficacy concerns the use of PLEX for the recovery of renal function...

  18. Clinical Outcomes of Remission Induction Therapy for Severe Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    NARCIS (Netherlands)

    Miloslavsky, E. M.; Specks, U.; Merkel, P. A.; Seo, P.; Spiera, R.; Langford, C. A.; Hoffman, G. S.; Kallenberg, C. G. M.; St Clair, E. W.; Tchao, N. K.; Viviano, L.; Ding, L.; Sejismundo, L. P.; Mieras, K.; Ikle, D.; Jepson, B.; Mueller, M.; Brunetta, P.; Allen, N. B.; Fervenza, F. C.; Geetha, D.; Keogh, K.; Kissin, E. Y.; Monach, P. A.; Peikert, T.; Stegeman, C.; Ytterberg, S. R.; Stone, J. H.

    Objective. To evaluate the reasons that complete remission is not achieved or maintained with original treatment in some patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) treated with rituximab (RTX) or with cyclophosphamide/azathioprine (CYC/AZA). Methods. The

  19. Rituximab for the Treatment of Relapses in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    NARCIS (Netherlands)

    Miloslavsky, E. M.; Specks, U.; Merkel, P. A.; Seo, P.; Spiera, R.; Langford, C. A.; Hoffman, G. S.; Kallenberg, C. G. M.; Clair, E. W. St.; Tchao, N. K.; Viviano, L.; Ding, L.; Ikle, D.; Villarreal, M.; Jepson, B.; Brunetta, P.; Allen, N. B.; Fervenza, F. C.; Geetha, D.; Keogh, K.; Kissin, E. Y.; Monach, P. A.; Peikert, T.; Stegeman, C.; Ytterberg, S. R.; Stone, J. H.

    2014-01-01

    Objective. Disease relapses are frequent in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). This study was undertaken to evaluate outcomes in patients with AAV who are re-treated with rituximab (RTX) and prednisone for severe disease relapses. Methods. The Rituximab in AAV trial

  20. Anti-neutrophil cytoplasmic antibody-associated vasculitis with renal involvement: Analysis of 89 cases.

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    Caravaca-Fontán, Fernando; Yerovi, Estefanía; Delgado-Yagu E, María; Galeano, Cristina; Pampa-Saico, Saúl; Tenorio, Maria Teresa; Liaño, Fernando

    2017-01-06

    The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with renal involvement are associated with high morbi-mortality. In this study we analyse if the prognosis of these diseases have improved in recent years, and which factors influence the outcomes. Retrospective single-centre observational study, which included all patients diagnosed with microscopic polyangiitis and granulomatosis with polyangiitis with renal involvement in the last 25 years. Demographic, clinical and biochemical parameters of prognostic interest were recorded. The differences between four chronological periods were analysed, along with the determinants of a poor outcome (death or end-stage renal disease). Eighty-nine patients were included (mean age 64±15 years). Sixty-four patients (72%) had microscopic polyangiitis and 25 (28%) granulomatosis with polyangiitis. During the study period, 37 (42%) patients died. Through Cox regression analysis, the best determinants of mortality were the initial glomerular filtration rate (HR 0.911; P=.003), Charlson comorbidity index (HR 1.513; P<.0001) and tobacco smoking (HR 1.816; P=.003). 35% developed end-stage renal disease, and the best determinants (by competing-risk regression) were: initial glomerular filtration rate (sub-hazard ratio [SHR]: 0.791; P<.0001), proteinuria (SHR: 1.313; P<.0001), and smoking status (SHR: 1.848; P=.023). No differences were found in patients' mortality or renal survival between the different study periods. Prognosis of anti-neutrophil cytoplasm antibodies vasculitis with renal involvement treated with conventional immunosuppressive therapy remains unsatisfactory, and continues to have increased long-term complications and mortality. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  1. Plasma exchange and glucocorticoid dosing in the treatment of anti-neutrophil cytoplasm antibody associated vasculitis (PEXIVAS)

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    Walsh, Michael; Merkel, Peter A; Peh, Chen Au

    2013-01-01

    Granulomatosis with polyangiitis (GPA, Wegener's) and microscopic polyangiitis (MPA) are small vessel vasculitides collectively referred to as anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). AAV is associated with high rates of morbidity and mortality due to uncontrolled disease...

  2. Clinical outcome of patients with coexistent antineutrophil cytoplasmic antibodies and antibodies against glomerular basement membrane.

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    Lindic, Jelka; Vizjak, Alenka; Ferluga, Dusan; Kovac, Damjan; Ales, Andreja; Kveder, Radoslav; Ponikvar, Rafael; Bren, Andrej

    2009-08-01

    Antineutrophil cytoplasmic antibodies (ANCA) and antibodies against glomerular basement membrane (anti-GBM) rarely coexist. Both antibodies may be associated with rapidly progressive glomerulonephritis and pulmonary hemorrhage. We describe the clinical, serological and histological features of our patients with dual antibodies. From 1977 to 2008, 48 patients with anti-GBM antibody-associated renal disease were observed. Eight out of the 30 tested patients (26.7%), all females, had positive myeloperoxidase (MPO)-ANCA coexistent with anti-GBM antibodies. The patients' mean age was 63.4 +/- 7.8 years. Five presented with pulmonary-renal syndrome, all but one were dialysis-dependent on admission. They had constitutional symptoms and different organ involvement. The kidney biopsies revealed intense linear staining for immunoglobulin G and C3 along the glomerular and distal tubular basement membrane associated with irregular diffuse or focal extracapillary crescentic glomerulonephritis with necrosis of varying extent. Lesions of varying ages were characteristically expressed. Seven patients were treated with methylprednisolone and plasma exchange, four with cyclophosphamide, and one with intravenous immunoglobulin. After 28-74 months, there were three dialysis-dependent survivors and one patient with stable chronic renal disease. Two clinical relapses with pulmonary involvement and MPO-ANCA positivity without anti-GBM antibodies occurred in two dialysis-dependent patients. In summary, screening for ANCA and anti-GBM antibodies should be undertaken in patients with clinical signs of systemic vasculitis. In dialysis-dependent patients, the goal of treatment is to limit the damage of other involved organs and not to preserve renal function. Careful follow-up is necessary due to the relapsing nature of the ANCA component of the disease.

  3. Hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis presenting with a vasculitic syndrome, acute nephritis and a puzzling skin rash: a case report

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    Keasberry Justin

    2013-01-01

    Full Text Available Abstract Introduction Anti-neutrophil cytoplasmic antibody-associated vasculitis has been associated with many drugs and it is a relatively rare side effect of the antihypertensive drug hydralazine. The diagnosis and management of patients who have anti-neutrophil cytoplasmic antibody-associated vasculitis may be challenging because of its relative infrequency, variability of clinical expression and changing nomenclature. The spectrum of anti-neutrophil cytoplasmic antibody-associated vasculitis is wide and can be fatal. This case documents a 62-year-old woman who presented with hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis with a puzzling cutaneous rash. Case presentation We report a rare case of hydralazine-induced anti-neutrophil cytoplasmic antibody-associated vasculitis in a 62-year-old Caucasian woman who presented with a vasculitic syndrome with a sore throat, mouth ulcers and otalgia after several months of constitutional symptoms. She then proceeded to develop a rash over her right lower limb. Clinically, the rash had features to suggest Sweet’s syndrome, but also had some appearances consistent with embolic phenomena and did not have the appearance of palpable purpure usually associated with cutaneous vasculitis. Differential diagnoses were hydralazine-associated Sweet’s syndrome, streptococcal-induced cutaneous eruption or an unrelated contact dermatitis. A midstream urine sample detected glomerular blood cells in the setting of anti-neutrophil cytoplasmic antibody-positive renal vasculitis and Streptococcus pyogenes bacteremia. A renal biopsy revealed a pauci-immune, focally necrotizing glomerulonephritis with small crescents. Her skin biopsy revealed a heavy neutrophil infiltrate involving the full thickness of the dermis with no evidence of a leucocytoclastic vasculitis, but was non-specific. She was initially commenced on intravenous lincomycin for her bloodstream infection and subsequently

  4. Hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis presenting with a vasculitic syndrome, acute nephritis and a puzzling skin rash: a case report.

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    Keasberry, Justin; Frazier, Jeremy; Isbel, Nicole M; Van Eps, Carolyn L; Oliver, Kimberley; Mudge, David W

    2013-01-14

    Anti-neutrophil cytoplasmic antibody-associated vasculitis has been associated with many drugs and it is a relatively rare side effect of the antihypertensive drug hydralazine. The diagnosis and management of patients who have anti-neutrophil cytoplasmic antibody-associated vasculitis may be challenging because of its relative infrequency, variability of clinical expression and changing nomenclature. The spectrum of anti-neutrophil cytoplasmic antibody-associated vasculitis is wide and can be fatal. This case documents a 62-year-old woman who presented with hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis with a puzzling cutaneous rash. We report a rare case of hydralazine-induced anti-neutrophil cytoplasmic antibody-associated vasculitis in a 62-year-old Caucasian woman who presented with a vasculitic syndrome with a sore throat, mouth ulcers and otalgia after several months of constitutional symptoms. She then proceeded to develop a rash over her right lower limb. Clinically, the rash had features to suggest Sweet's syndrome, but also had some appearances consistent with embolic phenomena and did not have the appearance of palpable purpure usually associated with cutaneous vasculitis. Differential diagnoses were hydralazine-associated Sweet's syndrome, streptococcal-induced cutaneous eruption or an unrelated contact dermatitis. A midstream urine sample detected glomerular blood cells in the setting of anti-neutrophil cytoplasmic antibody-positive renal vasculitis and Streptococcus pyogenes bacteremia. A renal biopsy revealed a pauci-immune, focally necrotizing glomerulonephritis with small crescents. Her skin biopsy revealed a heavy neutrophil infiltrate involving the full thickness of the dermis with no evidence of a leucocytoclastic vasculitis, but was non-specific. She was initially commenced on intravenous lincomycin for her bloodstream infection and subsequently commenced on immunosuppression after cessation of hydralazine

  5. CanVasc Recommendations for the Management of Antineutrophil Cytoplasm Antibody (ANCA-Associated Vasculitides – Executive Summary

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    Lucy McGeoch

    2015-11-01

    Full Text Available The Canadian Vasculitis research network (CanVasc is composed of physicians from different medical specialties, including rheumatology and nephrology and researchers with expertise in vasculitis. One of its aims was to develop recommendations for the diagnosis and management of antineutrophil cytoplasm antibody (ANCA-associated vasculitides in Canada. This executive summary features the 19 recommendations and 17 statements addressing general AAV diagnosis and management, developed by CanVasc group based on a synthesis of existing international guidelines, other published supporting evidence and expert consensus considering the Canadian healthcare context.

  6. [Anti-neutrophil cytoplasmic antibodies (ANCA) in patients with symptomatic and asymptomatic HIV infection].

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    Habegger de Sorrentino, A; Motta, P; Iliovich, E; Sorrentino, A P

    1997-01-01

    The cytopathic effect of HIV on CD4 T cells, as well as the active autoimmune mechanism occurring during infection, have been documented. Of the cytokines involved in the pathogenesis of AIDS, the main one produced by the monocyte-macrophage series is tumor necrosis factor alfa (TNF alpha). This cytokine induces antigens such as proteinase 3 (Pr 3) or mieloperoxidase (MPO). Anti-neutrophil cytoplasmic antibodies (ANCA) are directed against this type of PMN antigens. In the present paper, the role of anti-neutrophil cytoplasmic antibodies (ANCA) in HIV infected patients as responsible for autoimmune phenomena in relation to opportunistic infections, was studied. A total of 88 serum samples belonging to 49 asymptomatic and 39 symptomatic HIV infected patients were tested for ANCA by an indirect immunofluorescence (IIF) test over a neutrophil substrate. ANCA were detected in 53.8% of symptomatic patients as compared to 4.1% in asymptomatic cases (p tuberculosis is a frequent finding in HIV infected patients from Northeastern Argentina. When the presence of ANCA in TB(+) HIV(+) and TB(+) HIV(-) patients was studied, it was seen that positive-ANCA significantly correlated with the first group (p pulmonar TB, could indicate that the virus may not be responsible for the induction of these antibodies.

  7. Stress and Disease Onset in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

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    Christina V. Golemati

    2017-12-01

    Full Text Available ObjectiveTo explore the potential contribution of stress as a trigger for disease onset in patients with antineutrophil cytoplasmic antibody (ANCA associated vasculitis (AAV.Methods53 AAV and 85 rheumatoid arthritis (RA patients as well as 53 healthy controls (HC were thoroughly asked for the number and impact of stressful life events, coping strategies, and available social support 12 months prior to disease onset. Anxiety, depression, personality dimensions, insomnia, and fatigue were also determined.ResultsAAV patients reported higher scoring of the impact of stressful life events compared to the RA and HC group prior to disease onset (2.8 ± 3.1 vs 1.8 ± 2.1 vs 1.7 ± 2.3, p-values: 0.047 and 0.053, respectively. While the number of reported stressful events was found to be significantly higher in AAV vs RA patients but not HC, certain coping strategies and social support features were more commonly implemented by AAV patients compared to HC, but not RA patients. As far as personality and other psychosocial characteristics, AAV patients displayed significantly higher psychoticism traits compared to RA, with no other differences being detected between AAV patients and both RA and HC. After adjusting for potential cofounders, scoring of the impact of stressful life events >3 was independently associated with AAV development compared to both RA and HC [ORs (95% CI: 4.6 (1.6–13.4 and 4.4 (1.0–19.0, respectively].ConclusionThe perceived impact of stressful life events prior to disease onset emerged as a contributing factor for AAV development.

  8. [Maintainance treatment of anti-neutrophil cytoplasm associated antibodies (ANCA)].

    Science.gov (United States)

    Dábague Guzmán, Janet; Pedroza Granados, Jorge; Zúñiga Varga, Javier

    2011-12-01

    ANCA-associated vasculitides are rare and complex systemic diseases. Collaborative studies in both Europe and the United States of America have been particularly important in the development of randomized clinical trials that have studied the safety of maintenance therapy for these diseases. Although cyclophosphamide continues to be the main drug utilized during induction therapy, its long-term side effects have given rise to the study of other immunosuppressive drugs for the maintenance phase. We herein review these studies with particular attention to combination therapy and the duration of treatment. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  9. 18F-FDG PET/CT findings preceded elevation of serum proteinase 3 antineutrophil cytoplasmic antibodies in Wegener granulomatosis.

    Science.gov (United States)

    Ito, Kimiteru; Minamimoto, Ryogo; Yamashita, Hiroyuki; Morooka, Miyako; Okasaki, Momoko; Mimori, Akio; Kubota, Kazuo

    2014-01-01

    A 67-year-old woman underwent F-FDG PET/CT after developing a fever of unknown origin. PET/CT revealed intensive FDG uptake at the nasal and lung lesions. On the laboratory data, serum myeloperoxidase antineutrophil cytoplasmic antibodies (ANCA) titer was elevated, although serum directed against proteinase 3 (PR3) ANCA titer was within normal limits. One month after treatment, follow-up PET/CT revealed decreased FDG uptake at the lesions. One year later, serum PR3-ANCA titer elevated, which finally led to a diagnosis of Wegener granulomatosis (WG). WG lesions may be detected earlier by FDG PET/CT than by serum PR3-ANCA titers.

  10. Early Outcomes in Children With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    DEFF Research Database (Denmark)

    Morishita, Kimberly A; Moorthy, Lakshmi N; Lubieniecka, Joanna M

    2017-01-01

    OBJECTIVE: To characterize the early disease course in childhood-onset antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and the 12-month outcomes in children with AAV. METHODS: Eligible subjects were children entered into the Pediatric Vasculitis Initiative study who were...... (Pediatric Vasculitis Activity Score [PVAS] of 0) at 12 months with a corticosteroid dosage of ..., presence of damage at 12 months (measured by a modified Pediatric Vasculitis Damage Index [PVDI]; score 0 = no damage, score 1 = one damage item present), and relapse rates at 12 months. RESULTS: In total, 105 children with AAV were included in the study. The median age at diagnosis was 13.8 years...

  11. Anti-Neutrophil Cytoplasmic Antibody-associated Vasculitis (AAV) Restricted to the Limbs: A Case Report.

    Science.gov (United States)

    Ojima, Yoshie; Sawada, Kinya; Fujii, Hiroshi; Shirai, Tsuyoshi; Saito, Ayako; Kagaya, Saeko; Aoki, Satoshi; Takeuchi, Yoichi; Ishii, Tomonori; Nagasawa, Tasuku

    2017-12-27

    A previously healthy 58-year-old man was admitted for muscle pain and weakness (manual muscle testing [MMT] of 4/4 for upper and lower limbs). We detected elevated levels of inflammatory makers and PR3-anti-neutrophil cytoplasmic antibody (ANCA). Subsequently, the muscle weakness rapidly progressed to an MMT of 2 for all limbs. Magnetic resonance imaging indicated muscle edema, and the CK level increased to 29,998 U/L. mPSL and cyclophosphamide pulse therapy improved the patient symptoms. MMT recovered to 4 for all limbs. A muscle biopsy showed degenerated muscle fibers surrounded by neutrophil-predominant infiltration. In addition, lamina elastic breakdown and fibrinoid necrosis of arterioles were observed. A final diagnosis of microscopic polyangiitis (MPA) limited to the muscles was made.

  12. The Pharmacogenomic Association of Fc gamma Receptors and Cytochrome P450 Enzymes With Response to Rituximab or Cyclophosphamide Treatment in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    NARCIS (Netherlands)

    Cartin-Ceba, Rodrigo; Indrakanti, Divya; Specks, Ulrich; Stone, John H.; Hoffman, Gary S.; Kallenberg, Cees G. M.; Langford, Carol A.; Merkel, Peter A.; Spiera, Robert F.; Monach, Paul A.; St. Clair, E. William; Seo, Philip; Tchao, Nadia K.; Ytterberg, Steven R.; Brunetta, Paul G.; Song, Huijuan; Birmingham, Dan; Rovin, Brad H.; Grp, RAVE-Immune Tolerance Network Res

    Objective The Rituximab in ANCA-Associated Vasculitis (RAVE) trial compared rituximab to cyclophosphamide as induction therapy for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. We undertook the current study to determine whether known single-nucleotide

  13. Antibodies against linear epitopes on Goodpasture autoantigen in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.

    Science.gov (United States)

    Jia, Xiao-Yu; Yu, Jun-Tao; Hu, Shui-Yi; Li, Jian-Nan; Wang, Miao; Wang, Chen; Chen, Min; Cui, Zhao; Zhao, Ming-Hui

    2017-09-01

    In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr ≤ 133 μmol/L), 70% of patients with moderate renal dysfunction (133 μmol/L  600 μmol/L) (P = 0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of E A (P4) and E B (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r = 0.764, P = 0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P = 0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.

  14. Pediatric Inflammatory Bowel Disease with Cytoplasmic Staining of Antineutrophil Cytoplasmic Antibodies

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    Omar I. Saadah

    2013-01-01

    Full Text Available Background. It is unusual for the antineutrophil cytoplasmic antibody with cytoplasmic pattern (cANCA to present in patients with inflammatory bowel disease (IBD without vasculitis. The purpose of this study was to describe the occurrence and characteristics of pediatrics IBD with cANCA. Methods. A retrospective review of pediatric IBD associated with cANCA serology in patients from King Abdulaziz University Hospital, Saudi Arabia, between September 2002 and February 2012. Results. Out of 131 patients with IBD screened for cANCAs, cANCA was positive in 7 (5.3% patients of whom 4 had ulcerative colitis and 3 had Crohn's disease. The median age was 8.8 years (2–14.8 years. Six (86% were males. Of the 7 patients, 5 (71% were Saudi Arabians and 2 were of Indian ethnicity. The most common symptoms were diarrhea, abdominal pain, weight loss, and rectal bleeding. None had family history or clinical features suggestive of vasculitis involving renal and respiratory systems. No difference in the disease location or severity was observed between cANCA positive and cANCA negative patients apart from male preponderance in cANCA positive patients. Conclusion. The occurrence of cANCA in pediatric IBD is rare. Apart from male preponderance, there were no peculiar characteristics for the cANCA positive patients.

  15. C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody.

    Science.gov (United States)

    Xu, Peng-cheng; Hao, Jian; Yang, Xiao-wei; Chang, Dong-yuan; Chen, Min; Zhao, Ming-hui

    2012-10-01

    Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-α in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45 ± 16.87 vs. 58.65 ± 15.09, P Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Physical Fatigue, Fitness, and Muscle Function in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

    Science.gov (United States)

    McClean, Andrew; Morgan, Matthew D; Basu, Neil; Bosch, Jos A; Nightingale, Peter; Jones, David; Harper, Lorraine

    2016-09-01

    This study investigated differences in cardiorespiratory fitness, muscular function, perceived exertion, and anxiety/depression between patients and healthy controls (HCs) and assessed which of these variables may account for the fatigue experienced by patients. Fatigue was measured in 48 antineutrophil cytoplasmic antibody-associated vasculitis patients and 41 healthy controls using the Multidimensional Fatigue Inventory (MFI-20), focusing on the physical component. Quality of life, anxiety/depression, and sleep quality were assessed by validated questionnaires. Muscle mass was measured by dual-energy x-ray absorptiometry scan, strength as the maximal voluntary contraction (MVC) force, and endurance as sustained isometric contraction at 50% MVC of the quadriceps. Voluntary activation was assessed by superimposed electrical stimulation. Cardiorespiratory fitness ( ˙Vo2 max and oxygen pulse [O2 pulse]) and perceived exertion (Borg scale) were measured during progressive submaximal exercise. Patients reported elevated physical fatigue scores compared to HCs (patients MFI-20 physical 13 [interquartile range (IQR) 8-16], HCs MFI-20 physical 5.5 [IQR 4-8]; P Muscle mass was the same in both groups, but MVC and time to failure in the endurance test were lower due to reduced voluntary activation in patients. Estimated ˙Vo2 max and O2 pulse were the same in both groups. For the same relative workload, patients reported higher ratings of perceived exertion, which correlated with reports of MFI-20 physical fatigue (R(2)  = 0.2). Depression (R(2)  = 0.6), anxiety (R(2)  = 0.3), and sleep disturbance (R(2)  = 0.3) were all correlated with MFI-20 physical fatigue. These observations suggest that fatigue in patients is of a central rather than peripheral origin, supported by associations of fatigue with heightened perception of exertion, depression, anxiety, and sleep disturbance but normal muscle and cardiorespiratory function. © 2016, American College

  17. Poor renal outcome of antineutrophil cytoplasmic antibody negative Pauci-immune glomerulonephritis in Taiwanese.

    Science.gov (United States)

    Hung, Peir-Haur; Chiu, Yen-Ling; Lin, Wei-Chou; Chiang, Wen-Chih; Chen, Yung-Ming; Lin, Shuei-Liong; Wu, Kwan-Dun; Tsai, Tun-Jun

    2006-10-01

    Pauci-immune glomerulonephritis (GN) is an important cause of crescentic GN, acute renal failure and mortality. However, data are limited on the clinical presentation and outcome in antineutrophil cytoplasmic antibody (ANCA) negative patients, especially in Asians. This retrospective study analyzed medical records and pathology slides of patients who received renal biopsy between February 1998 and October 2004. Enzyme-linked immunosorbent assay was used routinely for ANCA testing in all patients. Among 637 patients with biopsy-proven GN included in this study, 88 (13.8%) had glomerular crescent formation. Among them, pauci-immune crescentic glomerulonephritis (PICGN) (42 patients, 47.2%) and lupus nephritis (25 patients, 28.4%) were the most common pathologic diagnoses. Lupus patients were younger (p = 0.028), while PICGN patients had more chronic lesions (p < 0.001), extensive glomerular crescents (p < 0.001), less severe proteinuria (p < 0.001) and poorer renal survival (p = 0.0017). Among the PICGN patients, 62.5% had a positive ANCA test, 80% had myeloperoxidase-ANCA and 20% had proteinase-3-ANCA. Subgroup analysis showed that ANCA negativity was associated with less crescent formation (p < 0.001) but more chronic glomerular lesions (p < 0.001) and a trend toward worse renal outcome (p = 0.055). This study illustrates the necessity for pathologic diagnosis of pauci-immune GN despite ANCA negativity. The poor prognosis associated with ANCA negativity in this study may be partly due to delayed diagnosis since these patients frequently lacked systemic involvement.

  18. Marfan syndrome with antineutrophil cytoplasmic antibody-associated systemic vasculitis presenting as severe anaemia and haematuria after the Bentall procedure.

    Science.gov (United States)

    Sijia, Li; Shuangxin, Liu; Wei, Shi; Yanhai, Cui

    2013-08-01

    One month previously, a 28-year old male underwent an emergency modified Bentall procedure because of Marfan syndrome with acute aortic dissection Stanford Class A. Computed tomography of the chest did not reveal severe graft stenosis of the anastomosis. To explore the cause of anaemia, renal dysfunction and macroscopic haematuria, the patient was tested for antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (AASV). Antimyeloperoxidase antibodies (MPO)-ANCA and antiproteinase 3 antibodies (PR3)-ANCA were strongly positive. Corticosteroid therapy was applied, followed by cyclophosphamide and azathioprine. In response to treatment, the MPO-ANCA and PR3-ANCA levels gradually decreased, proteinuria was alleviated and haemoglobin levels returned to normal after 6 months. This is the first report to highlight haemolytic anaemia and AASV with Marfan syndrome after surgery for aortic dissection.

  19. Antiproteinase 3 antineutrophil cytoplasmic antibodies and disease activity in Wegener granulomatosis.

    Science.gov (United States)

    Finkielman, Javier D; Merkel, Peter A; Schroeder, Darrell; Hoffman, Gary S; Spiera, Robert; St Clair, E William; Davis, John C; McCune, W Joseph; Lears, Andrea K; Ytterberg, Steven R; Hummel, Amber M; Viss, Margaret A; Peikert, Tobias; Stone, John H; Specks, Ulrich

    2007-11-06

    The utility of antineutrophil cytoplasmic antibody (ANCA) levels to guide the management of patients with Wegener granulomatosis remains controversial. To determine whether pro-proteinase 3 (PR3)-ANCA levels are a better measure of disease activity than mature-PR3-ANCA levels, whether decreases in either level are associated with shorter time to remission, and whether increases are followed by relapse. Prospective, observational cohort study. 8 United States medical centers that participated in a treatment trial for Wegener granulomatosis. 156 patients with Wegener granulomatosis enrolled during periods of active disease. PR3-ANCA levels (by capture enzyme-linked immunosorbent assay) and disease activity (by the Birmingham Vasculitis Activity Score for Wegener granulomatosis). The ANCA levels were only weakly associated with disease activity across patients. The longitudinal association within patients was stronger, but changes in ANCA levels explained less than 10% of the variation in disease activity. Decreases in mature- and pro-PR3-ANCA levels were not statistically significantly associated with shorter time to remission, and increases in mature-PR3-ANCA levels (adjusted hazard ratio, 0.8 [95% CI, 0.4 to 1.9]; P = 0.67) and pro-PR3-ANCA levels (adjusted hazard ratio, 1.0 [CI, 0.5 to 2.1]; P = 0.99) were not associated with relapse. The proportion of patients who had relapse within 1 year of an increase in PR3-ANCA levels was 40% for mature-PR3 (CI, 18% to 56%) and 43% for pro-PR3 (CI, 22% to 58%). Samples were collected approximately every 3 months. Sensitivity and specificity of ANCA levels for detecting remission and relapse could not be calculated because each patient had different follow-up times. Pro-PR3-ANCA is no better than mature-PR3-ANCA as a measure of Wegener granulomatosis activity. Decreases in PR3-ANCA levels are not associated with shorter time to remission, and increases are not associated with relapse. These findings suggest that ANCA levels

  20. Allosteric modulation of proteinase 3 activity by anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis.

    Science.gov (United States)

    Hinkofer, Lisa C; Hummel, Amber M; Stone, John H; Hoffman, Gary S; Merkel, Peter A; Spiera, E Robert F; St Clair, William; McCune, Joseph W; Davis, John C; Specks, Ulrich; Jenne, Dieter E

    2015-05-01

    Anti-neutrophil cytoplasmic antibodies (ANCA) with proteinase 3 (PR3) specificity are a useful laboratory biomarker for the diagnosis of Granulomatosis with Polyangiitis (GPA) and are believed to be implicated in the pathogenesis. It has been repeatedly suggested that disease activity of GPA is more closely related to the appearance and rise of PR3-inhibiting ANCA than to an increase of total ANCA. Previous studies on a limited number of patient samples, however, have yielded inconclusive results. To overcome the previous methodological limitations, we established a new ultrasensitive method to quantify the inhibitory capacity of PR3-ANCA using small volumes of plasma from patients with GPA. A large collection of longitudinally-collected samples from the Wegener Granulomatosis Etanercept Trial (WGET) became available to us to determine the functional effects of ANCA on PR3 in comparison to clinical disease manifestations. In these patient samples we not only detected PR3-ANCA with inhibitory capacity, but also PR3-ANCA with enhancing effects on PR3 activity. However no correlation of these activity-modulating PR3-ANCA with disease activity at either the time of enrollment or over the course of disease was found. Only patients with pulmonary involvement, especially patients with nodule formation in the respiratory tract, showed a slight, but not significant, decrease of inhibitory capacity. Epitope mapping of the activity-modulating PR3-ANCA revealed a binding on the active site surface of PR3. Yet these ANCA were able to bind to PR3 with an occupied active site cleft, indicating an allosteric mechanism of inhibition. The recently described signal ratio between the MCPR3-3 and MCPR3-2 capture ELISA was consistent with the binding of activity-modulating ANCA to the active site surface. Evidence for a shared epitope between activity-modulating PR3-ANCA and MCPR3-7, however, was very limited, suggesting that a majority of PR3-ANCA species do not inhibit PR3 by the same

  1. Spotlight on rituximab in the treatment of antineutrophil cytoplasmic antibody-associated vasculitis: current perspectives

    Directory of Open Access Journals (Sweden)

    Moog P

    2015-11-01

    Full Text Available Philipp Moog, Klaus Thuermel Abteilung für Nephrologie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Abstract: A 54-year-old patient presented to his general practitioner because of strong muscle pain in both thighs. Inflammatory parameters (CRP 16.3 mg/dL and white blood cells (15 g/L were elevated. The patient reported a weight loss of 10 kg in 4 weeks. There was no fever or any other specific symptoms. Urine dipstick examination and computed tomography of the chest were unremarkable. Because of increasing symptoms, the patient was referred to our department. Magnetic resonance tomography showed diffuse inflammatory changes of the muscles of both thighs. Neurological examination and electrophysiology revealed axonal sensorimotor neuropathy and ground-glass opacities of both lungs had occurred. Serum creatinine increased to 229 µmol/L within a few days, with proteinuria of 3.3 g/g creatinine. Kidney biopsy showed diffuse pauci-immune proliferative glomerulonephritis. Proteinase 3-specific antineutrophil cytoplasmic antibodies were markedly increased. Birmingham Vasculitis Activity Score was 35. Within 2 days, serum creatinine further increased to 495 µmol/L. Plasma exchange, high-dose glucocorticosteroids, and hemodialysis were started. The patient received cyclophosphamide 1 g twice and rituximab 375 mg/m2 four times according to the RITUXVAS protocol. Despite ongoing therapy, hemodialysis could not be withdrawn and had to be continued over 3 weeks until diuresis normalized. Glucocorticosteroids were tapered to 20 mg after 2 months, and serum creatinine was 133 µmol/L. However, nephritic urinary sediment reappeared. Another dose of 1 g cyclophosphamide was given, and glucocorticosteroids were raised for another 4 weeks. After 6 months, the daily prednisolone dose was able to be tapered to 5 mg. Serum creatinine was 124 µmol/L, proteinuria further decreased to 382 mg/g creatinine, and the Birmingham

  2. Risk Factors for Relapse of Antineutrophil Cytoplasmic Antibody-associated Vasculitis in Japan: A Nationwide, Prospective Cohort Study.

    Science.gov (United States)

    Hara, Akinori; Wada, Takashi; Sada, Ken-Ei; Amano, Koichi; Dobashi, Hiroaki; Harigai, Masayoshi; Takasaki, Yoshinari; Yamada, Hidehiro; Hasegawa, Hitoshi; Hayashi, Taichi; Fujimoto, Shouichi; Muso, Eri; Kawakami, Tamihiro; Homma, Sakae; Yoshida, Masaharu; Hirahashi, Junichi; Ogawa, Noriyoshi; Ito, Satoshi; Makino, Hirofumi; Arimura, Yoshihiro

    2018-02-01

    The aim was to elucidate the prognosis and risk factors associated with relapse during longterm remission maintenance therapy for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Patients with newly diagnosed AAV (n = 156) were registered in the Remission Induction Therapy in Japanese patients with ANCA-associated Vasculitides (RemIT-JAV) study, and among them, 83 patients who achieved remission were enrolled and followed up for 24 additional months in our nationwide, prospective cohort study (Co-RemIT-JAV; registration number UMIN 000006373). Patterns of maintenance therapy, effectiveness, and safety were evaluated from months 25 to 48 after the RemIT-JAV. The primary outcome measure was the rate of relapse. Secondary outcome measures included overall and renal survival, risk factors associated with relapse, and incidence rates of serious infections. The patients comprised 35 men and 48 women aged 65.3 ± 12.6 years. Between months 25 and 48, the survival rate was 95% (79/83). Causes of death included 1 thyroid cancer, 1 infection, and 2 unknown reasons. Four patients had developed endstage renal disease (ESRD) by Month 24; 1 developed ESRD beyond Month 25. The relapse rate was 24% (20/83) from months 25 to 48. Multivariable analysis revealed that oral prednisolone ≤ 2.5 mg/day at Month 24 was a significant risk factor for relapse between months 25 and 48 (HR = 3.1, 95% CI 1.1-8.5). One-quarter of patients with AAV relapsed during maintenance therapy, and relapse was associated with the dose of oral prednisolone 24 months after the initiation of remission induction therapy in Japan.

  3. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies for inflammatory bowel disease : High prevalence in patients with celiac disease

    NARCIS (Netherlands)

    Damoiseaux, JGMC; Bouten, B; Linders, AMLW; Austen, J; Roozendaal, C; Russel, MGVM; Forget, PP; Tervaert, JWC

    Both celiac disease and inflammatory bowel disease (IBD) are characterized by chronic diarrhea and the presence of distinct (auto)antibodies. In the present study we wanted to determine the prevalence of serological markers for inflammatory bowel disease, i.e., perinuclear antineutrophil cytoplasmic

  4. Perinuclear Antineutrophil Cytoplasmic Autoantibodies and Anti-Saccharomyces Cerevisiae Antibodies as Serological Markers Are Not Specific in the Identification of Crohn’s Disease and Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    Vishal Anand

    2008-01-01

    Full Text Available OBJECTIVE: To evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic autoantibodies (pANCAs and anti-Saccharomyces cerevisiae antibodies (ASCAs, as single agents and in combination, for the diagnosis of Crohn’s disease (CD and ulcerative colitis (UC, including in cases of indeterminate colitis (IC.

  5. Myeloperoxidase-Antineutrophil Cytoplasmic Antibody (ANCA)-Positive and ANCA-Negative Patients With Granulomatosis With Polyangiitis (Wegener's) Distinct Patient Subsets

    NARCIS (Netherlands)

    Miloslavsky, Eli M.; Lu, Na; Unizony, Sebastian; Choi, Hyon K.; Merkel, Peter A.; Seo, Philip; Spiera, Robert; Langford, Carol A.; Hoffman, Gary S.; Kallenberg, Cees G. M.; St Clair, E. William; Tchao, Nadia K.; Fervenza, Fernando; Monach, Paul A.; Specks, Ulrich; Stone, John H.

    2016-01-01

    Objective. To examine the relationship of antineutrophil cytoplasmic antibody (ANCA) type and ANCA-associated vasculitis (AAV) diagnosis with demographic features, disease manifestations, and clinical outcomes. We focused on patients who account for the differences between ANCA type and disease type

  6. Prevalence of reduced bone mineral density in patients with antineutrophil cytoplasmic antibody associated vasculitis and the role of immunosuppressive therapy : A cross-sectional study

    NARCIS (Netherlands)

    Boomsma, MM; Stegeman, CA; Kramer, AB; Karsijns, M; Piers, DA; Tervaert, JWC

    2002-01-01

    Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a relapsing-remitting disease, which is treated with corticosteroids (CS) in combination with cyclophosphamide. One of the major side-effects of this treatment is osteoporosis, which may result in the increased occurrence of

  7. Rapidly progressive antineutrophil cytoplasm antibodies associated with pulmonary-renal syndrome in a 10-year-old girl

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    Fermin Blanco Filho

    2001-01-01

    Full Text Available CONTEXT: The term pulmonary-renal syndrome has been used frequently to describe the clinical manifestations of a great number of diseases in which pulmonary hemorrhage and glomerulonephritis coexist. The classic example of this type of vasculitis is Goodpasture´s syndrome, a term used to describe the association of pulmonary hemorrhage, glomerulonephritis and the presence of circulating antiglomerular basement membrane antibodies (anti-GBM. Among the several types of systemic vasculitides that can present clinical manifestations of the pulmonary-renal syndrome, we focus the discussion on two types more frequently associated with antineutrophil cytoplasm antibodies (ANCA, microscopic polyangiitis and Wegener´s granulomatosis, concerning a 10 year old girl with clinical signs and symptoms of pulmonary-renal syndrome, with positive ANCA and rapidly progressive evolution. CASE REPORT: We describe the case of a 10-year-old girl referred to our hospital for evaluation of profound anemia detected in a primary health center. Five days before entry she had experienced malaise, pallor and began to cough up blood-tinged sputum that was at first attributed to dental bleeding. She was admitted to the infirmary with hemoglobin = 4 mg/dL, hematocrit = 14%, platelets = 260,000, white blood cells = 8300, 74% segmented, 4% eosinophils, 19% lymphocytes and 3% monocytes. Radiographs of the chest revealed bilateral diffuse interstitial alveolar infiltrates. There was progressive worsening of cough and respiratory distress during the admission day, when she began to cough up large quantities of blood and hematuria was noted. There was rapid and progressive loss of renal function and massive lung hemorrhage. The antineutrophil cytoplasm antibody (ANCA test with antigen specificity for myeloperoxidase (anti-MPO was positive and the circulating anti-GBM showed an indeterminate result.

  8. ELISA test for anti-neutrophil cytoplasm antibodies detection evaluated by a computer screen photo-assisted technique.

    Science.gov (United States)

    Filippini, D; Tejle, K; Lundström, I

    2005-08-15

    The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation.

  9. Circulating microRNA expression pattern separates patients with anti-neutrophil cytoplasmic antibody-associated vasculitis from healthy controls

    DEFF Research Database (Denmark)

    Skoglund, C.; Carlsen, A.; Weiner, M.

    2015-01-01

    patients from healthy subjects as well as from renal transplant recipients. Loadings plots indicated similar contribution of the same miRNAs in both cohorts to the PCA. Renal engagement was important for miRNA expression but consistent correlations between estimated glomerular filtration rate and mi......Objective. Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) has an unpredictable course and better biomarkers are needed. Micro-RNAs in body fluids are protected from degradation and might be used as biomarkers for diagnosis and prognosis, here we explore the potential in AAV...... individual miRNAs were differently expressed compared to controls in both cohorts; miR-29a, -34a, -142-3p and -383 were up-regulated and miR-20a, -92a and -221 were down-regulated. Cluster analysis as well as principal component analysis (PCA) indicated that patterns of miRNA expression differentiate AAV...

  10. Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial

    DEFF Research Database (Denmark)

    de Groot, Kirsten; Harper, Lorraine; Jayne, David R W

    2009-01-01

    BACKGROUND: Current therapies for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis are limited by toxicity. OBJECTIVE: To compare pulse cyclophosphamide with daily oral cyclophosphamide for induction of remission. DESIGN: Randomized, controlled trial. Random assignments were...... generalized ANCA-associated vasculitis with renal involvement but not immediately life-threatening disease. INTERVENTION: Pulse cyclophosphamide, 15 mg/kg every 2 to 3 weeks (76 patients), or daily oral cyclophosphamide, 2 mg/kg per day (73 patients), plus prednisolone. MEASUREMENT: Time to remission (primary.......7%). Thirteen patients in the pulse group and 6 in the daily oral group achieved remission by 9 months and subsequently had relapse. Absolute cumulative cyclophosphamide dose in the daily oral group was greater than that in the pulse group (15.9 g [interquartile range, 11 to 22.5 g] vs. 8.2 g [interquartile...

  11. Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial

    DEFF Research Database (Denmark)

    de Groot, Kirsten; Harper, Lorraine; Jayne, David R W

    2009-01-01

    BACKGROUND: Current therapies for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis are limited by toxicity. OBJECTIVE: To compare pulse cyclophosphamide with daily oral cyclophosphamide for induction of remission. DESIGN: Randomized, controlled trial. Random assignments were...... generalized ANCA-associated vasculitis with renal involvement but not immediately life-threatening disease. INTERVENTION: Pulse cyclophosphamide, 15 mg/kg every 2 to 3 weeks (76 patients), or daily oral cyclophosphamide, 2 mg/kg per day (73 patients), plus prednisolone. MEASUREMENT: Time to remission (primary...... regimen induced remission of ANCA-associated vasculitis as well as the daily oral regimen at a reduced cumulative cyclophosphamide dose and caused fewer cases of leukopenia. PRIMARY FUNDING SOURCE: The European Union....

  12. Anti-neutrophil cytoplasmic antibody negative crescentic paucimmune glomerulonephritis in a case of scleroderma with systemic lupus erythematosus overlap

    Directory of Open Access Journals (Sweden)

    Rohit Tewari

    2016-01-01

    Full Text Available Renal Involvement in scleroderma is a known problem and the manifestations are well described. Renal involvement in systemic lupus erythematosus (SLE is also well known. However, in scleroderma and SLE overlap syndrome, the renal findings may vary being a combination of features of immune complex mediated glomerulonephritis as well as thrombotic microangiopathy. We report a case in which the renal manifestation in such a situation was of a focal necrotising pauci-immune glomerulonephritis with crescents, anti-neutrophil cytoplasmic antibody negative. To the best of our knowledge, such manifestations have not been described before. Renal dysfunction in a normotensive setting in such a case should direct one towards evaluation for other causes and should prompt a kidney biopsy. This would be valuable in delineating the pathological process in the kidney and would help in guiding therapy.

  13. Systemic Lupus Erythematosus and Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Overlap Syndrome in Patients With Biopsy-Proven Glomerulonephritis

    Science.gov (United States)

    Jarrot, Pierre-Andre; Chiche, Laurent; Hervier, Baptiste; Daniel, Laurent; Vuiblet, Vincent; Bardin, Nathalie; Bertin, Daniel; Terrier, Benjamin; Amoura, Zahir; Andrés, Emmanuel; Rondeau, Eric; Hamidou, Mohamed; Pennaforte, Jean-Loup; Halfon, Philippe; Daugas, Eric; Dussol, Bertrand; Puéchal, Xavier; Kaplanski, Gilles; Jourde-Chiche, Noemie

    2016-01-01

    Abstract The aim of the study was to report the clinical, biological, and pathological characteristics of patients with glomerulonephritis (GN) secondary to systemic lupus erythematosus (SLE)/antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) overlap syndrome. A nationwide survey was conducted to identify cases of SLE/AAV overlap syndrome. Data were collected from SLE and AAV French research groups. Inclusion criteria were diagnosis of both SLE and AAV according to international classification criteria and biopsy-proven GN between 1995 and 2014. Additional cases were identified through a systematic literature review. A cohort of consecutive biopsy-proven GN was used to study the prevalence of overlapping antibodies and/or overlap syndrome. The national survey identified 8 cases of SLE/AAV overlap syndrome. All patients were female; median age was 40 years. AAV occurred before SLE (n = 3), after (n = 3), or concomitantly (n = 2). Six patients had rapidly progressive GN and 3/8 had alveolar hemorrhage. All patients had antinuclear antibodies (ANA); 7/8 had p-ANCA antimyeloperoxidase (MPO) antibodies. Renal biopsies showed lupus nephritis (LN) or pauci-immune GN. Remission was obtained in 4/8 patients. A literature review identified 31 additional cases with a similarly severe presentation. In the GN cohort, ANCA positivity was found in 30% of LN, ANA positivity in 52% of pauci-immune GN, with no correlation with pathological findings. The estimated prevalence for SLE/AAV overlap syndrome was 2/101 (2%). In patients with GN, SLE/AAV overlap syndrome may occur but with a low prevalence. Most patients have an aggressive renal presentation, with usually both ANA and anti-MPO antibodies. Further studies are needed to assess shared pathogenesis and therapeutic options. PMID:27258503

  14. Successful treatment of Bordetella bronchiseptica pneumonia by minocycline in anti-neutrophil cytoplasmic antibodies-associated vasculitis patient.

    Science.gov (United States)

    Ito, Yuji; Uemura, Keiichi

    2016-12-01

    Bordetella bronchiseptica is a bacterial pathogen usually isolated from animals and rarely causes human infections. There are, however, some reports that B. bronchiseptica causes human respiratory infections in immunocompromised patients or those with underlying respiratory diseases, although there is a lack of treatment guidelines. An 80-year-old woman was admitted to our hospital to treat anti-neutrophil cytoplasmic antibodies-associated vasculitis. On the 16th day after admission, she complained of a productive cough with right pleuritic pain and had low-grade fever. After chest CT scans, we diagnosed pneumonia. Gram stain of her sputum revealed moderate levels of gram-negative coccobacilli, which was later identified as B. bronchiseptica by mass spectrometry. According to the result of minimum inhibitory concentration, we successfully treated the pneumonia with minocycline. This case suggests that B. bronchiseptica pneumonia can be treated by minocycline if the minimum inhibitory concentration is less than 0.25 μg/mL. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  15. Long-term Prognosis of Anti-Neutrophil Cytoplasmic Antibody-Negative Renal Vasculitis: Cohort Study in Korea.

    Science.gov (United States)

    Lee, Sung Woo; Yu, Mi-Yeon; Baek, Seon Ha; Ahn, Shin-Young; Kim, Sejoong; Na, Ki Young; Chae, Dong-Wan; Chin, Ho Jun

    2016-04-01

    Few studies have reported on the long-term prognosis of anti-neutrophil cytoplasmic antibody (ANCA)-negative renal vasculitis. Between April 2003 and December 2013, 48 patients were diagnosed with renal vasculitis. Their ANCA status was tested using indirect immunofluorescence and enzyme-linked immunosorbent assays. During a median (interquartile range) follow-up duration of 933.5 (257.5-2,079.0) days, 41.7% of patients progressed to end stage renal disease (ESRD) and 43.8% died from any cause. Of 48 patients, 6 and 42 were ANCA-negative and positive, respectively. The rate of ESRD within 3 months was higher in ANCA-negative patients than in ANCA-positive patients (P = 0.038). In Kaplan-Meier survival analysis, ANCA-negative patients showed shorter renal survival than did ANCA-positive patients (log-rank P = 0.033). In univariate Cox-proportional hazard regression analysis, ANCA-negative patients showed increased risk of ESRD, with a hazard ratio 3.190 (95% confidence interval, 1.028-9.895, P = 0.045). However, the effect of ANCA status on renal survival was not statistically significant in multivariate analysis. Finally, ANCA status did not significantly affect patient survival. In conclusion, long-term patient and renal survival of ANCA-negative renal vasculitis patients did not differ from those of ANCA-positive renal vasculitis patients. Therefore, different treatment strategy depending on ANCA status might be unnecessary.

  16. A case of myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated hypertrophic pachymeningitis presenting with multiple cranial nerve palsies and diabetes insipidus.

    Science.gov (United States)

    Yasuda, Ken; Sainouchi, Makoto; Goto, Masahiro; Murase, Nagako; Ohtani, Ryo; Nakamura, Michikazu

    2016-05-31

    A 61-year-old woman developed hearing difficulties and became thirsty after experiencing cold symptoms. A neurological examination revealed a loss of odor sensation, facial palsy, dysphasia, and dysarthria. Vocal cord palsy was observed during pharyngoscopy. Brain magnetic resonance imaging (MRI) showed a thickened pituitary stalk and swelling of the pituitary gland, but no high signal intensity regions were seen in the posterior portion of the pituitary gland. Gadolinium-enhanced MRI demonstrated a thickened dura mater over the anterior cranial fossa. A biopsy specimen of the thickened dura mater showed fibrosis, granulomatous inflammation, and necrotic foci. Blood tests detected myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA). The patient's urine osmolarity was low even though she exhibited hypernatremia. We diagnosed her with hypertrophic pachymeningitis associated with MPO-ANCA and diabetes insipidus. The patient received two courses of 5-day high-dose intravenous methylprednisolone (1.0 g/day), and was subsequently administered oral prednisolone, which gradually relieved her symptoms. However, the patient's symptoms recurred despite the high-dose prednisolone treatment. It was difficult to control the patient's symptoms in this case with oral prednisolone monotherapy, but combined treatment with cyclosporine resulted in sustained remission. It is considered that patients with MPO-ANCA-positive hypertrophic pachymeningitis require combination therapy with prednisolone and immunosuppressive agents at an early stage.

  17. Health related quality of life in patients with newly diagnosed anti-neutrophil cytoplasm antibody associated vasculitis

    Science.gov (United States)

    Walsh, Michael; Mukhtyar, Chetan; Mahr, Alfred; Herlyn, Karen; Luqmani, Raashid; Merkel, Peter A.; Jayne, David R. W.

    2011-01-01

    Background Anti-neutrophil cytoplasm antibody-associated vasculitis (AAV) can present with a broad spectrum of signs and symptoms. The relative effects of different manifestations on health related quality of life (HRQOL) is unknown. Methods We conducted an individual patient data meta-analysis of baseline Short Form 36 (SF-36) scores from four randomized controlled trials of patients with newly diagnosed AAV. We determined the associations between organ manifestations at trial entry and the SF-36 Physical Composite Score (PCS) and Mental Composite Score (MCS) using mixed effects models adjusted for demographic factors. Associations with each of the 8 domains of the SF-36 were further explored using multivariate multiple regression. Results SF-36 data was available from 346 patients. Older age (−0.11 points/year; 95% Confidence Interval [CI] −0.21 to −0.012; p=0.029) and neurologic involvement (−5.84, p<0.001) at baseline were associated with lower Physical Composite Scores. Physical Function scores were the most affected and older age (−0.25 points per year, 95% Confidence Interval [CI] −0.38 to −0.11; p<0.001) scores and neurologic involvement (−8.48 points, 95% CI −12.90 to −4.06; p<0.001) had the largest effects. The MCS was negatively affected only by chest involvement (p=0.027) but this effect was not exerted in any particular domain. Conclusions HRQOL in patients with newly diagnosed AAV are complex and incompletely explained by their organ system manifestations. PMID:21452254

  18. Atualização do tratamento das vasculites associadas a anticorpo anticitoplasma de neutrófilos Treatment of antineutrophil cytoplasmic antibody-associated vasculitis: update

    Directory of Open Access Journals (Sweden)

    Alfredo Nicodemos Cruz Santana

    2011-12-01

    Full Text Available As vasculites antineutrophil cytoplasmic antibody (ANCA, anticorpo anticitoplasma de neutrófilos associadas (VAAs são caracterizadas por uma inflamação sistêmica das artérias de pequeno e médio calibre (especialmente no trato respiratório superior e inferior, e nos rins. As VAAs compreendem a granulomatose de Wegener (agora chamada de granulomatose com poliangeíte, poliangeíte microscópica, VAA limitada ao rim e a síndrome de Churg-Strauss. Neste artigo, discutiremos as fases de tratamento dessas vasculites, como fase de indução (com ciclofosfamida ou rituximab e fase de manutenção (com azatioprina, metotrexato ou rituximab. Além disso, discutiremos como manusear os casos refratários à ciclofosfamida.In its various forms, antineutrophil cytoplasmic antibody (ANCA-associated vasculitis (AAV is characterized by a systemic inflammation of the small and medium-sized arteries (especially in the upper and lower respiratory tracts, as well as in the kidneys. The forms of AAV comprise Wegener's granulomatosis (now called granulomatosis with polyangiitis, microscopic polyangiitis, renal AAV, and Churg-Strauss syndrome. In this paper, we discuss the phases of AAV treatment, including the induction phase (with cyclophosphamide or rituximab and the maintenance phase (with azathioprine, methotrexate, or rituximab. We also discuss how to handle patients who are refractory to cyclophosphamide.

  19. Targeted proteomics reveals promising biomarkers of disease activity and organ involvement in antineutrophil cytoplasmic antibody-associated vasculitis.

    Science.gov (United States)

    Ishizaki, Jun; Takemori, Ayako; Suemori, Koichiro; Matsumoto, Takuya; Akita, Yoko; Sada, Ken-Ei; Yuzawa, Yukio; Amano, Koichi; Takasaki, Yoshinari; Harigai, Masayoshi; Arimura, Yoshihiro; Makino, Hirofumi; Yasukawa, Masaki; Takemori, Nobuaki; Hasegawa, Hitoshi

    2017-09-29

    Targeted proteomics, which involves quantitative analysis of targeted proteins using selected reaction monitoring (SRM) mass spectrometry, has emerged as a new methodology for discovery of clinical biomarkers. In this study, we used targeted serum proteomics to identify circulating biomarkers for prediction of disease activity and organ involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). A large-scale SRM assay targeting 135 biomarker candidates was established using a triple-quadrupole mass spectrometer coupled with nanoflow liquid chromatography. Target proteins in serum samples from patients in the active and remission (6 months after treatment) stages were quantified using the established assays. Identified marker candidates were further validated by enzyme-linked immunosorbent assay using serum samples (n = 169) collected in a large-cohort Japanese study (the RemIT-JAV-RPGN study). Our proteomic analysis identified the following proteins as biomarkers for discriminating patients with highly active AAV from those in remission or healthy control subjects: tenascin C (TNC), C-reactive protein (CRP), tissue inhibitor of metalloproteinase 1 (TIMP1), leucine-rich alpha-2-glycoprotein 1, S100A8/A9, CD93, matrix metalloproteinase 9, and transketolase (TKT). Of these, TIMP1 was the best-performing marker of disease activity, allowing distinction between mildly active AAV and remission. Moreover, in contrast to CRP, serum levels of TIMP1 in patients with active AAV were significantly higher than those in patients with infectious diseases. The serum levels of TKT and CD93 were higher in patients with renal involvement than in those without, and they predicted kidney outcome. The level of circulating TNC was elevated significantly in patients with lung infiltration. AAV severity was associated with markers reflecting organ involvement (TKT, CD93, and TNC) rather than inflammation. The eight markers and myeloperoxidase (MPO

  20. Exploration, Development, and Validation of Patient-reported Outcomes in Antineutrophil Cytoplasmic Antibody–associated Vasculitis Using the OMERACT Process

    Science.gov (United States)

    Robson, Joanna C.; Milman, Nataliya; Tomasson, Gunnar; Dawson, Jill; Cronholm, Peter F.; Kellom, Katherine; Shea, Judy; Ashdown, Susan; Boers, Maarten; Boonen, Annelies; Casey, George C.; Farrar, John T.; Gebhart, Don; Krischer, Jeffrey; Lanier, Georgia; McAlear, Carol A.; Peck, Jacqueline; Sreih, Antoine G.; Tugwell, Peter; Luqmani, Raashid A.; Merkel, Peter A.

    2016-01-01

    Objective Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of linked multisystem life- and organ-threatening diseases. The Outcome Measures in Rheumatology (OMERACT) vasculitis working group has been at the forefront of outcome development in the field and has achieved OMERACT endorsement of a core set of outcomes for AAV. Patients with AAV report as important some manifestations of disease not routinely collected through physician-completed outcome tools; and they rate common manifestations differently from investigators. The core set includes the domain of patient-reported outcomes (PRO). However, PRO currently used in clinical trials of AAV do not fully characterize patients’ perspectives on their burden of disease. The OMERACT vasculitis working group is addressing the unmet needs for PRO in AAV. Methods Current activities of the working group include (1) evaluating the feasibility and construct validity of instruments within the PROMIS (Patient-Reported Outcome Measurement Information System) to record components of the disease experience among patients with AAV; (2) creating a disease-specific PRO measure for AAV; and (3) applying The International Classification of Functioning, Disability and Health to examine the scope of outcome measures used in AAV. Results The working group has developed a comprehensive research strategy, organized an investigative team, included patient research partners, obtained peer-reviewed funding, and is using a considerable research infrastructure to complete these interrelated projects to develop evidence-based validated outcome instruments that meet the OMERACT filter of truth, discrimination, and feasibility. Conclusion The OMERACT vasculitis working group is on schedule to achieve its goals of developing validated PRO for use in clinical trials of AAV. (First Release September 1 2015; J Rheumatol 2015;42:2204–9; doi:10.3899/jrheum.141143) PMID:26329344

  1. 18F-fluoro-deoxy-glucose positron emission tomography combined with computed tomography can reliably rule-out infection and cancer in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis suspected of disease relapse

    DEFF Research Database (Denmark)

    Frary, Evan C; Hess, Søren; Gerke, Oke

    2017-01-01

    Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by systemic inflammation in small- to medium-sized blood vessels. Although immunosuppressive therapy has greatly improved the prognosis for these patients, there are still...

  2. Borderline positive antineutrophil cytoplasmic antibodies (ANCA)-PR3/MPO detection in a large cohort tertiary center: lessons learnt from a real-life experience.

    Science.gov (United States)

    Watad, Abdulla; Bragazzi, Nicola L; Sharif, Kassem; Gilburd, Boris; Yavne, Yarden; McGonagle, Dennis; Amital, Howard; Shoenfeld, Yehuda

    2018-01-29

    Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) are the best strategies for antineutrophil cytoplasmic antibodies (ANCA) detection. In a minority of subjects, ELISA-based ANCA testing may result in a borderline positive titre. Therefore, we assessed the clinical significance of such a result. This is a retrospective study, which included all subjects screened for ANCA subtypes (myeloperoxidase (MPO) or proteinase-3 (PR3)) with subsequent identification of borderline positive results, as determined by ELISA and retested using IIF. The demographic, clinical and laboratory data of subjects with borderline positive ANCA test results were extracted from their medical records. A total of 14,555 PR3/MPO-ANCA tests were performed with ELISA during the study period (2006-2016). Of the 14,555 PR3-ANCA antibody tests that were performed, 94 were borderline positive (titre 0.9-1.1), and of 14,555 MPO-ANCA antibody tests, 43 were borderline positive (titre 0.9-1.1). The male-to-female ratio was 1:1.08 and the mean age was 50.95±21.79 years. Four MPO-ANCA (9.30%) and 11 PR3-ANCA (11.70%) antibody borderline samples resulted positive on IIF testing. Subjects with borderline positive MPO-ANCA were found to have a poorer outcome in terms of renal failure and the requirement of dialysis. Subjects with borderline positive MPO-ANCA and positive p-ANCA (IIF) seem to have a less favorable outcome. Physicians should be aware of these findings and possibly perform a closer follow-up and routine screening for these subjects.

  3. The role of α1 -antitrypsin Deficiency in the Pathogenesis of Antineutrophil Cytoplasmic Antibodies Associated Systemic Necrotizing Vasculitides

    Directory of Open Access Journals (Sweden)

    Alzouki Abdul-Nasser

    1999-01-01

    Full Text Available Alpha1-antitrypsin (D,1AT is the most abundant circulating protease inhibitor (Pi in human plasma. It has central function in controlling tissue degradation by inhibiting a large number of proteases including neutrophil elastase and proteinase 3 (PR3. PR3, the Wegner′s autoantigen, has been suggested to be involved in the pathogenesis of small-vessel systemic vasculitides. α1 AT deficiency (PiZ is frequent in Caucasian populations, and its homozygous state (PiZZ is known to predispose to lung emphysema and chronic liver disease. A strong correlation between heterozygous (PiZ and homozygous (PiZZ α1 AT deficiency and anti-neutrophil cytoplasmic autoantibodies (ANCA associated systemic nocrotizing vasculitides has recently been reported in various populations. In this review the pathogenesis of small-vessel vasculitides is outlined, focusing on the role of α1 AT deficiency. α1 AT has been suggested to have a crucial role as a protective protein in ANCA-associated vasculitic syndromes.

  4. Anti-neutrophil cytoplasm autoantibodies (ANCA) in autoimmune liver diseases

    NARCIS (Netherlands)

    Roozendaal, C.; Kallenberg, Cees

    1999-01-01

    Anti-neutrophil cytoplasm antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. ANCA have been detected in serum from patients with inflammatory bowel diseases (mainly ulcerative colitis) and autoimmune mediated liver diseases

  5. Pulmonary renal syndrome in a child with coexistence of anti-neutrophil cytoplasmic antibodies and anti-glomerular basement membrane disease: case report and literature review.

    Science.gov (United States)

    Bogdanović, Radovan; Minić, Predrag; Marković-Lipkovski, Jasmina; Stajić, Nataša; Savić, Nataša; Rodić, Milan

    2013-03-22

    Pulmonary renal syndrome (PRS), denoting the presence of diffuse alveolar hemorrhage and glomerulonephritis as manifestations of systemic autoimmune disease, is very rare in childhood. The coexistence of circulating anti-neutrophil cytoplasmic antibody (ANCA) and anti-glomerular basement membrane (GBM) disease in children affected by this syndrome is exceptional, with unfavorable outcome in five out of seven patients reported to date. We describe a child with PRS associated with both circulating anti-myeloperoxidase (anti-MPO) ANCA and anti-GBM disease on renal biopsy who was successfully treated with immunosuppressive therapy. A 10-year old girl presented with fever, fatigue, malaise, and pallor followed by hemoptysis and severe anemia. Diffuse alveolar hemorrhage was revealed on fiberoptic bronchoscopy. Renal findings consisted of microscopic hematuria, moderate proteinuria, and anti-GBM disease on renal biopsy. ANCA with anti-MPO specificity were present whereas anti-GBM antibodies were on borderline for positivity. Methyl-prednisolone pulses followed by prednisone led to cessation of hemoptysis, marked improvement of lung fuction, and normal finding on chest x-ray within 10 days. An immunosuppressive regimen was then given consisting of prednisone daily for 4 weeks with subsequent taper on alternate day, i.v. cyclophosphamide pulses monthly for 6 doses, followed by mycophenolate mofetil that resulted in normal lung function tests, hemoglobin concentration, and anti-MPO level within four subsequent weeks. During 10-months of follow-up she remained well, her blood pressure and renal function tests were normal, and proteinuria and hematuria gradually resolved. We report a child with an exceptionally rare coexistence of circulating ANCA and anti-GBM disease manifesting as PRS in whom renal disease was not the prominent part of clinical presentation, contrary to other reported pediatric patients. A review of literature on disease with double positive antibodies is

  6. The effects of duration of glucocorticoid therapy on relapse rate in anti-neutrophil cytoplasm antibody associated vasculitis: A meta-analysis

    Science.gov (United States)

    Walsh, Michael; Merkel, Peter A.; Mahr, Alfred; Jayne, David

    2010-01-01

    Objective Disease relapses are common for patients with anti-neutrophil cytoplasm antibody associated vasculitis (AAV). The role of low-dose glucocorticoids (GC) in relapse prevention is controversial. We undertook a systematic review and meta-analysis to determine if GC target doses influence relapses of AAV. Methods Medline, EMBASE and Cochrane databases were searched for observational studies and randomized controlled trials of treatment of AAV that included a predefined GC treatment plan. The association of GC target dose with the proportion of relapses in studies was assessed using meta-regression and multi-level generalized linear modeling. Results Thirteen studies (983 patients) were identified for inclusion. There were no studies directly comparing GC regimens. We classified 288 patients as having a non-zero GC target dose by study end and 695 patients as having a zero GC target dose by study end. The pooled proportion of patients with a relapse was 36% (95% confidence interval [CI] 25 to 47%). GC regimen was the most significant variable explaining the variability between the proportions of patients with relapses. The proportion of patients with a relapse was 14% (95% CI 10 to 19%) in non-zero GC target dose and 43% (95% CI 33 to 52%) in zero GC target dose studies. Differences other than GC regimens exist between studies that complicate the comparability of trials and isolation of the variability in relapses due to GC target alone. Conclusions Studies with longer courses of GC in AAV are associated with fewer relapses. These results have implications for study design and outcome assessment in clinical trials of AAV. PMID:20235186

  7. Perinuclear antineutrophil cytoplasmic autoantibodies and anti-Saccharomyces cerevisiae antibodies as serological markers are not specific in the identification of Crohn’s disease and ulcerative colitis

    Science.gov (United States)

    Anand, Vishal; Russell, Anthony S; Tsuyuki, Ross; Fedorak, Richard

    2008-01-01

    OBJECTIVE: To evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic autoantibodies (pANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCAs), as single agents and in combination, for the diagnosis of Crohn’s disease (CD) and ulcerative colitis (UC), including in cases of indeterminate colitis (IC). METHODS: The sera from a total of 98 patients were studied: 77 with CD, 16 with UC and five with IC. The medical records of these patients were reviewed for disease diagnosis, demographic data, and patient symptoms and medications. ELISAs were utilized to detect the presence of ASCAs and deoxyribonuclease-sensitive pANCAs, and these results were then compared with the patients’ clinical data. RESULTS: For UC, a positive pANCA test alone provided a sensitivity of 50% and a specificity of 82%. For CD, a positive ASCA test alone provided a sensitivity of 40% and a specificity of 100%. A combination of pANCA-positive and ASCA-negative results showed a sensitivity of 50% and specificity of 90% for the diagnosis of UC. Similarly, the combination of ASCA-positive and pANCA-negative results provided a sensitivity and specificity of 32% and 100% for the diagnosis of CD, respectively. Interestingly, 80% of IC patients showed serology results consistent with UC. CONCLUSIONS: Although this combination of serological markers provides a diagnostic tool with generally high specificities, the low sensitivities of these serological markers, most notably in terms of CD, preclude the possibility that they can replace the tools currently used for inflammatory bowel disease diagnosis and management. It is possible, however, that these serological markers may prove beneficial in the management of IC. PMID:18209778

  8. Perinuclear antineutrophil cytoplasmic autoantibodies and anti-Saccharomyces cerevisiae antibodies as serological markers are not specific in the identification of Crohn's disease and ulcerative colitis.

    Science.gov (United States)

    Anand, Vishal; Russell, Anthony S; Tsuyuki, Ross; Fedorak, Richard

    2008-01-01

    To evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic autoantibodies (pANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCAs), as single agents and in combination, for the diagnosis of Crohn's disease (CD) and ulcerative colitis (UC), including in cases of indeterminate colitis (IC). The sera from a total of 98 patients were studied: 77 with CD, 16 with UC and five with IC. The medical records of these patients were reviewed for disease diagnosis, demographic data, and patient symptoms and medications. ELISAs were utilized to detect the presence of ASCAs and deoxyribonuclease-sensitive pANCAs, and these results were then compared with the patients' clinical data. For UC, a positive pANCA test alone provided a sensitivity of 50% and a specificity of 82%. For CD, a positive ASCA test alone provided a sensitivity of 40% and a specificity of 100%. A combination of pANCA-positive and ASCA-negative results showed a sensitivity of 50% and specificity of 90% for the diagnosis of UC. Similarly, the combination of ASCA-positive and pANCA-negative results provided a sensitivity and specificity of 32% and 100% for the diagnosis of CD, respectively. Interestingly, 80% of IC patients showed serology results consistent with UC. Although this combination of serological markers provides a diagnostic tool with generally high specificities, the low sensitivities of these serological markers, most notably in terms of CD, preclude the possibility that they can replace the tools currently used for inflammatory bowel disease diagnosis and management. It is possible, however, that these serological markers may prove beneficial in the management of IC.

  9. Rapidly progressive antineutrophil cytoplasm antibodies associated with pulmonary-renal syndrome in a 10-year-old girl

    OpenAIRE

    Blanco Filho,Fermin; Ernesto,Luci Carla; Rosa,Mônica Assis; Stuginski,Luis Antônio; Zlochevsky,Eliana Regina; Blanco,Fernando

    2001-01-01

    CONTEXT: The term pulmonary-renal syndrome has been used frequently to describe the clinical manifestations of a great number of diseases in which pulmonary hemorrhage and glomerulonephritis coexist. The classic example of this type of vasculitis is Goodpasture´s syndrome, a term used to describe the association of pulmonary hemorrhage, glomerulonephritis and the presence of circulating antiglomerular basement membrane antibodies (anti-GBM). Among the several types of systemic vasculitides th...

  10. Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic antibodies in coeliac disease before and after gluten-free diet.

    Science.gov (United States)

    Granito, A; Zauli, D; Muratori, P; Muratori, L; Grassi, A; Bortolotti, R; Petrolini, N; Veronesi, L; Gionchetti, P; Bianchi, F B; Volta, U

    2005-04-01

    Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies are markers of Crohn's disease and ulcerative colitis respectively. To determine the prevalence of anti-S. cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies in a large series of coeliac disease patients before and after gluten free diet, and to correlate anti-S. cerevisiae-positivity with intestinal mucosal damage. One hundred and five consecutive coeliac disease patients and 141 controls (22 ulcerative colitis, 24 Crohn's disease, 30 primary sclerosing cholangitis, 15 postenteritis syndrome, 50 blood donors) were tested for anti-S. cerevisiae by enzyme-linked immunosorbent assay and for perinuclear anti-neutrophil cytoplasmic autoantibodies by indirect immunofluorescence. In coeliac disease anti-S. cerevisiae (immunoglobulin G and/or immunoglobulin A) were slightly less frequent (59%) than in Crohn's disease (75%, P = 0.16) and significantly more frequent than in ulcerative colitis (27%), primary sclerosing cholangitis (30%), postenteritis syndrome (26%) and blood donors (4%) (P = 0.009, P = 0.0002, P = 0.025, P < 0.0001). No correlation was found between anti-S. cerevisiae and degree of mucosal damage. Perinuclear anti-neutrophil cytoplasmic autoantibodies were detected only in one coeliac. After gluten free diet the disappearance of anti-S. cerevisiae-immunoglobulin A (93%) was more frequent than that of immunoglobulin G (17%, P = 0.0001); perinuclear anti-neutrophil cytoplasmic autoantibodies disappeared in the only coeliac positive at diagnosis. More than half of untreated coeliacs are anti-S. cerevisiae-positive irrespective of the severity of mucosal damage. Differently from immunoglobulin A, anti-S. cerevisiae-immunoglobulin G persisted in more than 80% after gluten free diet. The high prevalence of anti-S. cerevisiae in coeliac disease suggests that they may be the effect of a non-specific immune response in course of chronic small bowel disease.

  11. [The significances of peripheral neutrophils CD(55) and myeloperoxidase expression in patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody associated vasculitis].

    Science.gov (United States)

    Zhou, X L; Zheng, M J; Shuai, Z W; Zhang, L; Zhang, M M; Chen, S Y

    2017-06-01

    Objective: To investigate the expression of CD(55) and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation. Methods: Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study. The CD(55) on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry. Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA. The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3). The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis. Results: (1)The mean fluorescence intensity(MFI) of CD(55) expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9, P =0.033]. Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L, P <0.001; 35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L, P <0.001], respectively. However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0, P =0.003) . (2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO( r =-0.710, P <0.001) and Ba ( r =-0.589, P =0.001). Moreover, serum MPO was positively associated with serum Ba( r =0.691, P <0.001). Membrane intensity of CD(55) on neutrophils was positively correlated with patient age ( r =0.514, P =0.001), C reactive protein ( r =0.376, P =0.018), peripheral neutrophils count ( r =0.485, P =0.001) and BVAS-V3 ( r =0.484, P =0.002), whereas negative correlation between membrane CD(55) and disease duration was seen ( r =-0.403, P =0.01). (3) The result of multiple

  12. Clinical characteristics of and risk factors for serious infection in Japanese patients within six months of remission induction therapy for antineutrophil cytoplasmic antibody-associated vasculitis registered in a nationwide, prospective, inception cohort study.

    Science.gov (United States)

    Watanabe-Imai, Kaori; Harigai, Masayoshi; Sada, Ken-Ei; Yamamura, Masahiro; Fujii, Takao; Dobashi, Hiroaki; Amano, Koichi; Ito, Satoshi; Homma, Sakae; Kumagai, Shunichi; Banno, Shogo; Arimura, Yoshihiro; Makino, Hirofumi

    2017-07-01

    The purpose of this study was to identify the clinical characteristics and predictors of serious infections (SIs) in the RemIT-JAV, a nationwide, prospective, inception cohort study for Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). We analyzed SIs within six months of remission induction therapy in 156 AAV patients. Hazard ratios with 95% confidence intervals (CIs) for SIs were calculated using the COX proportional hazard model. Sixty-three SIs in 42 patients were identified. The incidence rate (IR) of SIs was 87.59/100 patient-years. The median length of time to the onset of first SIs was 54 days. Hazard ratios (95%CI) for SIs were 1.97 (0.99-3.95) for age >65 years, 0.47 (0.25-0.89) for female sex, 2.11 (1.05-4.27) for the severe form of AAV, and 2.88 (1.49-5.88) for initial PSL >0.8 mg/kg/day in the first model, and 2.64 (1.39-5.01) for smoking and 3.27 (1.66-6.45) for initial PSL >0.8 mg/kg/day in the second model. Lowering the IR of SIs in Japanese AAV patients is mandatory to improve the vital prognosis of these patients. For remission induction therapy of AAV patients with these risk factors, risk management of immunosuppressive treatment should be carefully considered.

  13. 18F-fluoro-deoxy-glucose positron emission tomography combined with computed tomography can reliably rule-out infection and cancer in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis suspected of disease relapse.

    Science.gov (United States)

    Frary, Evan C; Hess, Søren; Gerke, Oke; Laustrup, Helle

    2017-07-01

    Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by systemic inflammation in small- to medium-sized blood vessels. Although immunosuppressive therapy has greatly improved the prognosis for these patients, there are still significant comorbidities, such as cancer and infection, associated with AAV. These comorbidities are often indistinguishable from an underlying AAV disease relapse, and create a clinical conundrum, as these conditions are normally contraindications for immunosuppressive treatment. Thus, it is important to be able to rule out these comorbidities before initiation of immunosuppressive treatment. We examined F-fluoro-deoxy-glucose positron emission tomography combined with computed tomography (FDG-PET/CT)'s value in ruling out cancer or infection in patients with AAV.Data were obtained retrospectively for a clinically based cohort of AAV patients who underwent FDG-PET/CT during 2009 to 2014 owing to a suspicion of cancer, infection, or both cancer and infection indistinguishable from disease relapse. FDG-PET/CT conclusions were compared to the final diagnoses after follow-up analysis (mean 43 months).A total of 19 patients were included who underwent a total of 26 scans. The results of FDG-PET/CT outcome compared to final diagnosis were: 9 true positives, 3 false positives, 13 true negatives, and 1 false negative. The diagnostic probabilities for FDG-PET/CT with respect to overall comorbidity (i.e., cancer or infection) were: sensitivity 90% ( 95% confidence interval [CI] 60%-98%), specificity 81% ( 95% CI 57%-93%), positive predictive value 75% (95% CI 47%-91%), negative predictive value 93% (95% CI 68%-99%), and accuracy 84% (95% CI 66%-94%).FDG-PET/CT had a high negative predictive value and ruled out the comorbidities correctly in all but one case of urinary tract infection, a well-known limitation. Our study showed FGD-PET/CT's promise as an effective tool for ruling out

  14. Clinical features and treatment outcomes of otitis media with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (OMAAV): A retrospective analysis of 235 patients from a nationwide survey in Japan.

    Science.gov (United States)

    Harabuchi, Yasuaki; Kishibe, Kan; Tateyama, Kaori; Morita, Yuka; Yoshida, Naohiro; Kunimoto, Yasuomi; Matsui, Takamichi; Sakaguchi, Hiroshi; Okada, Masahiro; Watanabe, Takeshi; Inagaki, Akira; Kobayashi, Shigeto; Iino, Yukiko; Murakami, Shingo; Takahashi, Haruo; Tono, Tetsuya

    2017-01-01

    We aimed to analyze clinical features and treatment outcomes of otitis media caused by antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), i.e. otitis media with AAV (OMAAV). This survey was performed between December 2013 and February 2014. The study began with a preliminary survey to 123 otolaryngology institutions in Japan to inquire about their experiences with OMAAV patients during the past 10 years, and was followed by a questionnaire survey to investigate clinical and laboratory findings. OMAAV was defined using the criteria described in the text. Two hundred and thirty-five patients classified as OMAAV were enrolled in this study. They were characterized as follows: (1) disease onset with initial signs/symptoms due to intractable otitis media with effusion or granulation, which did not respond to ordinary treatments such as antibiotics and insertion of tympanic ventilation tubes, followed by progressive hearing loss; (2) predominantly female (73%) and older (median age: 68 years); (3) predominantly myeloperoxidase (MPO)-ANCA-positive (60%), followed by proteinase 3 (PR3)-ANCA-positive (19%) and both ANCAs-negative (16%); (4) frequently observed accompanying facial palsy (36%) and hypertrophic pachymeningitis (28%); and (5) disease often involving lung (35%) and kidney (26%) lesions. Four factors associated with OMAAV were found to be related to an unfavorable clinical course threatening the patient's hearing and/or lives, namely facial palsy, hypertrophic pachymeningitis, both ANCAs-negative phenotype, and disease relapse. The occurrence of hypertrophic pachymeningitis was associated with facial palsy (p < 0.05), both ANCAs-negative phenotype (p < 0.001), and headache (p < 0.001). The administration of corticosteroid together with an immunosuppressant was an independent predicting factor for lack of disease relapse (odds ratio [OR] = 1.90, p = 0.03) and an improvement in hearing loss (OR =2.58, p = 0.0002). Since

  15. Detection of antineutrophil cytoplasmic antibodies (ANCAs)

    DEFF Research Database (Denmark)

    Damoiseaux, Jan; Csernok, Elena; Rasmussen, Niels

    2017-01-01

    of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF...

  16. Anti-neutrophil cytoplasm autoantibodies (ANCA). The need for specific and sensitive assays

    DEFF Research Database (Denmark)

    Baslund, B; Petersen, J

    1998-01-01

    Anti-neutrophil cytoplasm antibodies (ANCA) are a group of autoantibodies primarily associated with systemic vasculitis. Hitherto, the method of choice for ANCA detection has been indirect immunofluorescence (IIF). By this method two major patterns can be seen: a cytoplasmic pattern (cANCA) or a ......Anti-neutrophil cytoplasm antibodies (ANCA) are a group of autoantibodies primarily associated with systemic vasculitis. Hitherto, the method of choice for ANCA detection has been indirect immunofluorescence (IIF). By this method two major patterns can be seen: a cytoplasmic pattern (c......ANCA) or a perinuclear pattern (pANCA). The cANCA pattern is most often caused by antibodies directed against proteinase-3 (PR3) and in rare cases it is caused by anti-myeloperoixdase (MPO) antibodies. The pANCA pattern can de caused by antibodies directed against a large group of proteins i.e. MPO, lactofenin...

  17. Antineutrophil cytoplasmic antibody–negative pauci-immune glomerulonephritis with massive intestinal bleeding

    Directory of Open Access Journals (Sweden)

    Miyeon Kim

    2015-09-01

    Full Text Available A 61-year-old woman was admitted to hospital because of generalized edema and proteinuria. Her renal function deteriorated rapidly. Serum immunoglobulin and complement levels were within normal ranges. An autoantibody examination showed negative for antinuclear antibody and antineutrophil cytoplasmic antibody. Histologic examination of a renal biopsy specimen revealed that all of the glomeruli had severe crescent formations with no immune deposits. The patient was treated with steroid pulse therapy with cyclophosphamide followed by oral prednisolone. Fifteen days later, she experienced massive recurrent hematochezia. Angiography revealed an active contrast extravasation in a branch of the distal ileal artery. We selectively embolized with a permanent embolic agent. On the 45th hospital day, the patient suddenly lost consciousness. Brain computed tomography showed intracerebral hemorrhage. We report a case of antineutrophil cytoplasmic antibody–negative pauci-immune glomerulonephritis with massive intestinal bleeding and cerebral hemorrhage.

  18. Antibody Repertoire Development in Swine

    Czech Academy of Sciences Publication Activity Database

    Butler, J. E.; Wertz, N.; Šinkora, Marek

    2017-01-01

    Roč. 5, FEB 17 (2017), s. 255-279 ISSN 2165-8102 R&D Projects: GA ČR GA15-02274S; GA ČR(CZ) GA16-09296S Institutional support: RVO:61388971 Keywords : swine * pre-immune antibody repertoire * ileal Peyer's patches Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.708, year: 2016

  19. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans

    2008-08-01

    Full Text Available Today’s proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

  20. Development and evaluation of Indirect Hemagglutination Antibody ...

    African Journals Online (AJOL)

    The study was conducted to develop and evaluate an Indirect Hemagglutination Antibody Test (IHAT) for the serological diagnosis of Cysticercus bovis in live animals. IHAT was set-up in-house and used to test serum samples of cattle against sheep red blood cell (SRBC) coated with crude extracts of C. bovis cyst. Serum ...

  1. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  2. Recent developments in monoclonal antibody radiolabeling techniques

    International Nuclear Information System (INIS)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs

  3. Development of Antibody Therapeutics against Flaviviruses

    Science.gov (United States)

    Sun, Haiyan; Chen, Qiang; Lai, Huafang

    2017-01-01

    Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates. PMID:29295568

  4. Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

    Science.gov (United States)

    Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

    2017-06-01

    Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

  5. Sequential development of pulmonary hemorrhage with MPO-ANCA complicating anti-glomerular basement membrane antibody-mediated glomerulonephritis.

    Science.gov (United States)

    Peces, R; Rodríguez, M; Pobes, A; Seco, M

    2000-05-01

    We report a case of rapidly progressive glomerulonephritis caused by anti-glomerular basement membrane (anti-GBM) antibodies that progressed to end-stage renal disease in a 67-year-old woman with diabetes. Intensive combined immunosuppressive therapy with methylprednisolone bolus, oral prednisone, and cyclophosphamide led to negativity of anti-GBM antibodies but was not able to restore renal function. After 28 months of hemodialysis, the patient suddenly presented with pulmonary hemorrhage. In this setting, high levels of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA) and negative anti-GBM antibodies were found. Therapy with oral prednisone and cyclophosphamide led to resolution of pulmonary hemorrhage and negativity of MPO-ANCA.

  6. Development of Polyclonal Antibody against Clenbuterol for Immunoassay Application

    Directory of Open Access Journals (Sweden)

    Nurul Ain A. Talib

    2018-03-01

    Full Text Available Development of an immunoassay for clenbuterol (CLB detection required an anti-CLB antibody as an important bioreceptor. In this study, we report our work on production and purification of a rabbit-derived polyclonal anti-CLB antibody. The antibody was then purified by nProtein A Sepharose affinity column and the antibody purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE analysis. The activities of purified antibody were evaluated based on high antibody titer determined from enzyme-linked immunosorbent assay (ELISA. The sensitivity and selectivity of this antibody was evaluated and exhibits negligible cross-reactivity to antibiotics other than β-agonist families. Evaluation of the antibody as bioreceptor in immunoassay was performed using direct competitive ELISA and exhibited linear calibration plot (R2 = 0.9484. The antibody was used to detect the content of CLB in spiked milk samples and the recovery of more than 92% indicating significant performance as bioreceptor for the development of a rapid and simple immunoassay.

  7. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  8. Development of scoring functions for antibody sequence assessment and optimization.

    Directory of Open Access Journals (Sweden)

    Daniel Seeliger

    Full Text Available Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity.

  9. Integration of Antibody Array Technology into Drug Discovery and Development.

    Science.gov (United States)

    Huang, Wei; Whittaker, Kelly; Zhang, Huihua; Wu, Jian; Zhu, Si-Wei; Huang, Ruo-Pan

    Antibody arrays represent a high-throughput technique that enables the parallel detection of multiple proteins with minimal sample volume requirements. In recent years, antibody arrays have been widely used to identify new biomarkers for disease diagnosis or prognosis. Moreover, many academic research laboratories and commercial biotechnology companies are starting to apply antibody arrays in the field of drug discovery. In this review, some technical aspects of antibody array development and the various platforms currently available will be addressed; however, the main focus will be on the discussion of antibody array technologies and their applications in drug discovery. Aspects of the drug discovery process, including target identification, mechanisms of drug resistance, molecular mechanisms of drug action, drug side effects, and the application in clinical trials and in managing patient care, which have been investigated using antibody arrays in recent literature will be examined and the relevance of this technology in progressing this process will be discussed. Protein profiling with antibody array technology, in addition to other applications, has emerged as a successful, novel approach for drug discovery because of the well-known importance of proteins in cell events and disease development.

  10. Antibodies in HIV-1 vaccine development and therapy.

    Science.gov (United States)

    Klein, Florian; Mouquet, Hugo; Dosenovic, Pia; Scheid, Johannes F; Scharf, Louise; Nussenzweig, Michel C

    2013-09-13

    Despite 30 years of study, there is no HIV-1 vaccine and, until recently, there was little hope for a protective immunization. Renewed optimism in this area of research comes in part from the results of a recent vaccine trial and the use of single-cell antibody-cloning techniques that uncovered naturally arising, broad and potent HIV-1-neutralizing antibodies (bNAbs). These antibodies can protect against infection and suppress established HIV-1 infection in animal models. The finding that these antibodies develop in a fraction of infected individuals supports the idea that new approaches to vaccination might be developed by adapting the natural immune strategies or by structure-based immunogen design. Moreover, the success of passive immunotherapy in small-animal models suggests that bNAbs may become a valuable addition to the armamentarium of drugs that work against HIV-1.

  11. Development of Broadly Neutralizing Antibody Mimitopes for Characterization of CRF01_AE HIV-1 Antibody Responses

    Directory of Open Access Journals (Sweden)

    Jesse V. Schoen

    2017-10-01

    Full Text Available Mapping humoral immune responses to HIV-1 over the course of natural infection is important in understanding epitope exposure in relation to elicitation of broadly neutralizing antibodies (bNAbs, which is considered imperative for effective vaccine design. When analyzing HIV-specific immune responses, the antibody binding profiles may be a correlate for functional antibody activity. In this study, we utilized phage display technology to identify novel mimitopes that may represent Env epitope structures bound by bNAbs directed at V1V2 and V3 domains, CD4 binding site (CD4bs and the membrane proximal external region (MPER of Env. Mimitope sequence motifs were determined for each bNAb epitope. Given the ongoing vaccine development efforts in Thailand, these mimitopes that represent CD4bs and MPER epitopes were used to map immune responses of HIV-1 CRF01_AE-infected individuals with known neutralizing responses from two distinct time periods, 1996-98 and 2012-15. The more contemporary cohort showed an increase in binding breadth with binding observed for all MPER and CD4bs mimitopes, while the older cohort showed only 75% recognition of the CD4bs mimitopes and no MPER mimotope binding. Furthermore, mimitope binding profiles correlated significantly with magnitude (p=0.0036 and breadth (p=0.0358 of neutralization of a multi-subtype Tier 1 panel of pseudoviruses. These results highlight the utility of this mimitope mapping approach for detecting human plasma IgG-specificities that target known neutralizing antibody epitopes, and may also provide an indication of the plasticity of antibody binding within HIV-1 Env neutralization determinants.

  12. Development of polyclonal antibodies for the detection of ...

    African Journals Online (AJOL)

    Development of polyclonal antibodies for the detection of recombinant human erythropoietin. Collares Thaís Farias, Monte Leonardo Garcia, Campos Vinicius Farias, Seixas Fabiana Kömmiling, Collares Tiago Veiras, Dellagostin Odir, Hartleben Cláudia Pinho ...

  13. Markers of mouse macrophage development detected by monoclonal antibodies

    NARCIS (Netherlands)

    P.J. Leenen (Pieter); M.F.T.R. de Bruijn (Marella F.T.R); J.S. Voerman (Jane); P.A. Campbell (Priscilla); W. van Ewijk (Willem)

    1994-01-01

    textabstractIn this review, we present and discuss a selected panel of antibody-defined markers expressed during different stages of mouse macrophage development. We distinguish four categories of markers, which are characteristic of: (1) macrophage precursors and immature macrophages (ER-MP12,

  14. Development of Antibody-Coated Magnetite Nanoparticles for Biomarker Immobilization

    Directory of Open Access Journals (Sweden)

    Christian Chapa Gonzalez

    2014-01-01

    Full Text Available Magnetic nanoparticles (MNPs have great potential in biomedical applications because of their magnetic response offers the possibility to direct them to specific areas and target biological entities. Magnetic separation of biomolecules is one of the most important applications of MNPs because their versatility in detecting cancer biomarkers. However, the effectiveness of this method depends on many factors, including the type of functionalization onto MNPs. Therefore, in this study, magnetite nanoparticles have been developed in order to separate the 5′-nucleotidase enzyme (5eNT. The 5eNT is used as a bio-indicator for diagnosing diseases such as hepatic ischaemia, liver tumor, and hepatotoxic drugs damage. Magnetic nanoparticles were covered in a core/shell type with silica, aminosilane, and a double shell of silica-aminosilane. A ScFv (fragment antibody and anti-CD73 antibody were attached to the coated nanoparticles in order to separate the enzyme. The magnetic separation of this enzyme with fragment antibody was found to be 28% higher than anti-CD73 antibody and the enzyme adsorption was improved with the double shell due to the increased length of the polymeric chain. Magnetite nanoparticles with a double shell (silica-aminosilane were also found to be more sensitive than magnetite with a single shell in the detection of biomarkers.

  15. Relevant shaking stress conditions for antibody preformulation development.

    Science.gov (United States)

    Eppler, Annette; Weigandt, Markus; Hanefeld, Andrea; Bunjes, Heike

    2010-02-01

    In protein formulation development, shaking stress is often employed to assess the physical stability of antibody formulations against aggregation. Since there are currently no guidelines describing suitable test conditions, very different shaking stress designs are used. These different designs may influence the resulting stability data. The aim of this study was to establish a shaking stress design within the protein range of 2-5mg/ml which can rapidly distinguish between antibody formulations of poor stability and those with potential for further development. Small scale shaking stress experiments were performed with different monoclonal IgG antibodies (as buffered solutions or marketed formulations). Variables were the filling degree of the sample containers, the container type and size and the shaking intensity. The stability of the samples was assessed by visual inspection, UV-VIS spectrophotometric turbidity measurements and size exclusion chromatography. All tested parameters had a strong influence on the stability results. The most discriminating conditions were obtained when shaking of the formulations was performed at 200rpm in a 2ml injection vial filled with 1ml protein solution. This experimental setup led to clearly different stability results for buffered solutions and marketed products. Moreover, this setup required only relatively small amounts of protein solution which is advantageous in prefomulation studies. Copyright (c) 2009 Elsevier B.V. All rights reserved.

  16. Churg-Strauss syndrome associated with antiphospholipid antibodies in a patient with retinal vasculitis.

    Science.gov (United States)

    Sánchez-Vicente, J L; Gálvez-Carvajal, S; Medina-Tapia, A; Rueda, T; González-García, L; Szewc, M; Muñoz-Morales, A

    2016-11-01

    We present the case of a 69-year-old woman with unilateral retinal vasculitis. Investigations showed asthma, rhinosinusitis, nasal polyposis, peripheral blood eosinophilia, increased sedimentation rate, proteinuria, and antiphospholipid antibodies. Anti-neutrophil cytoplasmic antibodies (ANCA) were negative. Although her anti-neutrophil cytoplasmatic antibody (ANCA) status was negative, taking into account the other clinical and laboratory features, retinal vasculitis was thought to be an ocular manifestation of Churg-Strauss syndrome. Treatment was started with high-dose corticosteroids and anticoagulant therapy. Copyright © 2016 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

  17. Triacetone triperoxide (TATP): hapten design and development of antibodies.

    Science.gov (United States)

    Walter, Maria Astrid; Pfeifer, Dietmar; Kraus, Werner; Emmerling, Franziska; Schneider, Rudolf J; Panne, Ulrich; Weller, Michael G

    2010-10-05

    Triacetone triperoxide (TATP), an improvised explosive, is a potential security threat because of its cost-efficient synthesis and the difficulty in detecting it. A highly selective antibody could provide the necessary specificity to the detection process. To obtain antibodies, a hapten made from acetone, hydrogen peroxide, and 7-oxooctanoic acid has been designed, synthesized, and confirmed by NMR that displays the utmost similarity to the analyte. The single-crystal X-ray structures of the solvated species TATP·methanol (1:1) and the TATP derivate were determined. In both compounds, the molecules exhibit D(3) symmetry and adopt a twisted boat-chair conformation. The hapten was coupled to bovine serum albumin, and mice were immunized. An immune response against TATP was elicited, and selective antibodies were detected in the mouse serum, which should be very useful for the development of a TATP biosensor system. An ELISA with a limit of detection for TATP of 65 μg L(-1) is shown.

  18. Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, G.C.

    1977-01-01

    A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated >99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.

  19. Pathogenetic Role of Thyrotropin Receptor Antibody in the Development of Hyperthyroidism Following Primary Hypothyroidism *

    OpenAIRE

    Shong, Young Kee; Cho, Bo Youn; Hong, Sung Kwan; Lee, Hong Kyu; Koh, Chang-Soon; Min, Hun Ki

    1989-01-01

    The authors measured thyrotropin binding inhibitory immunoglobulin (TBII), thyroid stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) sequentially in patients who developed hyperthyroidism following primary hypothyroidism, and compared changes in these various funcional parameters of thyrotropin receptor antibody (TRAb) with clinical manifestations, in order to investigate the role of TRAb in the development of hyperthyroidism following primary hypothyroidism. In a...

  20. Process development of a FGF21 protein-antibody conjugate.

    Science.gov (United States)

    Dirksen, Anouk; Davis, Keith A; Collins, Joe T; Bhattacharya, Keshab; Finneman, Jari I; Pepin, Erin L; Ryczek, Jeffrey S; Brown, Paul W; Wellborn, William B; Mangalathillam, Ratish; Evans, Brad P; Pozzo, Mark J; Finn, Rory F

    2017-09-26

    A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein-antibody conjugate, CVX-343, an extended half-life therapeutic for the treatment of metabolic disease. CVX-343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half-life extension. CVX-343 is representative of a growing number of complex novel peptide- and protein-based bioconjugate molecules currently being explored as therapeutic candidates. The complexity of these bioconjugates, assembled using well-established chemistries, can lead to very difficult production schemes requiring multiple starting materials and a combination of diverse technologies. Key improvements had to be made to the original CVX-343 Phase 1 manufacturing process in preparation for Phase 3 and commercial manufacturing. A strategy of minimizing FGF21 A129C dimerization and stabilizing the FGF21 A129C Drug Substance Intermediate (DSI), linker, and activated FGF21 intermediate was pursued. The use of tris(2-carboxyethyl)phosphine (TCEP) to prevent FGF21 A129C dimerization through disulfide formation was eliminated. FGF21 A129C dimerization and linker hydrolysis were minimized by formulating and activating FGF21 A129C at acidic instead of neutral pH. An activation use test was utilized to guide FGF21 A129C pooling in order to minimize misfolds, dimers, and misfolded dimers in the FGF21 A129C DSI. After final optimization of reaction conditions, a process was established that reduced the consumption of FGF21 A129C by 36% (from 4.7 to 3.0 equivalents) and the consumption of linker by 55% (from 1.4 to 0.95 equivalents for a smaller required amount of FGF21 A129C ). The overall process time was reduced from ∼5 to ∼3 days. The product distribution improved from containing ∼60% to ∼75% desired bifunctionalized (+2 FGF21) FGF21-antibody conjugate in the crude conjugation mixture and from ∼80% to ∼85% in the final CVX-343 Drug Substance

  1. Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies

    Directory of Open Access Journals (Sweden)

    Fa Yang

    2016-12-01

    Full Text Available With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

  2. Development of phage/antibody immobilized magnetostrictive biosensors

    Science.gov (United States)

    Fu, Liling

    There is an urgent need for biosensors that are able to detect and quantify the presence of a small amount of pathogens in a real-time manner accurately and quickly to guide prevention efforts and assay food and water quality. Acoustic wave (AW) devices, whose performance is defined by mass sensitivity (Sm) and quality factor (Q value), have been extensively studied as high performance biosensor platforms. However, current AW devices still face some challenges such as the difficulty to be employed in liquid and low Q value in practical applications. The objective of this research is to develop magnetostrictive sensors which include milli/microcantilever type (MSMC) and particle type (MSP). Compared to other AW devices, MSMC exhibits the following advantages: (1) wireless/remote driving and sensing; (2) easy to fabricate; (3) works well in liquid; (4) exhibits a high Q value (> 500 in air). The fundamental study of the damping effect on MSMCs from the surrounding media including air and liquids were conducted to improve the Q value of MSMCs. The experiment results show that the Q value is dependent on the properties of surrounding media (e.g. viscosity, density), the geometry of the MSMCs, and the harmonic mode on the resonance behavior of MSMCs, etc. The phage-coated MSMC has high specificity and sensitivity even while used in water with a low concentration of targeted bacteria. Two currently developed phages, JRB7 and E2, respectively respond to Bacillus anthracis spores and Salmonella typhimurium, were employed as bio-recognition elements in this research. The phage-immobilized MSMC biosensors exhibited high performance and detection of limit was 5 x 104 cfu/ml for the MSMC in size of 1.4 x 0.8 x 0.035 mm. The MSMC-based biosensors were indicated as a very potential method for in-situ monitoring of the biological quality in water. The MSP combine antibody was used to detect Staphylococcus aureus in this experiment. The interface between MSPs and antibody was

  3. Conference report: hot topics in antibody-drug conjugate development.

    Science.gov (United States)

    Thudium, Karen; Bilic, Sanela

    2013-12-01

    American Association of Pharmaceutical Scientists National Biotechnology Conference Sheraton San Diego Hotel and Marina, San Diego, CA, USA, 19-23 May 2013 The National Biotechnology Conference, is a premier meeting for biotechnology professionals covering a broad range of hot topics in the biotechnology industry. Attracting participants from academia, industry and regulatory, this meeting features sessions that aim to address emerging subjects of interest and allows for open exchange between scientists. The 2013 conference featured leading researchers in the fields of antibody-drug conjugates (ADCs) and immunogenicity. Herein, we present a summary of the ADC hot topics, including bioanalytical and PK considerations, quantitative evaluation of the impact of immunogenicity and ADME to understand ADC drug-drug interactions, and clinical considerations for ADC development. This article aims to summarize the recommendations that were made by the speakers during various sessions throughout the conference.

  4. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Nielsen, C; Hansen, J E

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection....... of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay...

  5. Development of muscarinic m3 and m4 receptor antibodies with pharmacological activities.

    Science.gov (United States)

    Wang, H Y; Zeng, S J; Qiu, P X

    1998-11-01

    To investigate the feasibility of developing subtype-selective anti-receptor antibodies with pharmacological activities for the study of subtypes of receptors. New Zealand white rabbits were immunized with synthesized subtype-selective peptide segments of m3 and m4 receptors to develop antibodies. The effects of the antibodies on ligand-binding to muscarinic receptors were studied by competitive radioligand assay. The effects of the prepared antibodies on the contraction or relaxation activity of ACh in isolated rat ilea and aortic rings were studied. Antibodies against synthesized m3 and m4 receptor subtype-selective peptides were successfully prepared. Both antibodies inhibited [3H]QNB binding to muscarinic receptors with different maximal inhibitions which may be the proportions of m3 or m4 subtypes among the total muscarinic receptors in the tissues. The maximal inhibitory rates in rat cerebral cortex, myocardium, and salivary glands were 12.1% +/- 2.1%, 15.7% +/- 1.1%, and 63.6% +/- 2.8% for m3 antibodies, whereas 28% +/- 6%, 19.3% +/- 2.6%, and 1.6% +/- 1.4% for m4 antibodies respectively. The m3 antibodies inhibited the contraction activity of ACh in isolated rat ilea and the relaxation activity of ACh in isolated rat aortic rings. It is feasible to develop subtype-selective anti-receptor antibodies as new tools in the study of the functions of m3 and m4 subtypes of muscarinic receptors.

  6. Development of recombinant human IgA for anticardiolipin antibodies assay standardization.

    Science.gov (United States)

    Knappik, Achim; Capuano, Francesco; Frisch, Christian; Ylera, Francisco; Bonelli, Fabrizio

    2009-09-01

    Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents.

  7. Comparison of disease activity measures for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis

    Science.gov (United States)

    Merkel, PA; Cuthbertson, DD; Hellmich, B; Hoffman, GS; Jayne, DRW; Kallenberg, CGM; Krischer, JP; Luqmani, R; Mahr, AD; Matteson, EL; Specks, U; Stone, JH

    2011-01-01

    Aim Currently, several different instruments are used to measure disease activity and extent in clinical trials of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, leading to division among investigative groups and difficulty comparing study results. An exercise comparing six different vasculitis instruments was performed. Methods A total of 10 experienced vasculitis investigators from 5 countries scored 20 cases in the literature of Wegener granulomatosis or microscopic polyangiitis using 6 disease assessment tools: the Birmingham Vasculitis Activity Score (BVAS), The BVAS for Wegener granulomatosis (BVAS/WG), BVAS 2003, a Physician Global Assessment (PGA), the Disease Extent Index (DEI) and the Five Factor Score (FFS). Five cases were rescored by all raters. Results Reliability of the measures was extremely high (intraclass correlations for the six measures all=0.98). Within each instrument, there were no significant differences or outliers among the scores from the 10 investigators. Test/retest reliability was high for each measure: range=0.77 to 0.95. The scores of the five acute activity measures correlated extremely well with one another. Conclusions Currently available tools for measuring disease extent and activity in ANCA-associated vasculitis are highly correlated and reliable. These results provide investigators with confidence to compare different clinical trial data and helps form common ground as international research groups develop new, improved and universally accepted vasculitis disease assessment instruments. PMID:18664546

  8. Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Juan C. Almagro

    2018-01-01

    Full Text Available The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life, which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

  9. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Nielsen, C; Hansen, J E

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of ...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.......The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...... of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay...

  10. Development of radiolabelling techniques of anti-CEA monoclonal antibody

    International Nuclear Information System (INIS)

    Castiglia, S.G. de

    1998-01-01

    The purpose of this work was to label monoclonal and polyclonal antibodies with 99 Tc m such as the ior-CEA-1 antibody and polyclonal IgG using a direct method, to check the radiochemical and biological behavior of labelled products, to prepare it under sterile and apyrogenic conditions as a lyophilized kit and to employ it in clinical trials. In addition, a photoactivation method was used to label polyclonal IgG with 99 Tc m and to compare with the established method using mercaptoethanol (2-ME) as the reducing agent. Finally polyclonal IgG was labelled using an indirect method in which a chelator was covalently attached to the protein and the 99 Tc m added as glucoheptonate complex. The properties of 99 Tc m when labelled with monoclonal and polyclonal antibodies by different methods were assessed by in vitro and in vivo studies

  11. Pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis and potential targets for biologic treatment

    NARCIS (Netherlands)

    Sanders, J.S.F.; Abdulahad, W H; Stegeman, C A; Kallenberg, C G M

    2014-01-01

    Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are autoimmune diseases in which the small vessels are inflamed. Clinical observations suggest a pathogenic role for ANCA. Such a role is supported by in vitro experimental data and animal models, particularly for

  12. Urinary matrix metalloproteinases reflect renal damage in anti-neutrophil cytoplasm autoantibody-associated vasculitis

    NARCIS (Netherlands)

    Sanders, J.S.F.; Huitema, M.G.; Hanemaaijer, R.; Goor, H. van; Kallenberg, C.G.M.; Stegeman, C.A.

    2007-01-01

    Renal expression of MMP-2, -9, and tissue inhibitor of MMP-1 (TIMP-1) correlates with histological disease activity in anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV). We studied whether urinary and plasma levels of MMP-2, -9, and TIMP-1 reflect renal expression of these

  13. Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection.

    Science.gov (United States)

    Lavoisier, Alexandra; Schlaeppi, Jean-Marc

    2015-01-01

    Therapeutic antibodies represent one of the fastest growing segments in the pharmaceutical market. They are used in a broad range of disease fields, such as autoimmune diseases, cancer, inflammation and infectious diseases. The growth of the segment has necessitated development of new analytical platforms for faster and better antibody selection and characterization. Early quality control and risk assessment of biophysical parameters help prevent failure in later stages of antibody development, and thus can reduce costs and save time. Critical parameters such as aggregation, conformational stability, colloidal stability and hydrophilicity, are measured during the early phase of antibody generation and guide the selection process of the best lead candidates in terms of technical developability. We report on the use of a novel instrument (ActiPix/Viscosizer) for measuring both the hydrodynamic radius and the absolute viscosity of antibodies based on Taylor dispersion analysis and UV area imaging. The looped microcapillary-based method combines low sample consumption, fast throughput and high precision compared to other conventional methods. From a random panel of 130 antibodies in the early selection process, we identified some with large hydrodynamic radius outside the normal distribution and others with non-Gaussian Taylor dispersion profiles. The antibodies with such abnormal properties were confirmed later in the selection process to show poor developability profiles. Moreover, combining these results with those of the viscosity measurements at high antibody concentrations allows screening, with limited amounts of materials, candidates with potential issues in pre-formulation development.

  14. The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

    Directory of Open Access Journals (Sweden)

    Christine Rasetti-Escargueil

    2017-10-01

    Full Text Available The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies. For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs. The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

  15. Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W

    2011-01-01

    Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing...

  16. Identification of Functional and Expression Polymorphisms Associated With Risk for Antineutrophil Cytoplasmic Autoantibody–Associated Vasculitis

    Science.gov (United States)

    Merkel, Peter A.; Xie, Gang; Monach, Paul A.; Ji, Xuemei; Ciavatta, Dominic J.; Byun, Jinyoung; Pinder, Benjamin D.; Zhao, Ai; Zhang, Jinyi; Tadesse, Yohannes; Qian, David; Weirauch, Matthew; Nair, Rajan; Tsoi, Alex; Pagnoux, Christian; Carette, Simon; Chung, Sharon; Cuthbertson, David; Davis, John C.; Dellaripa, Paul F.; Forbess, Lindsy; Gewurz‐Singer, Ora; Hoffman, Gary S.; Khalidi, Nader; Koening, Curry; Langford, Carol A.; Mahr, Alfred D.; McAlear, Carol; Moreland, Larry; Seo, E. Philip; Specks, Ulrich; Spiera, Robert F.; Sreih, Antoine; St.Clair, E. William; Stone, John H.; Ytterberg, Steven R.; Elder, James T.; Qu, Jia; Ochi, Toshiki; Hirano, Naoto; Edberg, Jeffrey C.; Falk, Ronald J.; Amos, Christopher I.

    2017-01-01

    Objective To identify risk alleles relevant to the causal and biologic mechanisms of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV). Methods A genome‐wide association study and subsequent replication study were conducted in a total cohort of 1,986 cases of AAV (patients with granulomatosis with polyangiitis [Wegener's] [GPA] or microscopic polyangiitis [MPA]) and 4,723 healthy controls. Meta‐analysis of these data sets and functional annotation of identified risk loci were performed, and candidate disease variants with unknown functional effects were investigated for their impact on gene expression and/or protein function. Results Among the genome‐wide significant associations identified, the largest effect on risk of AAV came from the single‐nucleotide polymorphism variants rs141530233 and rs1042169 at the HLA–DPB1 locus (odds ratio [OR] 2.99 and OR 2.82, respectively) which, together with a third variant, rs386699872, constitute a triallelic risk haplotype associated with reduced expression of the HLA–DPB1 gene and HLA–DP protein in B cells and monocytes and with increased frequency of complementary proteinase 3 (PR3)–reactive T cells relative to that in carriers of the protective haplotype. Significant associations were also observed at the SERPINA1 and PTPN22 loci, the peak signals arising from functionally relevant missense variants, and at PRTN3, in which the top‐scoring variant correlated with increased PRTN3 expression in neutrophils. Effects of individual loci on AAV risk differed between patients with GPA and those with MPA or between patients with PR3‐ANCAs and those with myeloperoxidase‐ANCAs, but the collective population attributable fraction for these variants was substantive, at 77%. Conclusion This study reveals the association of susceptibility to GPA and MPA with functional gene variants that explain much of the genetic etiology of AAV, could influence and possibly be predictors of the clinical

  17. Clinical Outcomes of Remission Induction Therapy for Severe Antineutrophil Cytoplasmic Antibody–Associated Vasculitis

    Science.gov (United States)

    Miloslavsky, E. M.; Specks, U.; Merkel, P. A.; Seo, P.; Spiera, R.; Langford, C. A.; Hoffman, G. S.; Kallenberg, C. G. M.; St.Clair, E. W.; Tchao, N. K.; Viviano, L.; Ding, L.; Sejismundo, L. P.; Mieras, K.; Iklé, D.; Jepson, B.; Mueller, M.; Brunetta, P.; Allen, N. B.; Fervenza, F. C.; Geetha, D.; Keogh, K.; Kissin, E. Y.; Monach, P. A.; Peikert, T.; Stegeman, C.; Ytterberg, S. R.; Stone, J. H.

    2016-01-01

    Objective To evaluate the reasons that complete remission is not achieved or maintained with original treatment in some patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) treated with rituximab (RTX) or with cyclophosphamide/azathioprine (CYC/AZA). Methods The Rituximab in AAV trial was a randomized, double-blind, placebo-controlled trial comparing the rate of remission induction among patients treated with RTX (n = 99) and patients treated with CYC followed by AZA (n = 98). Glucocorticoids were tapered over a period of 5 months. The primary outcome measure was lack of disease activity without glucocorticoid treatment at 6 months. To determine the most important reason for failure to achieve the primary outcome, 7 hierarchical categories of reasons were defined retrospectively (uncontrolled disease, adverse event leading to therapy discontinuation, severe flare, limited flare, Birmingham Vasculitis Activity Score for Wegener’s Granulomatosis >0, prednisone treatment at any dosage, and other). Results Although remission (lack of disease activity) was achieved in 170 of the 197 patients (86%) in the first 6 months, the primary outcome measure was not achieved in 42%. There were 3 deaths. Twenty-four percent of the patients failed to achieve the primary end point due to active disease: 10 (5%) experienced uncontrolled disease in the first month and 37 (19%) experienced flares after initial improvement. In the majority of such patients, treatment with blinded crossover or according to best medical judgment led to disease control. Ninety-one percent of patients who had uncontrolled disease or experienced a severe flare had proteinase 3 (PR3)–ANCA. When patients with uncontrolled disease were excluded from analysis, those who were PR3-ANCA positive were found to experience fewer flares when treated with RTX compared to CYC/AZA (8 of 59 [14%] versus 20 of 62 [32%]; P = 0.02). Neither ANCA titers nor B cell counts predicted disease

  18. Development and characterization of three novel monoclonal antibodies against CA-125.

    Science.gov (United States)

    Michurina, Tatiana; Kerzhner, Maxim; Klimovich, Boris

    2014-10-01

    Cancer antigen 125 (CA-125) is the most widely used tumor marker for ovarian cancer. Thus, monoclonal antibodies (MAbs) against CA-125 are valuable reagents for the development of diagnostic tests and immunotherapy. We describe here the generation and characterization of three novel hybridoma cell lines producing MAbs against CA-125. CA-125 purified from culture supernatant of ovarian carcinoma cell line OVCAR-3 by affinity chromatography, was used for immunization of BALB/c mice. Three stable cell lines (3C8, 2B6, and 5A12) were selected for production of antibodies against CA-125 and were expanded in mass culture. All three antibodies were shown to recognize linear epitopes. Antibodies 2B6 and 5A12 were determined to recognize epitope cluster B (M 11-like); MAb 3C8 was classified as group A-epitope binders (OC 125-like). The antibodies produced may be used for the development and improvement of CA-125 immunoassays.

  19. Site-Selective Orientated Immobilization of Antibodies and Conjugates for Immunodiagnostics Development

    Science.gov (United States)

    Rusling, James

    2016-01-01

    Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay. PMID:27876681

  20. Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

  1. Review of Current Cell-Penetrating Antibody Developments for HIV-1 Therapy

    Directory of Open Access Journals (Sweden)

    Muhamad Alif Che Nordin

    2018-02-01

    Full Text Available The discovery of highly active antiretroviral therapy (HAART in 1996 has significantly reduced the global mortality and morbidity caused by the acquired immunodeficiency syndrome (AIDS. However, the therapeutic strategy of HAART that targets multiple viral proteins may render off-target toxicity and more importantly results in drug-resistant escape mutants. These have been the main challenges for HAART and refinement of this therapeutic strategy is urgently needed. Antibody-mediated treatments are emerging therapeutic modalities for various diseases. Most therapeutic antibodies have been approved by Food and Drug Administration (FDA mainly for targeting cancers. Previous studies have also demonstrated the promising effect of therapeutic antibodies against HIV-1, but there are several limitations in this therapy, particularly when the viral targets are intracellular proteins. The conventional antibodies do not cross the cell membrane, hence, the pathogenic intracellular proteins cannot be targeted with this classical therapeutic approach. Over the years, the advancement of antibody engineering has permitted the therapeutic antibodies to comprehensively target both extra- and intra-cellular proteins in various infections and diseases. This review aims to update on the current progress in the development of antibody-based treatment against intracellular targets in HIV-1 infection. We also attempt to highlight the challenges and limitations in the development of antibody-based therapeutic modalities against HIV-1.

  2. Purification of aflatoxin B1 antibody for the development of aflatoxin biosensor

    Science.gov (United States)

    Prihantoro, E. A. B.; Saepudin, E.; Ivandini, T. A.

    2017-07-01

    Aflatoxin B1 (AFB1) is produced from agricultural products especially peanuts overgrown with aspergillus flavus during the post-harvest process. Aflatoxin is classified as a highly toxic and carcinogenic substance to humans by the International Agency for Research on Cancer (IARC), WHO. This research was conducted to develop the AFB1 biosensor using antibody that specifically binds to aflatoxin B1. This antibody was produced by injecting an AFB1 hapten-protein (immunogen) to a rabbit. Antibody was obtained from rabbit's blood serum and purified using Protein A affinity chromatography and precipitation at the isoelectric point. The result showed that purification using protein A contains antibody of 4.0 mg/mL, whereas purification using precipitation at isoelectric pH contains antibody of 0.3 mg/mL. The pure antibody was tested for its specificity against AFB1, tetrahydrofuran (THF), dimethyl formamide (DMF), bovine serum albumin (BSA), and ethanol. The result revealed that THF, BSA, and ethanol were bound to antibody, while DMF showed no interaction. It was concluded that the polyclonal antibody which have been successfully purified from rabbit's blood serum using protein A affinity chromatography and precipitation methods showed an unspecific identification.

  3. Antineutrophil Cytoplasmic Antibodies Testing in a Large Cohort of Unselected Greek Patients

    Directory of Open Access Journals (Sweden)

    Konstantinos Tsiveriotis

    2011-01-01

    Full Text Available Objective. To retrospectively evaluate ANCA testing in a cohort of unselected Greek in- and outpatients. Methods. In 10803 consecutive serum samples, ANCA were tested by indirect immunofluorescence (IIF and ELISA. ELISA in inpatients was performed only on IIF positive sera. Results. Low prevalence (6.0% of IIF positive samples was observed. Among these samples, 63.5% presented perinuclear (p-ANCA, 9.3% cytoplasmic (c-ANCA and 27.2% atypical (x-ANCA pattern. 16.1% of p-ANCA were antimyeloperoxidase (anti-MPO positive, whereas 68.3% of c-ANCA were antiproteinase-3 (anti-PR3 positive. Only 17 IIF negative outpatients' samples were ELISA positive. ANCA-associated vasculitides (AAV, connective tissue disorders and gastrointestinal disorders represented 20.5%, 23.9%, and 21.2% of positive results, respectively. AAV patients exhibited higher rates of MPO/PR3 specificity compared to non-AAV (93.8% versus 8%. Conclusions. This first paper on Greek patients supports that screening for ANCA by IIF and confirming positive results by ELISA minimize laboratory charges without sacrificing diagnostic accuracy.

  4. Poor Renal Outcome of Antineutrophil Cytoplasmic Antibody Negative Pauci-immune Glomerulonephritis in Taiwanese

    Directory of Open Access Journals (Sweden)

    Peir-Haur Hung

    2006-01-01

    Conclusion: This study illustrates the necessity for pathologic diagnosis of pauci-immune GN despite ANCA negativity. The poor prognosis associated with ANCA negativity in this study may be partly due to delayed diagnosis since these patients frequently lacked systemic involvement.

  5. Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains▿

    Science.gov (United States)

    Schanzer, Jürgen; Jekle, Andreas; Nezu, Junichi; Lochner, Adriane; Croasdale, Rebecca; Dioszegi, Marianna; Zhang, Jun; Hoffmann, Eike; Dormeyer, Wilma; Stracke, Jan; Schäfer, Wolfgang; Ji, Changhua; Heilek, Gabrielle; Cammack, Nick; Brandt, Michael; Umana, Pablo; Brinkmann, Ulrich

    2011-01-01

    In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains. PMID:21300827

  6. Systematic review of the role of rituximab in treatment of antineutrophil cytoplasmic autoantibody-associated vasculitis, hepatitis C virus-related cryoglobulinemic vasculitis, Henoch–Schönlein purpura, ankylosing spondylitis, and Raynaud's phenomenon

    Directory of Open Access Journals (Sweden)

    Taha R

    2017-12-01

    Full Text Available Rbab Taha,1 Hadeel El-Haddad,1 Abdulqader Almuallim,2 Fatma Alshaiki,3 Elaf Obaid,2 Hani Almoallim1,2,4 1Department of Medicine, Dr Soliman Fakeeh Hospital, Jeddah, 2Department of Medicine, Faculty of Medicine, Umm Al-Qura University, Mecca, 3Department of Medicine, East Jeddah Hospital, Jeddah, 4Rheumatic Diseases, Umm Al-Qura University, Mecca, Saudi Arabia Abstract: Rituximab (RTX is established for the treatment of rheumatoid arthritis. This systematic review of the literature since 2006 summarizes evidence for the use of RTX in the treatment of additional rheumatological diseases: antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV, hepatitis C virus-related cryoglobulinemic vasculitis, Henoch–Schönlein purpura, ankylosing spondylitis, and Raynaud’s phenomenon. Data from randomized controlled trials are available only for AAV, confirming efficacy for remission induction, including in disease resistant to conventional treatment, and maintenance of remission. Further studies are required to confirm optimal maintenance regimens in AAV, important questions needing to be addressed including protocol administration versus treatment in response to clinical relapse and the importance of maintaining B-cell depletion. Sufficient data are available in other diseases to suggest RTX to be useful and that randomized controlled trials should be conducted. Keywords: anti-CD20 monoclonal antibody, anti-neutrophil cytoplasmic antibody (ANCA-associated vasculitis, refractory ankylosing spondylitis, resistant cryoglobulinemic vasculitis, refractory rheumatological diseases 

  7. Paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development.

    Science.gov (United States)

    Acosta, Eliana G; Bartenschlager, Ralf

    2016-01-01

    Highly effective prophylactic vaccines for flaviviruses including yellow fever virus, tick-borne encephalitis virus and Japanese encephalitis virus are currently in use. However, the development of a dengue virus (DENV) vaccine has been hampered by the requirement of simultaneous protection against four distinct serotypes and the threat that DENV-specific antibodies might either mediate neutralization or, on the contrary, exacerbate disease through the phenomenon of antibody-dependent enhancement (ADE) of infection. Therefore, understanding the cellular, biochemical and molecular basis of antibody-mediated neutralization and ADE are fundamental for the development of a safe DENV vaccine. Here we summarize current structural and mechanistic knowledge underlying these phenomena. We also review recent results demonstrating that the humoral immune response triggered during natural DENV infection is able to generate neutralizing antibodies binding complex quaternary epitopes only present on the surface of intact virions.

  8. Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development.

    Science.gov (United States)

    Tomar, Dheeraj S; Kumar, Sandeep; Singh, Satish K; Goswami, Sumit; Li, Li

    2016-01-01

    Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored.

  9. [Monoclonal antibodies in [corrected] development in [corrected] multiple sclerosis].

    Science.gov (United States)

    Sastre-Garriga, J; Montalban, X

    2011-11-01

    The last fifteen years have see the gradual appearance of a number of different drugs that have been shown to be effective as disease modifying therapies in multiple sclerosis. The opening and subsequent widening of the therapeutic armamentarium in multiple sclerosis will continue on a expanding course in the next few years due to the already known positive results of phase III clinical trials with orally administered molecules. Along with these, we have also seen the appearance of a group of drugs which, instead of being defined by their route of administration, are considered together as a consequence of their similar design: the monoclonal antibodies. The principal safety and efficacy results of three of the monoclonal antibodies that have already obtained positive results in phase II studies will be reviewed in this paper: alemtuzumab, rituximab / ocrelizumab, and daclizumab. For the preparation of this paper, information was obtained from already published articles and from the following web pages: www.clinicaltrials.gov of the National Institute of Health of the U.S.A., the EMA (European Medicines Agency) web page and the Spanish Medicines Agency (Agencia Española del Medicamento) web page. Final results from the phase III clinical trials in progress are required to produce definitive statements on the efficacy and safety of the reviewed drugs. However, and subject to confirmation of the presently available data from phase II trials, it is likely that this group of drugs is to be placed one step beyond the currently available disease-modifying therapies in terms of efficacy, but with a safety pattern which will make careful monitoring of treated patients a mandatory requirement so as to obtain adequate risk/benefit profiles. Copyright © 2010 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

  10. Absence of cross-reactivity to myeloperoxidase of anti-thyroid microsomal antibodies in patients with autoimmune thyroid diseases

    NARCIS (Netherlands)

    Freire, BA; Paula, ID; Paula, F; Kallenberg, GGM; Limburg, PC; Queluz, TT

    Background: Thyroperoxidase is the major antigen of the thyroid microsomal antibodies (TMA) detected in autoimmune thyroid diseases. Its amino acid sequence has 44% homology with myeloperoxidase (MPO), an enzyme present in the primary granules of neutrophils and one of the major antineutrophil

  11. Antibody development in pediatric sickle cell patients undergoing erythrocytapheresis.

    Science.gov (United States)

    Godfrey, Gwendolyn J; Lockwood, William; Kong, Maiying; Bertolone, Salvatore; Raj, Ashok

    2010-12-01

    Erythrocytapheresis, or red blood cell exchange transfusion (RBCX), with donor red blood cell (RBC) units is now increasingly used in the treatment of acute and chronic complications of sickle cell disease (SCD). As in all transfusions, RCBX carries a risk of immunization against foreign antigen on transfused cells. However, by selecting donor units with RBC phenotypes similar to the patient, the risk of allo- and autoimmunization can be reduced. The formation of RBC alloantibodies and/or autoantibodies in 32 multitransfused pediatric SCD patients undergoing monthly RBCX over a 11-year period (12/1998 to 12/2009) was evaluated utilizing a retrospective patient chart review at Kosair Children's Hospital, Louisville, Kentucky. After starting C, E, K antigen-matched RBCX, the rate of clinically significant allo-immunization decreased from 0.189/100 to 0.053/100 U, with a relative risk of 27.9%. Likewise, the rate of autoimmunization decreased from 0.063/100 to 0.035/100 U, with a relative risk of 55.9%. After controlling for clinically insignificant antibodies, our auto- and alloimmunization rate was much less than previously reported values. In addition, the incidence of clinically significant allo- and autoimmunization decreased in our patient population after starting minor antigen-matched RBCX. These results suggest that by matching selected RBC phenotypes, there may be an association in the risk of allo- and autoimmunization of multi-transfused SCD patients.

  12. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

    Science.gov (United States)

    Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

    2018-04-01

    While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

  13. Development of QCM Biosensor with Specific Cow Milk Protein Antibody for Candidate Milk Adulteration Detection

    Directory of Open Access Journals (Sweden)

    Setyawan P. Sakti

    2016-01-01

    Full Text Available Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence of this protein can be used as an indicator of cow milk content in a target solution. To detect the PSS 208 kDa protein, antibody specific to the PSS 208 was developed. The purified antibody was immobilized on top of the sensor surface on a polystyrene layer. The fraction of the immobilized antibody on the sensor was found at 1.5% of the given antibody. Using a static reaction cell, the developed immunosensor could detect the specific cow milk protein in buffer solution. The detection limit is 1 ppm. A linear relationship between frequency change and specific protein of cow milk concentration is found from a concentration of 1 ppm to 120 ppm.

  14. Prion Protein-Specific Antibodies-Development, Modes of Action and Therapeutics Application

    Directory of Open Access Journals (Sweden)

    Tihana Lenac Rovis

    2014-10-01

    Full Text Available Prion diseases or Transmissible Spongiform Encephalopathies (TSEs are lethal neurodegenerative disorders involving the misfolding of the host encoded cellular prion protein, PrPC. This physiological form of the protein is expressed throughout the body, and it reaches the highest levels in the central nervous system where the pathology occurs. The conversion into the pathogenic isoform denoted as prion or PrPSc is the key event in prion disorders. Prominent candidates for the treatment of prion diseases are antibodies and their derivatives. Anti-PrPC antibodies are able to clear PrPSc from cell culture of infected cells. Furthermore, application of anti-PrPC antibodies suppresses prion replication in experimental animal models. Major drawbacks of immunotherapy are immune tolerance, the risks of neurotoxic side effects, limited ability of compounds to cross the blood-brain barrier and their unfavorable pharmacokinetic. The focus of this review is to recapitulate the current understanding of the molecular mechanisms for antibody mediated anti-prion activity. Although relevant for designing immunotherapeutic tools, the characterization of key antibody parameters shaping the molecular mechanism of the PrPC to PrPSc conversion remains elusive. Moreover, this review illustrates the various attempts towards the development of anti-PrP antibody compounds and discusses therapeutic candidates that modulate PrP expression.

  15. Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay

    International Nuclear Information System (INIS)

    Sobotka, A.K.; Valentine, M.D.; Ishizaka, K.; Lichtenstein, L.M.

    1976-01-01

    A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r/sub s/ = 0.96 p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective

  16. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Kui Zhu

    2014-04-01

    Full Text Available Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv, as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs, magnetic nanoparticles (MNPs, quantum dots (QDs and carbon nanomaterials (graphene and carbon nanotube, for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC, which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT, will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  17. Development of a functional antibody by using a green fluorescent protein frame as the template.

    Science.gov (United States)

    Wang, Rongzhi; Xiang, Shuangshuang; Zhang, Yonghui; Chen, Qiuyu; Zhong, Yanfang; Wang, Shihua

    2014-07-01

    Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  18. Urinary levels of high mobility group box-1 are associated with disease activity in antineutrophil cytoplasmic autoantibody-associated vasculitis.

    Directory of Open Access Journals (Sweden)

    Tian-Tian Ma

    Full Text Available High mobility group box-1 (HMGB1, a kind of pro-inflammatory mediator, is associated with inflammatory conditions and tissue damage. Our previous study demonstrated that the circulating levels of HMGB1 correlated with disease activity of antineutrophil cytoplasmic antibody (ANCA-associated vasculitis (AAV. In the current study, we aimed to measure urinary levels of HMGB1 in AAV patients, correlated them to clinical activity index and analysed the immunohistochemical HMGB1 staining in kidney specimens.50 patients with AAV in active stage and 56 patients with AAV in remission were recruited. The urinary levels of HMGB1 were determined by enzyme-linked immunosorbent assay. Moreover, renal biopsy specimens from 27 patients with active AAV were randomly collected to evaluate the deposition of HMGB1.Urinary HMGB1 levels in AAV patients in active stage were significantly higher than those in AAV patients in remission and healthy controls (1.46 [0.56-3.43] versus 0.38 [0.10-1.35] mg/μmolCr, P=0.001; 1.46 [0.56-3.43] versus 0.48 [0.40-0.60] mg/μmolCr, P=0.000, respectively. Further analysis found that urinary levels of HMGB1 correlated with erythrocyte sedimentation rate (r=0.354, p=0.012, C-reactive protein (r=0.289, p=0.042, and Birmingham Vasculitis Activity Score (r=0.350, p=0.013. Renal tissue of active AAV patients showed HMGB1 was mainly expressed in the cytoplasm and the extracellular space. The percentage of HMGB1-negative nuclei in renal tissue of patients with active AAV was significantly higher than that in normal controls (60.6±20.2 % versus 2.7±0.6 %, p<0.01.Urinary levels of HMGB1 may be associated with the disease activity in AAV patients.

  19. VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry.

    Science.gov (United States)

    Häsler, Julien; Flajnik, Martin F; Williams, Gareth; Walsh, Frank S; Rutkowski, J Lynn

    2016-07-01

    B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Antibodies to Lactobacilli and Bifidobacteria in young children with different propensity to develop islet autoimmunity.

    Science.gov (United States)

    Talja, Ija; Kubo, Anna-Liisa; Veijola, Riitta; Knip, Mikael; Simell, Olli; Ilonen, Jorma; Vähä-Mäkilä, Mari; Sepp, Epp; Mikelsaar, Marika; Utt, Meeme; Uibo, Raivo

    2014-01-01

    The intestinal microbiota is essential to the maturation and homeostasis of the immune system. Immunoblot assays were used to establish the prevalence of serum IgG, IgM, and IgA antibodies specific for Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus rhamnosus GG proteins in young children presenting with or without type 1 diabetes (T1D). We demonstrated that children between the ages of 6 and 12 months had a substantial increase in the frequency of IgG antibodies specific for L. rhamnosus GG proteins. We measured IgG, IgM, and IgA class antibody reactivity against B. adolescentis DSM 20083, B. adolescentis DSM 20086, and B. longum DSM 20088 proteins demonstrating significantly higher IgA responses against B. adolescentis DSM 20083 strain proteins in children who developed islet autoimmunity and T1D later in life. B. adolescentis strains showed more IgM type antibodies in children who developed T1D later in life, but the difference was not statistically significant. B. longum proteins were recognized by IgG and IgA antibodies to a higher extent compared to other bacteria studied. These results confirm that differences in immune reactivity against some commensal strains in young children may represent a different risk factor for developing T1D.

  1. Antibodies to Lactobacilli and Bifidobacteria in Young Children with Different Propensity to Develop Islet Autoimmunity

    Directory of Open Access Journals (Sweden)

    Ija Talja

    2014-01-01

    Full Text Available The intestinal microbiota is essential to the maturation and homeostasis of the immune system. Immunoblot assays were used to establish the prevalence of serum IgG, IgM, and IgA antibodies specific for Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus rhamnosus GG proteins in young children presenting with or without type 1 diabetes (T1D. We demonstrated that children between the ages of 6 and 12 months had a substantial increase in the frequency of IgG antibodies specific for L. rhamnosus GG proteins. We measured IgG, IgM, and IgA class antibody reactivity against B. adolescentis DSM 20083, B. adolescentis DSM 20086, and B. longum DSM 20088 proteins demonstrating significantly higher IgA responses against B. adolescentis DSM 20083 strain proteins in children who developed islet autoimmunity and T1D later in life. B. adolescentis strains showed more IgM type antibodies in children who developed T1D later in life, but the difference was not statistically significant. B. longum proteins were recognized by IgG and IgA antibodies to a higher extent compared to other bacteria studied. These results confirm that differences in immune reactivity against some commensal strains in young children may represent a different risk factor for developing T1D.

  2. Development of novel monoclonal antibodies against starch and ulvan - Implications for antibody production against polysaccharides with limited immunogenicity

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Kračun, Stjepan K.; Fangel, Jonatan U.

    2017-01-01

    Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody...

  3. [The development of biochip to detect anti-cholera antibodies in human blood serum].

    Science.gov (United States)

    Utkin, D V; Osina, N A; Spitsin, A N; Kireev, M N; Gromova, O V; Zakharova, T L; Naidenova, E V; Kuklev, V E

    2015-02-01

    The full-scaled agglutinating immunoassay is commonly applied to detect content of antibodies to cholera agent Vibrio cholerae human in blood serum under application of serological diagnostic. The time of analysis implementation amounts to 18 hours. To shorten time of detection of antibodies a biological microchip (biochip) was developed. The biochip represents an activated slide with immobilized corpuscle and soluble antigen cholera agent (O-antigens, cholera toxin). The experimental work resulted in development of scheme of biochip and selection of optimal conditions of sorption and implementation of immunologic analysis using biochip. The application of biochip facilitated to detect specific antibodies to antigens of cholera agent in commercial experimental animal serums and blood serums of ill patients. The time of analysis implementation amounted to 2-3 hours. The results are substantiated by bacteriological and serological methods.

  4. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  5. Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains▿

    OpenAIRE

    Schanzer, Jürgen; Jekle, Andreas; Nezu, Junichi; Lochner, Adriane; Croasdale, Rebecca; Dioszegi, Marianna; Zhang, Jun; Hoffmann, Eike; Dormeyer, Wilma; Stracke, Jan; Schäfer, Wolfgang; Ji, Changhua; Heilek, Gabrielle; Cammack, Nick; Brandt, Michael

    2011-01-01

    In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linker...

  6. Development of a Recombinant Antibody-Based Treatment of Snakebites

    DEFF Research Database (Denmark)

    Engmark, Mikael; Laustsen, Andreas Hougaard; Andersen, Mikael Rørdam

    Antivenom for snakebites is produced by immunization of large mammals with snake venom using atraditional and expensive method developed in the 1890’s. Due to the animal origin, the products arehighly immunogenic and come with a high risk of adverse side effects such as serum sickness...

  7. Development of polyclonal antibodies for the detection of ...

    African Journals Online (AJOL)

    2013-09-11

    Sep 11, 2013 ... uncontrolled drugs has resulted in a need for the development of reliable rHuEPO detection ... EPO and for the sensitive detection of EPO in biological fluids (Bornemann et al., 2003; Mi et al., 2005; ... affinity chromatography using a protein A-Sepharose CL-4B column. (GE Healthcare Company, USA) ...

  8. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Directory of Open Access Journals (Sweden)

    Pan Kyeom Kim

    Full Text Available Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC. Two kinds of chimeric human antibodies (chimeric #7 and #17 were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  9. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Science.gov (United States)

    Kim, Pan Kyeom; Keum, Sun Ju; Osinubi, Modupe O V; Franka, Richard; Shin, Ji Young; Park, Sang Tae; Kim, Man Su; Park, Mi Jung; Lee, Soo Young; Carson, William; Greenberg, Lauren; Yu, Pengcheng; Tao, Xiaoyan; Lihua, Wang; Tang, Qing; Liang, Guodong; Shampur, Madhusdana; Rupprecht, Charles E; Chang, Shin Jae

    2017-01-01

    Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  10. Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis

    International Nuclear Information System (INIS)

    Benedictis, P. de; Minola, A.; Rota, E.; Aiello, R.; Zecchin, B.; Salomoni, A.; Foglierini, M.; Agatic, G.; Vanzetta, F.; Lavenir, R.; Lepelletier, A.; Bentley, E.; Weiss, R.; Cattoli, G.

    2016-01-01

    Full text: Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. (author)

  11. Updates on antibody functions in Mycobacterium tuberculosis infection and their relevance for developing a vaccine against tuberculosis.

    Science.gov (United States)

    Achkar, Jacqueline M; Prados-Rosales, Rafael

    2018-04-12

    A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    OpenAIRE

    Uchigashima, Mikiko; Watanabe, Eiki; Ito, Shigekazu; Iwasa, Seiji; Miyake, Shiro

    2012-01-01

    Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with cl...

  13. B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues.

    Science.gov (United States)

    Mage, Rose G; Lanning, Dennis; Knight, Katherine L

    2006-01-01

    The antibody repertoire of rabbits has interested immunologists for decades, in part because of the ease with which large quantities of high affinity antibodies can be obtained in serum, and in part because of the presence of genetic variants, allotypes, within V(H), C(H) and C(L) regions. Studies of these allotypes led to the initial descriptions of allelic exclusion, and neonatal suppression of serum Ig production (allotype suppression), and were instrumental in demonstrating that V and C regions are encoded by separate genes and are usually expressed in cis. The immune system of rabbit continues to be of interest primarily because of the use of both gene conversion and somatic hypermutation to diversify rearranged heavy and light chain genes and the role that gut-associated lymphoid tissues (GALT) and intestinal flora play in developing the primary (preimmune) antibody repertoire.

  14. The history of monoclonal antibody development – Progress, remaining challenges and future innovations

    Directory of Open Access Journals (Sweden)

    Justin K.H. Liu

    2014-12-01

    This review will focus on the history of monoclonal antibody development – how it has increasingly moved away from using laborious animal models to a more effective phage display system, some of the major drawbacks from a clinical and economical point of view and future innovations that are currently being researched to maximise their effectiveness for future clinical use.

  15. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Science.gov (United States)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  16. Development of a dipstick assay for detection of Leishmania-specific canine antibodies

    NARCIS (Netherlands)

    Schallig, Henk D. F. H.; Cardoso, Luís; Hommers, Marieke; Kroon, Nel; Belling, Guus; Rodrigues, Manuela; Semião-Santos, Saul J.; Vetter, Hans

    2004-01-01

    A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the

  17. Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

    NARCIS (Netherlands)

    Pal, Pankaj; Dowd, Kimberly A.; Brien, James D.; Edeling, Melissa A.; Gorlatov, Sergey; Johnson, Syd; Lee, Iris; Akahata, Wataru; Nabel, Gary J.; Richter, Mareike K. S.; Smit, Jolanda M.; Fremont, Daved H.; Pierson, Theodore C.; Heise, Mark T.; Diamond, Michael S.

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit

  18. Development and Characterization of Mouse Monoclonal Antibodies Reactive with Chicken CD83

    Science.gov (United States)

    This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein con...

  19. High-throughput antibody development and retrospective epitope mapping

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro

    , there are noteworthy differences in the less evolved species of algae as compared to land plants. The dynamic process orchestrating the deposition of these biopolymers both in algae and higher plants, is complex and highly heterogeneous, yet immensely important for the development and differentiation of the cell...... understanding of green plants, thus aiding their industrial applicability, as well as the fundamental biological understanding......Plant cell walls are composed of an interlinked network of polysaccharides, glycoproteins and phenolic polymers. When addressing the diverse polysaccharides in green plants, including land plants and the ancestral green algae, there are significant overlaps in the cell wall structures. Yet...

  20. Development of a peptide conjugate vaccine for inducing therapeutic anti-IgE antibodies.

    Science.gov (United States)

    Licari, Amelia; Castagnoli, Riccardo; De Sando, Elisabetta; Marseglia, Gian Luigi

    2017-04-01

    Given the multifaceted effector functions of IgE in immediate hypersensitivity, late-phase reactions, regulation of IgE receptor expression and immune modulation, IgE antibodies have long represented an attractive target for therapeutic agents in asthma and other allergic diseases. Effective pharmacologic blockade of the binding of IgE to its receptors has become one of most innovative therapeutic strategies in the field of allergic diseases in the last 10 years. Areas covered: The latest strategies targeting IgE include the development of a therapeutic vaccine, able to trigger our own immune systems to produce therapeutic anti-IgE antibodies, potentially providing a further step forward in the treatment of allergic diseases. The aim of this review is to discuss the discovery strategy, preclinical and early clinical development of a peptide conjugate vaccine for inducing therapeutic anti-IgE antibodies. Expert opinion: Outside the area of development of humanized anti-IgE monoclonal antibodies, the research field of therapeutic IgE-targeted vaccines holds potential benefits for the treatment of allergic diseases. However, most of the experimental observations in animal models have not yet been translated into new treatments and evidence of human efficacy and safety of this new therapeutic strategy are still lacking.

  1. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV

    Directory of Open Access Journals (Sweden)

    Hust Michael

    2008-09-01

    Full Text Available Abstract Background Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV and Eastern equine encephalitis virus (EEEV antigenic complex, nor did they react with Chikungunya virus (CHIKV, if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

  2. Challenges to the development of vaccines to hepatitis C virus that elicit neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Heidi Edelgard Drummer

    2014-07-01

    Full Text Available Despite 20 years of research, a vaccine to prevent hepatitis C virus (HCV infection has not been developed. A vaccine to prevent HCV will need to induce broadly reactive immunity able to prevent infection by the 7 genetically and antigenically distinct genotypes circulating world-wide. Hepatitis C virus encodes two surface exposed glycoproteins, E1 and E2 that function as a heterodimer to mediate viral entry. Neutralizing antibodies (NAbs to both E1 and E2 have been described with the major NAb target being E2. The function of E2 is to attach virions to host cells via cell surface receptors that include, but is not limited to, the tetraspanin CD81 and scavenger receptor B class I. However, E2 has developed a number of immune evasion strategies to limit the effectiveness of the NAb response and possibly limit the ability of the immune system to generate potent NAbs in natural infection. Hypervariable regions that shield the underlying core domain, subdominant neutralization epitopes and glycan shielding combine to make E2 a difficult target for the immune system. This review summarizes recent information on the role of neutralizing antibodies to prevent HCV infection, the targets of the neutralizing antibody response and structural information on glycoprotein E2 in complex with neutralizing antibodies. This new information should provide a framework for the rational design of new vaccine candidates that elicit highly potent broadly reactive NAbs to prevent HCV infection.

  3. Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines

    Directory of Open Access Journals (Sweden)

    Madhuri Koti

    2014-05-01

    Full Text Available Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3, which is exceptionally long (up to 66 amino acids, unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor, results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs.

  4. Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.

    Science.gov (United States)

    Boro, Robin C; Singh, K Vikas; Suri, C Raman

    2009-01-01

    The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

  5. Development of antibody-based c-Met inhibitors for targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Lee D

    2015-02-01

    Full Text Available Dongheon Lee, Eun-Sil Sung, Jin-Hyung Ahn, Sungwon An, Jiwon Huh, Weon-Kyoo You Hanwha Chemical R&D Center, Biologics Business Unit, Daejeon, Republic of Korea Abstract: Signaling pathways mediated by receptor tyrosine kinases (RTKs and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics. Keywords: hepatocyte growth factor, ligands, receptor tyrosine kinase, signaling pathway, therapeutic agent

  6. Development of human monoclonal antibodies against diseases caused by emerging and biodefense-related viruses.

    Science.gov (United States)

    Zhu, Zhongyu; Dimitrov, Antony S; Chakraborti, Samitabh; Dimitrova, Dimana; Xiao, Xiaodong; Broder, Christopher C; Dimitrov, Dimiter S

    2006-02-01

    Polyclonal antibodies have a century-old history of being effective against some viruses; recently, monoclonal antibodies (mAbs) have also shown success. The humanized mAb Synagis (palivizumab), which is still the only mAb against a viral disease approved by the US FDA, has been widely used as a prophylactic measure against respiratory syncytial virus infections in neonates and immunocompromised individuals. The first fully human mAbs against two other paramyxoviruses, Hendra and Nipah virus, which can cause high (up to 75%) mortality, were recently developed; one of them, m101, showed exceptional potency against infectious virus. In an amazing pace of research, several potent human mAbs targeting the severe acute respiratory syndrome coronavirus S glycoprotein that can affect infections in animal models have been developed months after the virus was identified in 2003. A potent humanized mAb with therapeutic potential was recently developed against the West Nile virus. The progress in developing neutralizing human mAbs against Ebola, Crimean-Congo hemorrhagic fever, vaccinia and other emerging and biodefense-related viruses is slow. A major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies, is the viruses' heterogeneity and mutability. A related problem is the low binding affinity of crossreactive antibodies able to neutralize a variety of primary isolates. Combinations of mAbs or mAbs with other drugs, and/or the identification of potent new mAbs and their derivatives that target highly conserved viral structures, which are critical for virus entry into cells, are some of the possible solutions to these problems, and will continue to be a major focus of antiviral research.

  7. Pros and cons of different therapeutic antibody formats for recombinant antivenom development

    DEFF Research Database (Denmark)

    Laustsen, Andreas H.; Gutiérrez, José María; Knudsen, Cecilie

    2018-01-01

    Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different...

  8. High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation

    Directory of Open Access Journals (Sweden)

    Kathrin Tissot

    2011-05-01

    Full Text Available Since the initial description of phage display technology for the generation of human antibodies, a variety of selection methods has been developed. The most critical parameter for all in vitro-based approaches is the quality of the antibody library. Concurrent evolution of the libraries has allowed display and selection technologies to reveal their full potential. They come in different flavors, from naïve to fully synthetic and differ in terms of size, quality, method of preparation, framework and CDR composition. Early on, the focus has mainly been on affinities and thus on library size and diversity. Subsequently, the increased awareness of developability and cost of goods as important success factors has spurred efforts to generate libraries with improved biophysical properties and favorable production characteristics. More recently a major focus on reduction of unwanted side effects through reduced immunogenicity and improved overall biophysical behavior has led to a re-evaluation of library design.

  9. Cytoplasmic-anti-neutrophil cytoplasmic antibodies targeting myeloperoxidase in Wegener′s granulomatosis: A rare phenomenon

    Directory of Open Access Journals (Sweden)

    Bhavana M Venkatesh

    2014-01-01

    Full Text Available Wegener′s granulomatosis (WG patients can rarely have antineutrophil cytoplasmic antibodies (ANCAs directed against myeloperoxidase (MPO, producing a cytoplasmic pattern on indirect immunofluorescence (IIF. This has important implications in the diagnosis and pathophysiology of the disease. We present to you a report of three cases of WG, demonstrating a cytoplasmic-ANCA pattern on indirect IIF, but directed against MPO. It is necessary to diagnose a patient taking into account both the autoimmune test results and the clinical features.

  10. Complexity of Human Antibody Response to Dengue Virus: Implication for Vaccine Development

    Directory of Open Access Journals (Sweden)

    Wen-Yang Tsai

    2017-07-01

    Full Text Available The four serotypes of dengue virus (DENV are the leading cause of arboviral diseases in humans. Decades of efforts have made remarkable progress in dengue vaccine development. Despite the first dengue vaccine (dengvaxia from Sanofi Pasteur, a live-attenuated tetravalent chimeric yellow fever-dengue vaccine, has been licensed by several countries since 2016, its overall moderate efficacy (56.5–60.8% in the presence of neutralizing antibodies during the Phase 2b and 3 trials, lower efficacy among dengue naïve compared with dengue experienced individuals, and increased risk of hospitalization among young children during the follow-up highlight the need for a better understanding of humoral responses after natural DENV infection. Recent studies of more than 300 human monoclonal antibodies (mAbs against DENV have led to the discovery of several novel epitopes on the envelope protein recognized by potent neutralizing mAbs. This information together with in-depth studies on polyclonal sera and B-cells following natural DENV infection has tremendous implications for better immunogen design for a safe and effective dengue vaccine. This review outlines the progress in our understanding of mouse mAbs, human mAbs, and polyclonal sera against DENV envelope and precursor membrane proteins, two surface proteins involved in vaccine development, following natural infection; analyses of these discoveries have provided valuable insight into new strategies involving molecular technology to induce more potent neutralizing antibodies and less enhancing antibodies for next-generation dengue vaccine development.

  11. The development of D antibodies after D-mismatched kidney transplantation in a setting of reduced immunosuppression.

    Science.gov (United States)

    Habets, Thomas H P M; Vanderlocht, Joris; Straat, Ron J M H E; van Smaalen, Tim C; Bos, Gerard M J; Beckers, Erik A; Christiaans, Maarten H L; Henskens, Yvonne M C

    2018-01-01

    D antigens are not taken into account in the allocation of solid organs. Female transplant recipients with D antibodies as a consequence of D-mismatched kidney transplantation may develop hemolytic disease of the fetus and newborn in future pregnancies. We examined D antibody development in transplant recipients who received D-mismatched kidney transplantation in absence of D prophylaxis and in a setting of reduced immunosuppression. From 1993 until 2015, a total of 1355 kidney patients received transplantations in our center of whom 156 received a D-mismatched graft. A retrospective analysis was conducted; frozen stored sera obtained from transplant recipients 3 months after transplantation were tested for irregular red blood cell (RBC) antibodies using a three-cell screening and an identification panel. In the case of D antibody positivity, additional testing was performed 1 month before transplantation. In seven of 156 (4.5%) transplant recipients we found irregular RBC antibodies after transplantation, of which five (3.2%) were determined to be D antibodies. We observed only one (0.6%) recipient without D antibodies before transplantation. Although the risk of D antibody development is considerably lower after D-mismatched kidney transplantation than D-mismatched pregnancy, anti-D prophylaxis may still be advisable for female transplant recipients of childbearing age. © 2017 AABB.

  12. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  13. Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

    Directory of Open Access Journals (Sweden)

    Phelps Amanda L

    2009-11-01

    Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

  14. Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A.

    Science.gov (United States)

    Cannavò, Antonino; Valsecchi, Carla; Garagiola, Isabella; Palla, Roberta; Mannucci, Pier Mannuccio; Rosendaal, Frits R; Peyvandi, Flora

    2017-03-09

    The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is the major complication in hemophilia A. Nonneutralizing antibodies (NNAs) have been detected in hemophilia patients and also in unaffected individuals. The aim of this study was to assess the prevalence of NNAs and to evaluate whether their presence is associated with the development of inhibitors in a cohort of previously untreated or minimally treated patients with hemophilia A; plasma samples of 237 patients with severe hemophilia A enrolled in the SIPPET trial were collected before any exposure to FVIII concentrates and analyzed for the presence of anti-FVIII NNAs. Patients were observed for the development of neutralizing antibodies. NNAs were found in 18 (7.6%) of 237 patients at screening, and there was a clear age gradient. Of those with NNAs, 7 patients subsequently developed an inhibitor for a cumulative incidence of 45.4% (95% confidence interval [CI], 19.5% to 71.3%); among the 219 patients without NNAs, 64 (29%) developed an inhibitor (cumulative incidence, 34.0%; 95% CI, 27.1%-40.9%). In Cox regression analyses, patients with NNAs at screening had an 83% higher incidence of inhibitor development than patients without NNAs (hazard ratio [HR], 1.83; 95% CI, 0.84-3.99). For high-titer inhibitors, the incidence rate had an almost threefold increase (HR, 2.74; 95% CI, 1.23-6.12). These associations did not materially change after adjustment. The presence of anti-FVIII NNAs in patients with severe hemophilia A who were not previously exposed to FVIII concentrates is associated with an increased incidence of inhibitors. © 2017 by The American Society of Hematology.

  15. Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Kaneko, Mika K; Nakamura, Takuro; Ichii, Osamu; Konnai, Satoru; Kato, Yukinari

    2017-04-01

    Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We previously established clone PMab-1 of rat IgG 2a as a specific monoclonal antibody (mAb) against mouse PDPN. PMab-1 is also very sensitive in immunohistochemical analysis; however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies in commercially available kits for immunohistochemical analysis. In this study, we develop a mouse-rat chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from rat PMab-1 mAb. Immunohistochemical analysis shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy from rat mAb to mouse mAb could be applicable to other mAbs.

  16. Development of immunoassay based on monoclonal antibody reacted with the neonicotinoid insecticides clothianidin and dinotefuran.

    Science.gov (United States)

    Uchigashima, Mikiko; Watanabe, Eiki; Ito, Shigekazu; Iwasa, Seiji; Miyake, Shiro

    2012-11-15

    Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  17. Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

    Directory of Open Access Journals (Sweden)

    Seiji Iwasa

    2012-11-01

    Full Text Available Enzyme-linked immunosorbent assay (ELISA based on a monoclonal antibody (MoAb was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl-1,3-thiazol-2-ylthio] propionic acid was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA. The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64% to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

  18. Clinical development of monoclonal antibody-based drugs in HIV and HCV diseases

    Directory of Open Access Journals (Sweden)

    Flego Michela

    2013-01-01

    Full Text Available Abstract Today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. Compared to conventional antiviral drugs, monoclonal antibodies (mAbs used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. Despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. The current high cost of mAbs' production, storage and administration (by injection only and the consequent obstacles to development are outweighed by mAbs' clinical advantages. These are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses. This review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mAb-based drugs in clinical trials for HIV and HCV diseases. For each clinical trial the available data are reported and the emerging conceptual problems of the employed mAbs are highlighted. This overview helps to give a clear picture of the efficacy and challenges of the mAbs in the field of these two infectious diseases which have such a global impact.

  19. Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera.

    Science.gov (United States)

    Vrublevskaya, V V; Afanasyev, V N; Grinevich, A A; Skarga, Yu Y; Gladyshev, P P; Ibragimova, S A; Krylsky, D V; Morenkov, O S

    2017-02-01

    Three lateral flow assays (LFAs) for the detection of antibodies against glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera: a competitive double antibody sandwich LFA without a preincubation step (CDAS-gB-LFA), a CDAS-gB-LFA with a preincubation step (pCDAS-gB-LFA), and a competitive direct gB-LFA have been developed and were compared with each other and with a gB-ELISA. The assays are based on monoclonal antibodies to immunodominant epitopes of ADV gB. The pCDAS-gB-LFA proved to be the most specific and sensitive assay to detect antibodies directed to ADV gB. The specificity and sensitivity of the pCDAS-gB-LFA with the use of an LFA reader for test line intensity measurements were 97.6 and 94.9%, respectively. The lower diagnostic sensitivity of the pCDAS-gB-LFA compared to a gB-ELISA reflects its reduced analytical sensitivity, which was shown in titration experiments with positive sera. The pCDAS-gB-LFA, using the reader-based and visual detection modes, showed good agreement in respect to specificity; however, the LFA reader detection provided a higher diagnostic and analytical sensitivity compared to visual detection. The developed pCDAS-gB-LFA is a rapid, sensitive, and specific method for the detection of antibodies to ADV gB and can be used for screening ADV-infected swine in unvaccinated herds. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Intermediate steroid withdrawal after renal transplantation and anti-HLA antibodies (HLA-Abs) development.

    Science.gov (United States)

    Monfá, Elena; San Segundo, David; San Millán, Juan Carlos Ruiz; Sanabria, Judith; Albines, Zoila; Rodrigo, Emilio; Romón, Iñigo; Asensio, Esther; Arias, Manuel; López-Hoyos, Marcos

    Steroid withdrawal in renal transplantation is desirable to avoid their adverse effects. However, by decreasing the immunosuppression, could lead to an increased risk for the development of HLA-Abs. Evaluate the relationship between steroid withdrawal and development of HLA-Abs in renal transplantation. We analyzed sera by Luminex from 182 kidney transplants performed from 1998 to 2011, before and two years after transplantation. All the patients had a pretransplant PRA (panel reactive of antibodies) 20%, we detected HLA-Abs pretransplant by Luminex in 11.5% of patients in both groups, of which, 66.6%, versus 53% (p 0.058), developed new specificities, with a similar percentage of donor specific antibodies (DSA) in both groups (33.33% vs 36.36%), pNS. In the subgroup without pretransplant HLA-Abs (group-I; n=115, group-II; n=45), 6.08% developed de novo HLA-Abs, being DSA 3.4% (Group-I) versus 7.69% in group II with 3.84% DSA (pNS). Steroid withdrawal at 7 months of renal transplantation does not entail a higher risk in terms of HLA-Abs development in patients without pretransplant HLA-Abs and treatment with tacrolimus and MMF, although larger studies are needed to confirm these findings. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  1. Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

    Science.gov (United States)

    Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

    2014-12-01

    An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Biosensor technology for the detection of illegal drugs II: antibody development and detection techniques

    Science.gov (United States)

    Hilpert, Reinhold; Bauer, Christian; Binder, Florian; Grol, Michael; Hallermayer, Klaus; Josel, Hans-Peter; Klein, Christian; Maier, Josef; Makower, Alexander; Oberpriller, Helmut; Ritter, Josef

    1994-10-01

    In a joint project of Deutsche Aerospace, Boehringer Mannheim and the University of Potsdam portable devices for the detection of illegal drugs, based on biosensor technology, are being developed. The concept enrichment of the drug from the gas phase and detection by immunological means. This publication covers the development of specific antibodies and various detection procedures. Antibodies with a high affinity for cocaine have been developed with the aid of specially synthesized immunogens. A competitive detection procedure with biosensors based on optical grating couplers and applying particulate labels has been established, showing a lower detection limit of 10-10 mol/l for cocaine. Additionally, a combination of a displacement-immunoreactor and an enzymatically amplified electrode was investigated, which at present still suffers from insufficient sensitivity of the immunoreactor. An alternative, fleece-matrix based test procedure, where enrichment and detection steps are integrated in a single unit, is promising in terms of simplicity and sensitivity. A simple swab-test for the detection of cocaine at surfaces has been developed, which has a lower detection limit of about 10 ng and which can be performed within one minute.

  3. Internal image anti-idiotypic antibody: A new strategy for the development a new category of prolactin receptor (PRLR) antagonist.

    Science.gov (United States)

    Lan, Hainan; Hong, Pan; Li, Ruonan; L, Suo; Anshan, Shan; Li, Steven; Zheng, Xin

    2017-07-01

    Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2β). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Development of a fluorescent antibody method for the detection of Enterococcus faecium and its potential for coastal aquatic environment monitoring.

    Science.gov (United States)

    Caruso, Gabriella; Monticelli, L S; Caruso, R; Bergamasco, A

    2008-02-01

    A direct, microscopic fluorescent antibody method was developed to detect the occurrence of Enterococcus faecium in coastal aquatic environments and was compared with the conventional membrane filtering method. The "in situ" application of the antibody-based protocol in the analysis of water samples collected from coastal polyhaline habitats demonstrated good sensitivity and ease of implementation. Data obtained with the microscopic technique were in agreement with those obtained from culture counts. The fluorescent antibody method proved to be a rapid and reliable technique for the detection of E. faecium. The advantages and limitations intrinsic to the method are discussed, highlighting the potential of this new technique for monitoring coastal aquatic environments.

  5. Development and Characterization of New Species Cross-Reactive Anti-Sialoadhesin Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Marjorie De Schryver

    2016-03-01

    Full Text Available Sialoadhesin (Sn is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs to target Sn-positive (Sn+ cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species.

  6. Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

    Science.gov (United States)

    Kamachi, Yasuharu; Omasa, Takeshi

    2018-04-01

    Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection

    DEFF Research Database (Denmark)

    Pedersen, C; Nielsen, C M; Vestergaard, B F

    1987-01-01

    A total of 276 sequential serum samples from 34 men with antibodies to the human immunodeficiency virus (HIV) followed up for two to seven years were analysed for HIV antigen and antibodies to the viral core and envelope proteins. Results were correlated with clinical outcome and CD4 T lymphocyte...... count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients...... and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test...

  8. The development of an Enzyme Linked Immunosorbent Assay for detecting Injectious laryngotrachitis viral antibodies in chicken serum

    Directory of Open Access Journals (Sweden)

    Risa Indriani

    2002-06-01

    Full Text Available The aim of this study was to develop an Enzyme-linked immunosorbent assay (ELISA for detection of antibody against gallid herpes virus, the causal agent of infectious laryngotracheitis (ILT in chicken. Its application in experimental chicken under laboratory condition was also evaluated. Results showed that ELISA for ILT was developed successfully with sensitivity and specificity was 98% and 97,14% respectively. The ELISA was able to determine the dynamic of antibodies respond in experimental chickens following vaccination and artificial infection with ILT virus. It was concluded that this ELISA offers a simple, sensitive and specific antibody assay for detection of antibodies against ILT virus in chickens arising from vaccination or infection.

  9. Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection

    DEFF Research Database (Denmark)

    Pedersen, C; Nielsen, C M; Vestergaard, B F

    1987-01-01

    A total of 276 sequential serum samples from 34 men with antibodies to the human immunodeficiency virus (HIV) followed up for two to seven years were analysed for HIV antigen and antibodies to the viral core and envelope proteins. Results were correlated with clinical outcome and CD4 T lymphocyte...... and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test...... count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients...

  10. Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

    OpenAIRE

    Seppänen, H

    1990-01-01

    An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural in...

  11. Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator.

    Science.gov (United States)

    Gakhal, Amandeep K; Jensen, Timothy J; Bozoky, Zoltan; Roldan, Ariel; Lukacs, Gergely L; Forman-Kay, Julie; Riordan, John R; Sidhu, Sachdev S

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.

  12. Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

    Science.gov (United States)

    Moreno, María J; D'Arienzo, Pasquale; Manclús, Juan J; Montoya, Angel

    2011-01-01

    The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

  13. Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies.

    Directory of Open Access Journals (Sweden)

    Anil Thachil

    Full Text Available Porcine deltacoronavirus (PDCoV, also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV, transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV.

  14. Development of monoclonal-antibody-based products for medical research and diagnostic imaging. Technical report, 28 January 1987-31 December 1988 (Final)

    International Nuclear Information System (INIS)

    Rhodes, B.A.; Pant, K.D.; Chauhan, N.; Buckelew, J.; Budd, P.

    1989-04-01

    Two major areas of application of monoclonal antibodies were examined: the development of products to support the 'Antibody Delivery System', a parent-specific and variable antibody formula drug system for use in imaging and treatment of cancer, and the development of an antibody-based radiopharmaceutical for imaging occult abscesses and other conditions involving high concentrations of white blood cells. In development of the Antibody Delivery System components, methods for characterization and purification of monoclonal antibodies were developed and validated; a dot immunoassay test, under the name RhoDot (TM) Immunoassay, was developed for matching antibodies to putative tumor specimen: a radioimmunoassay, under the name PhoChek (TM) Quality Control Test Kit for Radiolabeled Antibodies, was developed and commercialized for measuring the immunoreactive fraction of radiolabeled antibodies specific to colorecal cancer; and a patient-specific quality control test was developed. In development of the antibody-based radiopharmaceutical for imaging occult abscesses, a candidate antibody was identified and produced under U.S. Food and Drug Administration standards preparatory to human clinical trials

  15. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    Science.gov (United States)

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-07

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Exploiting the proteomics revolution in biotechnology: from disease and antibody targets to optimizing bioprocess development.

    Science.gov (United States)

    Heffner, Kelley M; Hizal, Deniz Baycin; Kumar, Amit; Shiloach, Joseph; Zhu, Jie; Bowen, Michael A; Betenbaugh, Michael J

    2014-12-01

    Recent advancements in proteomics have enabled the generation of high-quality data sets useful for applications ranging from target and monoclonal antibody (mAB) discovery to bioprocess optimization. Comparative proteomics approaches have recently been used to identify novel disease targets in oncology and other disease conditions. Proteomics has also been applied as a new avenue for mAb discovery. Finally, CHO and Escherichia coli cells represent the dominant production hosts for biopharmaceutical development, yet the physiology of these cells types has yet to be fully established. Proteomics approaches can provide new insights into these cell types, aiding in recombinant protein production, cell growth regulation, and medium formulation. Optimization of sample preparations and protein database developments are enhancing the quantity and accuracy of proteomic results. In these ways, innovations in proteomics are enriching biotechnology and bioprocessing research across a wide spectrum of applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

    Science.gov (United States)

    Isaacs, Alison T; Jasinskiene, Nijole; Tretiakov, Mikhail; Thiery, Isabelle; Zettor, Agnès; Bourgouin, Catherine; James, Anthony A

    2012-07-10

    Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

  18. Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Sjoerd Rijpkema

    2012-01-01

    Full Text Available Lethal toxin (LT of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8 is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb with toxin-neutralising (TN activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

  19. Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay. [/sup 125/I tracer techniques

    Energy Technology Data Exchange (ETDEWEB)

    Sobotka, A.K.; Valentine, M.D.; Ishizaka, K.; Lichtenstein, L.M.

    1976-07-01

    A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r/sub s/ = 0.96 p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective.

  20. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  1. Preliminary studies of Technetium-99m-labeled antimyosin monoclonal antibody: development of radiopharmaceutical for cardiac evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho, Guilherme Luiz de Castro; Spencer, Patrick Jack; Muramoto, Emiko; Araujo, Elaine Bortoleti de [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: glcarval@ipen.br

    2007-07-01

    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. Specific antibodies to cardiac myosin can therefore bind to the acutely necrotic myocyte, allowing the noninvasive localization and dimension of myocardial infarction. Because of its favorable physical characteristics, low cost, and ready availability, technetium-99m ({sup 99m}Tc) is the radionuclide of choice for scintigraphy. The purpose of this work was to study the labeling of the antimyosin monoclonal antibody with ({sup 99m}Tc for development of a radiopharmaceutical with high sensitivity and specificity used in the diagnostic of the myocardial infarction. The intact monoclonal antibody (IgG{sub 1}) was reduced by treatment with dithiothreitol (DTT) with the consequent generation of free thiol groups (- SH), responsible for the labeling of the antibody with ({sup 99m}Tc. The radiochemical yield was determined using Sephadex G-25 column (PD-10). The percentage of ({sup 99m}Tc-antibody was 90,06% and after purification procedure the radiochemical yield was > 98%. The biodistribution studies showed low uptake in the stomach and thyroid at different times (1, 4 e 24 hours) representing a small amount of unbounded ({sup 99m}Tc and a good stability of the purified ({sup 99m}Tc-antibody. The uptake in the normal heart was relatively low as expected. Based on these results, we concluded that the direct labeling procedure applied to the antimyosin monoclonal antibody allowed the easy preparation of the radiopharmaceutical with good stability to be used in the noninvasive diagnostic of the myocardial infarction. (author)

  2. Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

    Science.gov (United States)

    Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

    2017-12-01

    Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, prabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

  3. Development, persistence, and significance of type 2, poliomyelitis complement-fixing antibody in man.

    Science.gov (United States)

    CASALS, J; OLITSKY, P K; SABIN, A B

    1952-07-01

    Sera from 81 patients with a diagnosis of paralytic or non-paralytic poliomyelitis, and from 159 individuals of similar age groups giving no history of the disease, were tested with a high titered, complement-fixing poliomyelitis antigen of Type 2 (Lansing-like). The antigen consisted of brain tissue from newborn mice injected with the MEF1 strain of virus as previously adapted to these animals. The presence or absence of Type 2 neutralizing antibody in the sera under test was found not to affect the complement fixation. Positive reactions were obtained with 57 per cent of the sera deriving from non-paralytic patients and in 70 per cent from paralytics, when the specimens were tested at a dilution of 1:16. The complement-fixing antibody was often present in highest titer as early as 24 hours after the onset of poliomyelitis, and in almost all instances within 7 days. In about half of the patients a 4-fold or greater drop in titer occurred within 3 months, with little or no change in the others. The incidence of titers of 1:16 or higher with the control sera varied with the season of the year at which they were procured, 3 per cent of the winter samples proving positive and 13 per cent of the summer. The tests of sera from the group of patients from whom poliomyelitis virus was recovered, disclosed no significant differences between those having the paralytic and those having the non-paralytic disease. Type 1 (Brunhilde-like) strains of virus were recovered from many of the patients yielding positive tests, although they presented no evidence of previous or concurrent infection with Type 2 virus. This finding shows that Type 1 virus can give rise in patients to Type 2 complement-fixing antibody. The application of these data to the serologic diagnosis of poliomyelitis infection in man will of necessity be limited until information is obtained on the development, persistence, and significance of complement-fixation reactions with antigens deriving from Type 1 and

  4. A multicentre study to improve clinical interpretation of proteinase-3 and myeloperoxidase anti-neutrophil cytoplasmic antibodies

    DEFF Research Database (Denmark)

    Bossuyt, Xavier; Rasmussen, Niels; van Paassen, Pieter

    2017-01-01

    Objective: The objective of this multicentre study was to improve the clinical interpretation of PR3- and MPO-ANCAs as an adjunct for the diagnosis of ANCA-associated vasculitis (AAV) by defining thresholds and test result intervals based on predefined specificities and by calculating test result...

  5. Comparison of the epidemiology of anti-neutrophil cytoplasmic antibody-associated vasculitis between Japan and the U.K.

    Science.gov (United States)

    Fujimoto, Shouichi; Watts, Richard A; Kobayashi, Shigeto; Suzuki, Kazuo; Jayne, David R W; Scott, David G I; Hashimoto, Hiroshi; Nunoi, Hiroyuki

    2011-10-01

    The epidemiological manifestations of ANCA-associated vasculitis (AAV) differ geographically. However, there have been no prospective studies comparing the incidence of AAV between Japan and Europe over the same time period using the same case definitions. The incidence of AAV was determined by a population-based method in Miyazaki prefecture, Japan, and Norfolk, U.K., between 2005 and 2009. Patients with AAV were defined and classified according to the European Medicines Agency (EMEA) algorithm. The number of incident cases of AAV in Japan and the U.K. were 86 and 50, respectively, and the average annual incidence over the 5-year period was 22.6/million (95% CI 19.1, 26.2) and 21.8/million (95% CI 12.6, 30.9) in Japan and the U.K., respectively. The average age was higher in patients in Japan than in patients in the U.K. [mean (median), 69.7 (72) vs. 60.5 (61) years]. Microscopic polyangiitis (MPA) was the predominant subtype in Japan (83%), while granulomatosis with polyangiitis (Wegener's) was more frequent in the U.K. (66%). As for the pattern of ANCA positivity, >80% of Japanese patients were pANCA/MPO positive, whereas two-thirds of U.K. patients were cANCA/PR3 positive. Renal involvement in MPA was very common in both countries, but was much less common in granulomatosis with polyangiitis in Japan compared with the U.K. There was no major difference in AAV incidence between Japan and the U.K., but this prospective study found MPA and MPO-ANCA to be more common in Japan and granulomatosis with polyangiitis and PR3-ANCA to be more common in the U.K., in line with earlier reports.

  6. Treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis with high-dose intravenous immunoglobulin.

    Science.gov (United States)

    Richter, C; Schnabel, A; Csernok, E; De Groot, K; Reinhold-Keller, E; Gross, W L

    1995-07-01

    In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30 g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.

  7. Development of a Fully Human Anti-PDGFRβ Antibody That Suppresses Growth of Human Tumor Xenografts and Enhances Antitumor Activity of an Anti-VEGFR2 Antibody

    Directory of Open Access Journals (Sweden)

    Juqun Shen

    2009-06-01

    Full Text Available Platelet-derived growth factor receptor β (PDGFRβ is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRβ from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRβ and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRβ and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRβ antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

  8. Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification.

    Science.gov (United States)

    Larkin, J; Lohr, T A; Elefante, L; Shearin, J; Matico, R; Su, J-L; Xue, Y; Liu, F; Genell, C; Miller, R E; Tran, P B; Malfait, A-M; Maier, C C; Matheny, C J

    2015-08-01

    Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Experiments toward the development of a radioimmunoassay for the detection of serum antibodies for the respiratory syncytial virus

    International Nuclear Information System (INIS)

    Heizmann, W.R.

    1982-01-01

    In order to detect an infection by the respiratory syncytial virus (RSV) quickly and safely, a radioimmunassay (RIA) should be developed. Various antigen preparations were compared to one another. The immune serums used in the RIA came from guinea pigs with a RSV antibody titer of up to 320 in the complement binding reaction. A number of observations lead to the discussion of the possibility of the formation (incomplete) of cross-reactive antibodies between virus and host cell. This hypothesis could be well supported through references in the literature. Under the assumption of the existence of cross-reactive antibodies, a further model of the pathogenesis of the RSV illness allows itself to be developed, which could be preceived as an illness with autoimmune components. With this model the varying courses of this disease in different age groups can be easily explained. (orig.) [de

  10. Recent achievements in the development of radiolabeled monoclonal antibodies for diagnosis, therapy and biologic characterization of human tumors

    International Nuclear Information System (INIS)

    Larson, S.M.; Macapinlac, H.A.; Scott, A.M.; Divgi, C.R.

    1993-01-01

    Human tumors express antigenic sites that can serve as targets for radiolabeled monoclonal antibodies for diagnosis, therapy and biologic characterization of human tumors in vivo. Over the last decade, nearly 200 clinical trials have been performed which demonstrate that tumors can be detected with excellent sensitivity and specificity. Tumors which are otherwise occult, particularly for colorectal (anti-CEA and anti-TAG-72 antibodies) and ovarian cancer (anti-TAG-72 and anti-HMFG), are detected in a significant fraction of problem patients. Therapy using radiolabeled antibodies has been effective in lymphomas, leukemias and neuroblastomas, and is beginning to show promise in other solid tumors. Biologic characterization of tumors is likely to become more and more important in the future as monoclonal antibodies against oncogene products, such as her-2-neu, are developed. Development of new antibody forms through genetic engineering techniques, and the continual evolution toward higher resolution imaging instruments, such as PET and SPECT, will lead to further clinical improvements in cancer detection. (orig.)

  11. Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

    Science.gov (United States)

    Säll, Anna; Sjöholm, Kristoffer; Waldemarson, Sofia; Happonen, Lotta; Karlsson, Christofer; Persson, Helena; Malmström, Johan

    2015-11-06

    Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

  12. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    Science.gov (United States)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  13. Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1 and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

  14. Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors

    Science.gov (United States)

    Calpe, Silvia; Correia, Ana C. P.; Sancho-Serra, Maria del Carmen; Krishnadath, Kausilia K.

    2016-01-01

    ABSTRACT Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications. PMID:26967714

  15. Development of Two Murine Antibodies against Neospora caninum Using Phage Display Technology and Application on the Detection of N. caninum

    Science.gov (United States)

    Dong, Jinhua; Otsuki, Takahiro; Kato, Tatsuya; Kohsaka, Tetsuya; Ike, Kazunori; Park, Enoch Y.

    2013-01-01

    Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection. PMID:23308179

  16. Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Tran, Quang Huy; Hanh Nguyen, Thi Hong; Phan, Thi Nga; Mai, Anh Tuan; Nguyen, Thi Thuy; Vu, Quang Khue

    2012-01-01

    This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml −1 to 1 μg ml −1 , and the limit of detection was about 10 ng ml −1 . This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks

  17. Investigation of Anti-Myeloperoxidase Antibodies in a Dog with Bilateral Necrotizing Scleritis

    Directory of Open Access Journals (Sweden)

    Guillaume Cazalot

    2015-09-01

    Full Text Available Necrotizing scleritis is uncommon in dogs and presumed to be immune-mediated. Its clinical pattern and histopathology are similar to ocular lesions observed in humans suffering from granulomatosis with polyangiitis (GPA, formerly named Wegener’s granulomatosis, where the pathogenesis revolves around anti-neutrophil antibodies (e.g., anti-myeloperoxidase. These antibodies are used to diagnose and follow-up the disease in humans, but variants that only affect the eyes often test negative. Here, we present the first case of canine necrotizing scleritis where measurement of anti-myeloperoxidase antibodies was attempted. A 1.5 year-old female Scottish Terrier was presented with bilateral deep multifocal scleromalacia, severe inflammation of corneal/uveal/retrobulbar tissues, perilimbal corneal oedema and neovascularization, hypotony, and mild exophthalmos. Corticosteroids and antibiotics had been administrated (topically and orally without success. Due to painful multifocal scleral perforation with vitreal haemorrhage, the left eye underwent enucleation, so did the right eye one week later. The histopathology of the left eye revealed a neutrophilic and histiocytic scleral infiltration with extension of pyogranulomatous inflammation to the cornea, choroid, ciliary body, and orbital fat. Levels of plasma anti-myeloperoxidase antibodies were not statistically significant to those of 13 healthy dogs. Further research is warranted to investigate the presence and role of anti-neutrophil antibodies in canine necrotizing scleritis.

  18. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Science.gov (United States)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

  19. Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa

    DEFF Research Database (Denmark)

    Ito, T.; Olesen, Niels Jørgen; Skall, Helle Frank

    2010-01-01

    IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected...

  20. Immuno-PET : A navigator in monoclonal antibody development and applications

    NARCIS (Netherlands)

    van Dongen, Guus A. M. S.; Visser, Gerard W. M.; Hooge, Marjolijn N. Lub-de; Perk, Lars R.; de Vries, Elisabeth G. E.

    2007-01-01

    Monoclonal antibodies (mAbs) have been approved for use as diagnostics and therapeutics in a broad range of medical indications, but especially in oncology. In addition, hundreds of new mAbs, engineered mAb fragments, and nontraditional antibody-like scaffolds directed against either validated or

  1. Development of recombinant antibody technology for application in plant pathogen diagnosis

    NARCIS (Netherlands)

    Griep, R.

    1999-01-01

    This thesis describes the applicability of the novel phage display technique to select plant-pathogen-specific monoclonal antibodies (MAbs) from combinatorial antibody libraries. The retrieved MAbs are so specific that they can be used as diagnostic tools in sensitive immunoassays for the

  2. Class II Eplet Mismatch Modulates Tacrolimus Trough Levels Required to Prevent Donor-Specific Antibody Development.

    Science.gov (United States)

    Wiebe, Chris; Rush, David N; Nevins, Thomas E; Birk, Patricia E; Blydt-Hansen, Tom; Gibson, Ian W; Goldberg, Aviva; Ho, Julie; Karpinski, Martin; Pochinco, Denise; Sharma, Atul; Storsley, Leroy; Matas, Arthur J; Nickerson, Peter W

    2017-11-01

    Despite more than two decades of use, the optimal maintenance dose of tacrolimus for kidney transplant recipients is unknown. We hypothesized that HLA class II de novo donor-specific antibody ( dn DSA) development correlates with tacrolimus trough levels and the recipient's individualized alloimmune risk determined by HLA-DR/DQ epitope mismatch. A cohort of 596 renal transplant recipients with 50,011 serial tacrolimus trough levels had HLA-DR/DQ eplet mismatch determined using HLAMatchmaker software. We analyzed the frequency of tacrolimus trough levels below a series of thresholds mismatch. HLA-DR/DQ eplet mismatch was a significant multivariate predictor of dn DSA development. Recipients treated with a cyclosporin regimen had a 2.7-fold higher incidence of dn DSA development than recipients on a tacrolimus regimen. Recipients treated with tacrolimus who developed HLA-DR/DQ dn DSA had a higher proportion of tacrolimus trough levels mismatch. Mean tacrolimus trough levels in the 6 months before dn DSA development were significantly lower than the levels >6 months before dn DSA development in the same patients. Recipients with a high-risk HLA eplet mismatch score were less likely to tolerate low tacrolimus levels without developing dn DSA. We conclude that HLA-DR/DQ eplet mismatch and tacrolimus trough levels are independent predictors of dn DSA development. Recipients with high HLA alloimmune risk should not target tacrolimus levels <5 ng/ml unless essential, and monitoring for dn DSA may be advisable in this setting. Copyright © 2017 by the American Society of Nephrology.

  3. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum.

    Science.gov (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai

    2013-02-01

    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

  5. Radiolabeled monoclonal antibodies. Development of a new method to remove circulating activity - diagnostic applications and implications for therapy

    International Nuclear Information System (INIS)

    Norrgren, K.

    1993-04-01

    The aim of this thesis was to develop and investigate the usefulness of extracorporeal immunoadsorption (ECIA) to remove circulating activity after the localization of radiolabeled monoclonal antibodies to tumors. A compartment model, based on the biokinetics of 125 I-labeled antibodies 96.5 was developed to estimate the effect of ECIA on the tumor-to-normal tissue ratios. ECIA was simulated at different times after injection of the antibodies and the calculations showed an increased diagnostic ratio for several hours after the ECIA procedure, and that an enhancement of the therapeutic ratio was possible. These results led to the development of animal models where the ECIA could be evaluated and the biokinetic behaviour of different radiolabeled monoclonal antibodies investigated with and without the application of ECIA. A general ECIA method, based on biotinylated antibodies and an avidin agarose column as adsorbent, was developed. Studies in tumor bearing nude rats showed that ECIA enhanced of the tumor-to-normal tissue activity ratios by a factor of 4 for the liver, kidneys and bone marrow. For the L6 antibody, the image contrast of tumors localized in one kidney of the rats, was increased from 1.1 to 1.6. A software anthropomorphic phantom was used in Monte Carlo simulations of clinically realistic scintillation camera image acquisition. The effect of ECIA on the contrast enhancement and on the detectability of simulated tumors located centrally in the liver was studied. The contrast increased linearly with an increasing tumor/liver ratio. The contrast was higher for SPECT than for planar images and a contrast of 1.15 required a tumor/liver activity ratio of 1.9 for SPECT and 4.5 for planar images. ECIA in combination with SPECT imaging of radiolabeled antibodies has a great potential in increasing the detectability of tumors. These studies have shown the possibility with ECIA to increase the contrast in radioimmunoimaging and to enhance the therapeutic ratio

  6. Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65.

    Science.gov (United States)

    Liu, Maojun; DU, Gaimei; Zhang, Yue; Wu, Yuzi; Wang, Haiyan; Li, Bin; Bai, Yun; Feng, Zhixin; Xiong, Qiyan; Bai, Fangfang; Browning, Glenn F; Shao, Guoqing

    2016-09-01

    Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

  7. Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole.

    Science.gov (United States)

    Leivo, Janne; Mäkelä, Joonas; Rosenberg, Jaana; Lamminmäki, Urpo

    2016-01-01

    The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 μg L(-1), which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Development of a fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in dogs

    NARCIS (Netherlands)

    Schallig, H. D. F. H.; Schoone, G. J.; Beijer, E. G. M.; Kroon, C. C. M.; Hommers, M.; Ozbel, Y.; Ozensoy, S.; da Silva, E. S.; Cardoso, L. M.; da Silva, E. D.

    2002-01-01

    A fast agglutination screening test (FAST) for the detection of anti-Leishinania antibodies in serum samples from dogs with visceral leishmamosis was developed. The test is based on the direct agglutination test (DAT), but combines a higher parasite concentration with a smaller test volume. In

  9. Monoclonal Antibodies Against IL-13 and IL-31RA in Development for Atopic Dermatitis

    DEFF Research Database (Denmark)

    Hamann, Carsten R; Thyssen, Jacob P

    2017-01-01

    phase two trial has been completed for a humanized antibody against IL-31 receptor alpha, one subunit of the IL-31 receptor complex. This study showed significant dose-dependent reductions in pruritus, EASI scores, and markers of sleep quality. Initial clinical trials for monoclonal antibodies against......The IL-13 and IL-31 cytokines and inflammatory pathways have been identified as important for atopic dermatitis (AD) pathophysiology. Monoclonal antibodies against IL-13 have been studied for the treatment of asthma since 2011. More recently, two phase two trials have been completed...

  10. Mapping the AAV capsid host antibody response towards the development of second generation gene delivery vectors

    Directory of Open Access Journals (Sweden)

    Yu-Shan eTseng

    2014-01-01

    Full Text Available The recombinant Adeno-associated virus (rAAV gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2. Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from monoclonal antibodies, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

  11. Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

    Science.gov (United States)

    Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

    1989-01-01

    Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

  12. Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

    Science.gov (United States)

    Tian, Baomin; Wong, Wah Yau; Uger, Marni D; Wisniewski, Pawel; Chao, Heman

    2017-01-01

    Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MS E peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[( N -maleimidopropionamido)-diethyleneglycol] ester (SM(PEG) 2 ), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol

  13. Development of OMP based indirect ELISA to gauge the antibody titers in bovines against Pasteurella multocida

    Science.gov (United States)

    Dogra, V; Verma, S; Singh, G; Wani, A. H; Chahota, R; Dhar, P; Verma, L; Sharma, M

    2015-01-01

    Pasteurella multocida (P. multocida) is an important pathogen of various domestic animals. The outer membrane proteins (OMPs) play a major role in pathogenesis and immunogenicity of P. multocida. The aim of the study was to develop indirect enzyme linked immuno sorbant assay (ELISA) based on OMPs to ascertain the antibody titers in animals post-infection or to gauge the potency of vaccine. The OMPs were extracted and purified from P. multocida P:52 (vaccine strain) and P. multocida B:2 isolated from natural outbreak of Haemorrhagic septicaemia (HS) and analyzed on SDS PAGE and through western blot. The OMPs profile of the vaccine strain and the isolate from the natural outbreak of HS were found to be similar. Optimization of various components viz. coating antigens, anti-species conjugate, etc. were carried out against both anti-P. multocida hyper immune and pre immune serum. Validation of OMP based indirect ELISA assay to measure immune response against P. multocida in bovine revealed 91% diagnostic sensitivity (DSN) and about 100% diagnostic specificity (DSP) at 25% cut off. OMP based indirect ELISA was found to be more specific, but less sensitive as compared to WCL based assay. PMID:27175202

  14. Quality Control System for Beer Developed with Monoclonal Antibodies Specific to Barley Lipid Transfer Protein

    Directory of Open Access Journals (Sweden)

    Yukie Murakami-Yamaguchi

    2012-10-01

    Full Text Available Non-specific lipid transfer protein (LTP in barley grain reacted with the IgE in sera drawn from food allergy patients. A sandwich-type of enzyme-linked immunosorbent assay (ELISA was developed with mouse monoclonal antibodies raised against LTP purified with barley flour. This ELISA showed a practical working range of 0.3–3 ng/mL and no cross-reactivity with wheat, adlay and rye. Using this ELISA, LTP was determined in several types of barley-foods, including fermented foods such as malt vinegar, barley-malt miso and beer. LTP content in beer of the same kind was approximately constant, even if manufacturing factory and production days were different. Not only as a factor of foam formation and stability but also as an allergen, controlling and monitoring of LTP in beer should be considered. Taken together, our LTP-detecting ELISA can be proposed as an appropriate system for the quality control of beer.

  15. Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes.

    Science.gov (United States)

    Vandevenne, Evelien; Van Buggenhout, Sandy; Duvetter, Thomas; Brouwers, Els; Declerck, Paul J; Hendrickx, Marc E; Van Loey, Ann; Gils, Ann

    2009-09-30

    The enzyme pectin methylesterase (PME) was purified from red ripe tomatoes (Lycopersicon esculentum) and through affinity chromatography two isoenzymes were fractionated (t1PME and t2PME). Further analysis of these two isoenzymes, both having a molar mass of 34.5kDa, revealed a difference in the N-terminal sequence and in amino acid composition. t1PME was identified as the major isoenzyme of PME in tomato fruit. In this study the aim was to develop a toolbox, consisting of monoclonal antibodies, that allows to gain insight into the in situ localization of PME in plant based food systems like tomatoes. A panel of six interesting monoclonal antibodies was raised against both isoenzymes, designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. The differences in epitopes between these monoclonal antibodies were determined using affinity tests towards denatured PME, cross-reactivity tests and inhibition tests. Characterization of these antibodies indicated an immunological difference between t1PME and t2PME, also revealing a conserved epitope on t2PME, carrot PME and strawberry PME. Different epitopes are recognized by the generated antibodies making them excellent probes for immunolocalization of PME by tissue printing. In tomato, t1PME and t2PME showed a pronounced co-localization, especially in the pericarp and the radial arms of the pericarp. Three of the generated antibodies could be used for immunolocalization of PME in carrots (Daucus carota L.), which was only present in the cortex and not in the vascular cylinder of carrots.

  16. Development of a simple and quick immunochromatography method for detection of anti-HPV-16/-18 antibodies.

    Directory of Open Access Journals (Sweden)

    Fumiko Endo

    Full Text Available Immunochromatography (IC is widely used to detect target molecules in biological fluids. Since this method can be performed without a special technique or device, IC is a convenient way to assess the existence of antibodies or pathogens such as viruses and bacteria, simply and quickly. In this study, we established an IC method to detect serum antibodies against oncogenic human papillomavirus (HPV-16 and HPV-18 L1 proteins using recombinant L1 proteins produced by silkworms as antigens. Infection of oncogenic HPVs is a major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. We first measured blood sera of two groups by magnetic beads enzyme-linked immunosorbent assay (MB-ELISA. For the first group, sera were collected prospectively from young women who planned to receive HPV vaccination. The second group consisted of children under 20 years of age, non-vaccinated healthy women, vaccinated healthy women, dysplasia, cervical intraepithelial neoplasia III, and cervical cancer patients. We confirmed that standard vaccination doses significantly increased serum HPV antibody concentrations, and the level was sustained at least more than 30 months after vaccination. In contrast, an increase in antibody concentration was not observed in patients with precancerous cervical changes and cervical cancer. We next measured the samples in both groups using the IC method we originally developed, and found that the measurement values of IC highly correlated with those of MB-ELISA. The simple and quick IC method would be a useful tool for rapid monitoring of L1 specific antibody levels in a non-laboratory environment. With less than one drop of serum, our IC can easily detect serum HPV-16/-18 antibodies within 15 minutes, without the need for electronic devices or techniques.

  17. Development of active immunity in newborn pigs with colostral antibodies by vaccination with gIII-deleted PRV.

    Science.gov (United States)

    Kit, S; Kit, M; McConnell, S; Lawhorn, B

    1994-01-01

    Maternal antibodies interfere with active immunization of swine by gI-deleted pseudorabies virus [(PRV); Aujeszky's disease virus] vaccines. To test the hypothesis that modified-live (MLV) vaccines retaining the PRV gI and with deletions in the PRV glycoprotein gIII and thymidine kinase (TK) genes might be efficacious in circumventing colostral antibody interference, the OMNI-MARK-PRV (gI+ gIII- TK-) vaccine was administered intramuscularly to 13 newborn pigs with colostral antibodies, while 10 pigs from the same litters served as nonvaccinated controls. At 49 days of age, when PRV virus neutralization (VN) antibodies were negative and all nonvaccinated pigs as well as 10 vaccinates were latex agglutination test (LAT)-negative, the pigs were challenged intranasally with the virulent PRV(SHOPE) strain. In support of the hypothesis, it was found that several central nervous system and respiratory disease signs developed in 6 of 10 nonvaccinates, with one fatality, while 2 of 13 vaccinates showed only very mild and transient disease signs. Nonvaccinates lost weight until post challenge day (PCD) 6, did not regain prechallenge weight until PCD 8, and at PCD 11 had gained only 4.9 pounds/pig. Vaccinates gained weight after challenge and at PCD 11 showed a 9.4 pounds/pig weight gain. On PCD 11, the geometric mean titer (GMT) for VN antibodies of the nonvaccinates was 9.3, while the GMT of the vaccinates for VN antibodies was 49.0, showing that vaccinated pigs had been immunologically primed.

  18. Development of a monoclonal antibody-based colony blot immunoassay for detection of thermotolerant Campylobacter species.

    Science.gov (United States)

    Huang, Hongsheng; Phipps-Todd, Beverley; McMahon, Tanis; Elmgren, Catherine L; Lutze-Wallace, Cheryl; Todd, Zoe A; Garcia, Manuel M

    2016-11-01

    Campylobacter species, particularly thermotolerant Campylobacter spp., such as C. jejuni, are major human foodborne pathogens. Culture methods have been routinely used for the detection of this organism in various types of samples. An alternative, simple and rapid confirmation test(s) without further tedious biochemical tests would be useful. Meanwhile, Campylobacter-like colonies can be difficult to identify on agar plates overgrown with competitive bacteria, which can lead to false-negative results. This study was to develop a simple colony blot immunoassay using a new monoclonal antibody (Mab) produced in the present study for rapid screening, confirmation and quantification of campylobacters on culture agar plates. The procedure developed in this study was able to specifically detect thermotolerant Campylobacter spp., but not other non-thermotolerant Campylobacter and non-Campylobacter reference strains tested. This assay could detect 10 5 cells in a single dot. This assay showed 100% correlation with the culture method for the blotted membranes from 21 either chicken meat or vegetable samples experimentally inoculated with thermotolerant campylobacters. Among 101 natural samples of chicken meat (n=44), chicken feces (n=20) and vegetables (n=37), this assay also showed positive for 23 chicken meat and 14 fecal samples that were positive for thermotolerant campylobacters by culture method, and identified four additional suspects that were culture negative. Membranes stored at 4°C for at least 4years could also be used for this assay. The assay developed in this study can be used in quantitative study for immediate or archival usage, and for diagnostic test to preliminarily confirm the presence of thermotolerant Campylobacter on agar plates. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  19. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Kim Yeon-Gu

    2012-05-01

    Full Text Available Abstract Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES was introduced for using two green fluorescence protein (GFP fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

  20. Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity.

    Science.gov (United States)

    Tustian, Andrew D; Laurin, Linus; Ihre, Henrik; Tran, Travis; Stairs, Robert; Bak, Hanne

    2018-02-21

    There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Co-expression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (V H ). This paper details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bispecific antibodies. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

  1. Hapten synthesis and antibody production for the development of a melamine immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Lei Hongtao; Shen Yudong; Song Lijun; Yang Jinyi [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Chevallier, Olivier P.; Haughey, Simon A. [Institute of Agri-Food and Land Use, Queen' s University Belfast, Belfast BT9 5AG, Northern Ireland (United Kingdom); Wang Hong [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Sun Yuanming, E-mail: ymsun@scau.edu.cn [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Elliott, Christopher T., E-mail: chris.elliott@qub.ac.uk [Institute of Agri-Food and Land Use, Queen' s University Belfast, Belfast BT9 5AG, Northern Ireland (United Kingdom)

    2010-04-14

    The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by {sup 1}H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC{sub 50} of 70.6 ng mL{sup -1}, a LOD of 2.6 ng mL{sup -1} and a LOQ of 7.6 ng mL{sup -1}. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.

  2. Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

    Directory of Open Access Journals (Sweden)

    Marisa J Fortunato

    Full Text Available Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

  3. Peripheral CD5+ B Cells in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis

    Science.gov (United States)

    Unizony, Sebastian; Lim, Noha; Phippard, Deborah J.; Carey, Vincent J.; Miloslavsky, Eli M.; Tchao, Nadia K.; Iklé, David; Asare, Adam L.; Merkel, Peter A.; Monach, Paul A.; Seo, Philip; St Clair, E. William; Langford, Carol A.; Spiera, Robert; Hoffman, Gary S.; Kallenberg, Cees G. M.; Specks, Ulrich; Stone, John H.

    2015-01-01

    Objective CD5+ B cells have been conceptualized as a possible surrogate for Breg cells. The aim of the present study was to determine the utility of CD5+ B cells as biomarkers in antineutrophil cytoplasmic antibody–associated vasculitis (AAV). Methods The absolute and relative numbers (percentages) of CD5+ B cells (explanatory variables) were measured longitudinally during 18 months in 197 patients randomized to receive either rituximab (RTX) or cyclophosphamide (CYC) followed by azathioprine (AZA) for the treatment of AAV (Rituximab in ANCA-Associated Vasculitis [RAVE] trial). Outcome variables included disease activity (status of active disease versus complete remission), responsiveness to induction therapy, disease relapse, disease severity, and, in RTX-treated patients, relapse-free survival according to the percentage of CD5+ B cells detected upon B cell repopulation. Results CD5+ B cell numbers were comparable between the treatment groups at baseline. After an initial decline, absolute CD5+ B cell numbers progressively increased in patients in the RTX treatment arm, but remained low in CYC/AZA-treated patients. In both groups, the percentage of CD5+ B cells increased during remission induction and slowly declined thereafter. During relapse, the percentage of CD5+ B cells correlated inversely with disease activity in RTX-treated patients, but not in patients who received CYC/AZA. No significant association was observed between the numbers of CD5+ B cells and induction treatment failure or disease severity. The dynamics of the CD5+ B cell compartment did not anticipate disease relapse. Following B cell repopulation, the percentage of CD5+ B cells was not predictive of time to flare in RTX-treated patients. Conclusion The percentage of peripheral CD5+ B cells might reflect disease activity in RTX-treated patients. However, sole staining for CD5 as a putative surrogate marker for Breg cells did not identify a subpopulation of B cells with clear potential for

  4. Development of new immunoradiometric assay for CA 125 antigen using two monoclonal antibodies produced by immunizing lung cancer cells

    International Nuclear Information System (INIS)

    Kunimatsu, Mihoko; Endo, Keigo; Awaji, Toshikazu

    1988-01-01

    CA 125 is an antigen associated with non-mucinous epithelial ovarian cancer, which is defined by OC 125 antibody developed by immunizing ovarian cancer cells. We have produced two monoclonal antibodies, 130-22 and 145-9, by using the human lung adenocarcinoma cell line PC-9. Both 130-22 and 145-9 antibodies recognized CA 125 antigen. However, the binding sites seemed to be separate from those of OC 125. Testing by 9 immunoradiometric assays (IRMA), using different combinations of the 3 monoclonal antibodies 130-22, 145-9 and OC 125 demonstrated that the best standard curve for detecting CA 125 could be obtained by a 'simultaneous sandwich' assay based on a mixture of 125 I-labeled OC 125 and 130-22 or 145-9 coated beads. One-step IRMA, using 130-22 as a tracer and 145-9 as an immunoadsorbent, also showed good reproducibility and sensitivity for measuring CA 125. Antigens were detectable in the culture supernatants of PC-9 cells and 5 of 6 ovarian cancer and endometrial adenocarcinoma cells. These results indicate that one-step IRMA using 130-22 and 145-9 is useful for detecting CA 125 antigen. (author)

  5. In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability

    Science.gov (United States)

    Sharma, Vikas K.; Patapoff, Thomas W.; Kabakoff, Bruce; Pai, Satyan; Hilario, Eric; Zhang, Boyan; Li, Charlene; Borisov, Oleg; Kelley, Robert F.; Chorny, Ilya; Zhou, Joe Z.; Dill, Ken A.; Swartz, Trevor E.

    2014-01-01

    For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery. PMID:25512516

  6. Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.

    Science.gov (United States)

    Chapman, Kathryn; Adjei, Akosua; Baldrick, Paul; da Silva, Antonio; De Smet, Karen; DiCicco, Richard; Hong, Seung Suh; Jones, David; Leach, Michael W; McBlane, James; Ragan, Ian; Reddy, Praveen; Stewart, Donald I H; Suitters, Amanda; Sims, Jennifer

    2016-01-01

    Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors

  7. Optimized nonclinical safety assessment strategies supporting clinical development of therapeutic monoclonal antibodies targeting inflammatory diseases.

    Science.gov (United States)

    Brennan, Frank R; Cauvin, Annick; Tibbitts, Jay; Wolfreys, Alison

    2014-05-01

    An increasing number of immunomodulatory monoclonal antibodies (mAbs) and IgG Fc fusion proteins are either approved or in early-to-late stage clinical trials for the treatment of chronic inflammatory conditions, autoimmune diseases and organ transplant rejection. The exquisite specificity of mAbs, in combination with their multi-functional properties, high potency, long half-life (permitting intermittent dosing and prolonged pharamcological effects), and general lack of off-target toxicity makes them ideal therapeutics. Dosing with mAbs for these severe and debilitating but often non life-threatening diseases is usually prolonged, for several months or years, and not only affects adults, including sensitive populations such as woman of child-bearing potential (WoCBP) and the elderly, but also children. Immunosuppression is usually a therapeutic goal of these mAbs and when administered to patients whose treatment program often involves other immunosuppressive therapies, there is an inherent risk for frank immunosuppression and reduced host defence which when prolonged increases the risk of infection and cancer. In addition when mAbs interact with the immune system they can induce other adverse immune-mediated drug reactions such as infusion reactions, cytokine release syndrome, anaphylaxis, immune-complex-mediated pathology and autoimmunity. An overview of the nonclinical safety assessment and risk mitigation strategies utilized to characterize these immunomodulatory mAbs and Fc fusion proteins to support first-in human (FIH) studies and futher clinical development in inflammatory disease indications is provided. Specific emphasis is placed on the design of studies to qualify animal species for toxicology studies, early studies to investigate safety and define PK/PD relationships, FIH-enabling and chronic toxicology studies, immunotoxicity, developmental, reproductive and juvenile toxicity studies and studies to determine the potential for immunosuppression and

  8. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  9. Hacking into the granuloma: could antibody antibiotic conjugates be developed for TB?

    Science.gov (United States)

    Ekins, Sean

    2014-12-01

    Alternatives to small molecule or vaccine approaches to treating tuberculosis are rarely discussed. Attacking Mycobacterium tuberculosis in the granuloma represents a challenge. It is proposed that the conjugation of small molecules onto a monoclonal antibody that recognizes macrophage or lymphocytes cell surface receptors, might be a way to target the bacteria in the granuloma. This antibody drug conjugate approach is currently being used in 2 FDA approved targeted cancer therapies. The pros and cons of this proposal for further research are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  11. Process Research and Development of Antibodies as Countermeasures for C. botulinum

    National Research Council Canada - National Science Library

    Meagher, Michael

    2004-01-01

    ...) cells against serotype A botulinum neurotoxin. We have been able to clone and successful express the full intact S-25 antibody provided by Dr. Jim Marks of UCSF at 30 mg/L in 250 mL spinner shake flasks. A manuscript has been submitted on this work.

  12. Effects of a novel anti-exospore monoclonal antibody on microsporidial development in vitro

    Czech Academy of Sciences Publication Activity Database

    Sak, Bohumil; Saková, Kamila; Ditrich, Oleg

    2004-01-01

    Roč. 92, č. 1 (2004), s. 74-80 ISSN 0932-0113 R&D Projects: GA AV ČR IAA6022101 Institutional research plan: CEZ:AV0Z6022909 Keywords : Microsporidia * Encephalitozoon cuniculi * humoral antibodies Subject RIV: EC - Immunology Impact factor: 1.060, year: 2004

  13. Monoclonal antibodies against interleukin 13 and interleukin 31RA in development for atopic dermatitis.

    Science.gov (United States)

    Hamann, Carsten R; Thyssen, Jacob P

    2018-03-01

    The interleukin 13 (IL-13) and IL-31 cytokines and inflammatory pathways have been identified as important for the pathophysiology of atopic dermatitis (AD). Monoclonal antibodies against IL-13 have been studied for the treatment of asthma since 2011. More recently, 2 phase 2 trials have been completed with these antibodies in AD treatment. In both trials, significant reductions of Eczema Area and Severity Index scores were seen. IL-31 is thought to play a role transmitting itch sensation to the central nervous system, and blocking IL-31 activity reduces itch in patients with AD. One phase 2 trial has been completed for a humanized antibody against IL-31 receptor alpha, which is 1 subunit of the IL-31 receptor complex. This study showed significant dose-dependent reductions in pruritus, Eczema Area and Severity Index scores, and markers of sleep quality. Initial clinical trials for monoclonal antibodies against IL-13 and IL-31 receptor A all show promise, although long-term safety and efficacy data are lacking. Nevertheless, these medications will likely play a role in the treatment of moderate-to-severe AD. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  14. Development of the anti-citrullinated protein antibody repertoire prior to the onset of rheumatoid arthritis

    NARCIS (Netherlands)

    van de Stadt, Lotte A.; de Koning, Margret H. M. T.; van de Stadt, Rob J.; Wolbink, Gertjan; Dijkmans, Ben A. C.; Hamann, Dörte; van Schaardenburg, Dirkjan

    2011-01-01

    To examine how anti-citrullinated protein antibody (ACPA) epitope spreading takes place prior to the onset of clinical rheumatoid arthritis (RA), and to analyze the pattern of autoantigen reactivity at the beginning of the immune response. Multiple consecutive serum samples from 79 RA patients who

  15. Development of an immunoanalytical method for the detection of β- and γ- Crystallins and anti-crystallin antibodies. A molecular biomarker for cataract

    Directory of Open Access Journals (Sweden)

    Nayak Sujatha

    2002-01-01

    Full Text Available Purpose: To develop and evaluate an immunoanalytical method for the detection of β - and g-crystallins and anti-crystallin antibodies. Materials and Methods: Beta and g-crystallins isolated from rat lens were used as immunogens to raise polyclonal antibodies in rabbits. Antibody capture assay and western blot analysis showed that the antibodies to β - and g-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA developed to quantitate β - and g-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified β -crystallin antibodies showed that 33 ng of the purified antibody gave an absorbance of 1.1 at 450 nm, indicating the sensitivity of the method. Results: Antibodies to β - and g-crystallins were not detected in serum samples of the cataractous CFY/NIN rats (used as an animal model for induction of experimental cataract by feeding high galactose diet. However, the cataractous rat serum samples effectively displaced β - and g-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of β - and g-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6 - 81.6 mg/µl and 12.4- 19.6 µg/ml, respectively. Conclusions: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract

  16. Development of a new anti-prolactin receptor (PRLR) antibody, F56, which can serve as a PRLR antagonist.

    Science.gov (United States)

    Cui, Huanzhong; Ma, Yun Zhi; Wang, Yanrong; Song, Meng; Zhang, Hui

    2017-02-01

    In this work, we developed a new prolactin receptor (PRLR) antagonist using the hybridoma technique. A series of monoclonal antibodies against prolactin receptor (PRLR) was prepared, from which we characterized and selected one anti-PRLR antibody, F56. Epitome mapping showed that F56 and prolactin (PRL) share a common binding epitope on PRLR, and therefore, F56 could compete with prolactin (PRL) for binding to PRLR. Subsequent experiments indicated that F56 could effectively neutralize PRLR-mediated intracellular signalling molecules, such as signal transducer and activator of transcription (STAT) and extracellular signal-regulated kinase-1 and kinase 2 (ERK1/2), either by endogenously expressed PRLR or in a cell model transfected with PRLR. In addition, further experiments showed that F56 could effectively inhibit PRL-induced cell proliferation. The current study suggests that F56 has potential applications in PRLR-related studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Cross-reactive neutralizing antibody responses to enterovirus 71 infections in young children: implications for vaccine development.

    Directory of Open Access Journals (Sweden)

    Mei-Liang Huang

    Full Text Available BACKGROUND: Recently, enterovirus 71 (EV71 has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C and 11 genotypes (A, B1∼B5 and C1∼C5. In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2, 2000-2001 (B4, 2004-2005 (C4, and 2008 (B5. In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes. METHODS: We collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses. RESULTS: Serology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference. The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1 are specific for genogroup A and may be related to the observed antigenic variations. CONCLUSIONS: This study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions.

  18. An improved and robust DNA immunization method to develop antibodies against extra-cellular loops of multi-transmembrane proteins

    Science.gov (United States)

    Hazen, Meredith; Bhakta, Sunil; Vij, Rajesh; Randle, Steven; Kallop, Dara; Chiang, Vicki; Hötzel, Isidro; Jaiswal, Bijay S; Ervin, Karen E; Li, Bing; Weimer, Robby M; Polakis, Paul; Scheller, Richard H; Junutula, Jagath R; Hongo, Jo-Anne S

    2014-01-01

    Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation. PMID:24121517

  19. Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

    Science.gov (United States)

    Memon, Atta Muhammad; Bhuyan, Anjuman Ara; Chen, Fangzhou; Guo, Xiaozhen; Menghwar, Harish; Zhu, Yinxing; Ku, Xugang; Chen, Shuhua; Li, Zhonghua; He, Qigai

    2017-05-01

    Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 10 2.5 TCID 50 /mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

  20. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A

    2012-01-01

    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  1. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  2. Development of recombinant antivenoms based on mixtures of human antibodies against D. polylepis toxins

    DEFF Research Database (Denmark)

    Knudsen, Cecilie; Andersen, Mikael Rørdam; Lohse, Brian

    With an annual 150,000 deaths and countless amputations and disfigurements, snakebite envenoming is an ever-present threat in many parts of the rural tropical world1. In sub-Saharan Africa, only 1-2% of victims are treated with antivenom, which is currently based on animal-derived antibodies. Due...... to their heterologous origin, antivenoms often provoke serious side effects in human recipients, such as serum sickness and anaphylaxis, which in some cases leads to death.......With an annual 150,000 deaths and countless amputations and disfigurements, snakebite envenoming is an ever-present threat in many parts of the rural tropical world1. In sub-Saharan Africa, only 1-2% of victims are treated with antivenom, which is currently based on animal-derived antibodies. Due...

  3. Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

    Science.gov (United States)

    Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

    2017-01-01

    The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Process Research and Development of Antibodies as Countermeasures for C. botulinum

    Science.gov (United States)

    2009-02-01

    serotypes (A– G ) that cause the human disease botulism [5]. Botulism is characterized by flaccid paralysis and often results in death. The paralytic...of antibody purification is affinity chromatography based on Protein A or Protein G [18–23]. These purification methods are effective, but the sorbent... ProteinA –Sepharose Fast Flow resin (Amersham Biosciences). Alternatively, culture supernatant was di- rectly loaded onto the rProtein A–Sepharose Fast Flow

  5. Immunological development in nestling American kestrels Falco sparverius: post-hatching ontogeny of the antibody response.

    Science.gov (United States)

    Smits, Judit E G; Bortolotti, Gary R

    2008-12-01

    Avian research involving examination of immune function or testing of immunocompetence in wild birds has been based upon information on Galliforms, (chicken and quail) even though they are precocial, whereas most wild species with which ecologists, biologists and toxicologists work are altricial; blind, naked and completely dependent at hatching. Here we begin to address this gap in knowledge, offering insight into the early, post-hatching, humoral immune response in an altricial bird, the American kestrel (Falco sparverius). Over two breeding seasons, nestling kestrels were immunized with a non-pathogenic antigen, dinitrophenol keyhole limpet hemocyanin (DNP-KLH), between 3 and 9 days post-hatching and boostered 6 days later. Background levels, primary and secondary immune responses were measured using an enzyme linked immunosorbent assay. The specificity of our laboratory produced rabbit, anti-kestrel antibody was determined using a double immunodiffusion assay. Results showed the rabbit antiserum to have specific anti-kestrel IgG activity. Birds as young as three days old could successfully mount an antibody response, the magnitude of which increased with age at first vaccination. Early immunization did not compromise growth rate, nor did it affect the maximum secondary response. Comparatively, adult kestrels immunized during the same season and following the same protocol, had antibody levels four times higher than those of the nestlings.

  6. Oculocutaneous albinism: developing novel antibodies targeting the proteins associated with OCA2 and OCA4.

    Science.gov (United States)

    Kondo, Taisuke; Namiki, Takeshi; Coelho, Sergio G; Valencia, Julio C; Hearing, Vincent J

    2015-01-01

    Patients with oculocutaneous albinism (OCA) have severely decreased pigmentation of their skin, hair and eyes. OCA2 and OCA4 result from mutations of the OCA2 and SLC45A2 genes, respectively, both of which disrupt the trafficking of the critical melanogenic enzyme tyrosinase to melanosomes. Both proteins encoded by those loci (termed P and MATP, respectively) have 12 putative transmembrane regions and are thought to function as transporters, although their functions and subcellular localizations remain to be characterized. To generate specific antibodies against unique synthetic peptides encoded by P and MATP that could be used to characterize their functions and subcellular localizations. Western blotting and immunohistochemistry were used to assess the specificity of antibodies and to colocalize P and MATP proteins with various subcellular markers. Specific antibodies to the P and MATP proteins were generated that work well for Western blotting and immunohistochemistry. The localizations of P and MATP with various subcellular organelles were characterized using confocal microscopy, which revealed that they colocalize to some extent with LAMP2, but do not significantly colocalize with markers of the ER, Golgi or melanosomes. Interestingly, both P and MATP colocalize significantly with BLOC-1, a sorting component involved in the intracellular trafficking of melanosomal/lysosomal constituents. These results provide a basis to understand how disrupted functions of P or MATP result in the misrouting of tyrosinase and cause the hypopigmentation seen in OCA2 and OCA4. Copyright © 2014. Published by Elsevier Ireland Ltd.

  7. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  8. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    International Nuclear Information System (INIS)

    Duan, Hongying; Yoshimura, Kazunori; Kobayashi, Nobuharu; Sugiyama, Kazuo; Sawada, Jun-ichi; Saito, Yoshiro; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    2012-01-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  9. Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development

    Directory of Open Access Journals (Sweden)

    Victor Greiff

    2017-05-01

    Full Text Available Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1 B cell development (pre-B cell, naive B cell, plasma cell, (2 antigen exposure (three structurally different proteins, and (3 four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size extracted from antibody repertoire sequencing data (400 million reads. Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells and antigen-driven (e.g., 40% for clonal diversity in plasma cells predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.

  10. Maternal antibodies from mothers of children with autism alter brain growth and social behavior development in the rhesus monkey.

    Science.gov (United States)

    Bauman, M D; Iosif, A-M; Ashwood, P; Braunschweig, D; Lee, A; Schumann, C M; Van de Water, J; Amaral, D G

    2013-07-09

    Antibodies directed against fetal brain proteins of 37 and 73 kDa molecular weight are found in approximately 12% of mothers who have children with autism spectrum disorder (ASD), but not in mothers of typically developing children. This finding has raised the possibility that these immunoglobulin G (IgG) class antibodies cross the placenta during pregnancy and impact brain development, leading to one form of ASD. We evaluated the pathogenic potential of these antibodies by using a nonhuman primate model. IgG was isolated from mothers of children with ASD (IgG-ASD) and of typically developing children (IgG-CON). The purified IgG was administered to two groups of female rhesus monkeys (IgG-ASD; n=8 and IgG-CON; n=8) during the first and second trimesters of pregnancy. Another control group of pregnant monkeys (n=8) was untreated. Brain and behavioral development of the offspring were assessed for 2 years. Behavioral differences were first detected when the macaque mothers responded to their IgG-ASD offspring with heightened protectiveness during early development. As they matured, IgG-ASD offspring consistently deviated from species-typical social norms by more frequently approaching familiar peers. The increased approach was not reciprocated and did not lead to sustained social interactions. Even more striking, IgG-ASD offspring displayed inappropriate approach behavior to unfamiliar peers, clearly deviating from normal macaque social behavior. Longitudinal magnetic resonance imaging analyses revealed that male IgG-ASD offspring had enlarged brain volume compared with controls. White matter volume increases appeared to be driving the brain differences in the IgG-ASD offspring and these differences were most pronounced in the frontal lobes.

  11. Covalent immobilization of rabbit-antiaflatoxin-antibodies onto the poly-acrylamideacrylonitrile as well as hybrid material UREASIL and developing an optical immunosensor

    Directory of Open Access Journals (Sweden)

    Slavova M.

    2017-03-01

    Full Text Available The aim of this work is to describe a covalent immobilization of antibodies onto the poly- acrylamide-acrylonitrile or hybrid material UREASIL and creation of optical immunosensor for determination of aflatoxin Bl. For this purpose, mouse-anti-aflatoxin B1 antibodies with oxidized carbohydrate moieties were covalently immobilized on the membranes of polyacrylamide- polyacrylonitrile copolymer, as well as the hybrid material UREASIL. To determine the affinity> binding of the immobilized antibody with afatoxin Bl was used "sandwich" method. Associated with the immobilized antibody sought ingredients interact with a surplus of secondary’ signal antibodies. The described method has been developed as a model system, which can easily be applied for the determination of aflatoxins in samples of different origin. To the best of our knowledge, this is the first study to show that in the establishment of biosensor was used hybrid material UREASIL.

  12. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    International Nuclear Information System (INIS)

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders

  13. Evolution of anti-Trypanosoma cruzi antibody production in patients with chronic Chagas disease: Correlation between antibody titers and development of cardiac disease severity.

    Directory of Open Access Journals (Sweden)

    Ingebourg Georg

    2017-07-01

    Full Text Available Chagas disease is one of the most important endemic infections in Latin America affecting around 6-7 million people. About 30-50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005 between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF in chronic Chagas cardiomyopathy (CCC patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND of the disease (p = 0.0637. As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy.

  14. Evolution of anti-Trypanosoma cruzi antibody production in patients with chronic Chagas disease: Correlation between antibody titers and development of cardiac disease severity

    Science.gov (United States)

    Georg, Ingebourg; Hasslocher-Moreno, Alejandro Marcel; Xavier, Sergio Salles; de Holanda, Marcelo Teixeira; Bonecini-Almeida, Maria da Gloria

    2017-01-01

    Chagas disease is one of the most important endemic infections in Latin America affecting around 6–7 million people. About 30–50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005) between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF) in chronic Chagas cardiomyopathy (CCC) patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND) of the disease (p = 0.0637). As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy. PMID:28723905

  15. The future of monoclonal antibody technology

    OpenAIRE

    Zider, Alexander; Drakeman, Donald L

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  16. Production of polyclonal antibody against madecassoside and development of immunoassay methods for analysis of triterpene glycosides in Centella asiatica.

    Science.gov (United States)

    Tassanawat, Patcharin; Putalun, Waraporn; Yusakul, Gorawit; Sritularak, Boonchoo; Juengwatanatrakul, Thaweesak; Tanaka, Hiroyuki

    2013-01-01

    Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory. To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica. An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy. The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39-50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5-500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography. These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Development of the nanotiter plate for use in antibody and cell array technologies

    Science.gov (United States)

    Ramdutt, Devin; Lui, Rodney; Davies, Kerrie; Boswell, Rod W.; dos Remedios, Cristobal G.; Charles, Christine; Bilek, Marcela M.; McKenzie, David R.

    2005-02-01

    The design and fabrication of biomedical tools using techniques common in microelectronics is becoming established procedure. In our research, we use gaseous plasma dry etching to form microstructures on silicon wafers. These are intended for use in capturing and binding antibodies and live cells in an array to be used in High Throughput Screening (HTS) and High Content Screening (HCS) of new pharmaceuticals. We call this new arraying plate the "Nanotiter" plate. The benefit of our design (100 x 100 wells in a 25 x 25 mm array) over current 96-, 384- and 1056-well microtiter plates are that the number of samples (wells) that can be tested in one plate scan can be substantially increased, the wells can be rapidly and effectively washed, and the well surfaces can be modified to modulate ligand binding. Simple crowding of wells on a plate can result in cross contamination of samples in adjacent wells during the washing. Furthermore, motile cells may migrate between the wells. 1056 microtiter plates currently cannot be washed, and washing 384 plates is problematic. Our design incorporates plasma-deposited polymers that functionally bind antibodies (or other proteins) in but not between wells. Furthermore, the wells can be shaped to minimize cell migration. Inverting the plate on a wash solution allows unbound cells to simply fall away under gravity thus minimising the contamination of adjacent wells. Thus, our Nanotiter plate represents a substantial improvement over existing technology.

  18. Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

    DEFF Research Database (Denmark)

    Boesen, Henriette T.; Jensen, Tim Kåre; Jungersen, Gregers

    2005-01-01

    Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic...... with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment...

  19. TSOL18 Vaccine Antigen of Taenia solium: Development of Monoclonal Antibodies and Field Testing of the Vaccine in Cameroon

    Directory of Open Access Journals (Sweden)

    Assana, E.

    2010-01-01

    lack of knowledge of the taeniasis-cysticercosis complex and the absence of a pig pen in the household were associated with pig cysticercosis. Chapter 3 reports the investigations that were undertaken to characterise whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N— and the two truncations (TSOL18N—-1 and TSOL18N—-2 were used in inhibition ELISA to determine their ability to inhibit the binding of protective pig antibodies to TSOL18. TSOL18N— was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N— antibodies to TSOL18N— in ELISA. However, neither TSOL18N—-1 nor TSOL18N—-2, either alone or combined, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N— antibodies with TSOL18N—. It is concluded that the dominant antibody specificities, and likely the host-protective specificities, of TSOL18 are conformational epitopes. Chapter 4 describes the development of an antibody detection test for the specific diagnosis of porcine cysticercosis. A fraction with a major band of 14 kDa was obtained from crude cyst fluid (CF of T. solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration was purified using an anion exchange column on High Performance Liquid Chromatography (HPLC. Evaluation of the analytic sensitivity of this fraction (F3 was carried out in an antibody detection enzyme-linked immunosorbent assay (Ab-ELISA-F3 using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica

  20. Development and evaluation of a double antibody sandwich ELISA for the detection of human sDC-SIGN.

    Science.gov (United States)

    Chen, Shang-Liang; Li, Yan-Li; Tang, Yuan; Chen, Zhi-Cheng; Zhou, Jing; Zhou, Jia; Lu, Xiao; Zhao, Na; Chen, Zheng-Liang; Zuo, Daming

    2016-09-01

    sDC-SIGN is the soluble form of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, CD209), which is a molecule involved with pathogen recognition and immune regulation. However, there is no commercially available ELISA kit for detecting human sDC-SIGN, and the normal range of this molecule is unknown. Here, we describe an ELISA for detecting human sDC-SIGN with high specificity. First, sDC-SIGN protein was expressed and purified. Monoclonal and polyclonal antibodies were then raised against the purified protein and subsequently characterized. A sandwich ELISA was developed using polyclonal antibodies specific for sDC-SIGN for capture and a biotin-labeled monoclonal antibody specific for sDC-SIGN for detection of protein. This method has sensitivity up to 0.2 ng/ml. Using this ELISA, we found that the concentration of sDC-SIGN in sera of healthy volunteers ranges from 0-319 ng/ml with a mean concentration of 27.14 ng/ml. Interestingly, the concentration of sDC-SIGN in sera from patients with cancer or chronic hepatitis B virus (CHB) infection was lower than that of health controls. The mean concentrations of sDC-SIGN in cancer patients and chronic hepatitis B virus infection patients were 3.2 ng/ml and 3.8 ng/ml, respectively. We developed a sandwich ELISA for detecting human sDC-SIGN and demonstrated its use by assessing sera concentrations of sDC-SIGN in patients with cancer and chronic CHB infection compared to that of healthy controls. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. How Can HIV-Type-1-Env Immunogenicity Be Improved to Facilitate Antibody-Based Vaccine Development?

    NARCIS (Netherlands)

    Klasse, Per Johan; Sanders, Rogier W.; Cerutti, Andrea; Moore, John P.

    2012-01-01

    No vaccine candidate has induced antibodies (Abs) that efficiently neutralize multiple primary isolates of HIV-1. Preexisting high titers of neutralizing antibodies (NAbs) are essential, because the virus establishes infection before anamnestic responses could take effect. HIV-1 infection elicits

  2. Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter

    1996-01-01

    Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were...... techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity......) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also...

  3. Developing a Noninvasive Procedure Using Labeled Monoclonal Antibody Anti-VEGF (Bevacizumab) for Detection of Endometriosis

    Science.gov (United States)

    Machado, Daniel Escorsim; Perini, Jamila Alessandra; Orlando, Margarida Maria Camoes

    2015-01-01

    The off-label use of bevacizumab labeled with 99mTc as a new radiopharmaceutical for imaging of endometriosis is a promising noninvasive, new clinical procedure. The bevacizumab in monoclonal antibodies targeted at vascular endothelial growth factor (VEGF) is superexpressed in cases of endometriosis. In this study we evaluate the imaging of endometriosis lesion in rats (induced to endometriosis) using bevacizumab-99mTc. The results showed that bevacizumab-99mTc imaged the lesion and support his use for Nuclear Medicine applied to gynecology. Also the results appointed that this radiopharmaceutical has a hepatobiliary excretion. It is important to notice that the dose used was almost 0,01% of the usual dose for the bevacizumab. PMID:26240826

  4. The development and applications of polyclonal and monoclonal antibodies for the detection of illicit drugs in saliva samples

    OpenAIRE

    Fanning, Lorna M.

    2002-01-01

    Anti-tetrahydrocannabinol (THC), anti-cocaine and anti-morphine polyclonal antibodies were produced. These antibodies were successfully applied to an ELISA format for the detection of THC, cocaine, and morphine in saliva samples. Monoclonal antibodies against amphetamine and its derivatives were produced using two different conjugates, amphetamine-bovine serum albumin and methamphetaminebovine serum albumin. Two successful clones were produced, and the antibodies were applied to an ELISA ...

  5. Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis.

    Science.gov (United States)

    Wang, Xiaolei; Wang, Yan; Ma, Limei; Zhang, Ran; De, Yanyan; Yang, Xiaowen; Wang, Chuanqing; Wu, Qingmin

    2015-05-21

    Brucellosis is the most common bacterial zoonosis, and serological tests are routinely used in brucellosis control and eradication programs. In order to improve the accuracy of serological diagnostic method used in bovine brucellosis detection, this study developed an improved competitive ELISA with higher specificity and good sensitivity. This study prepared 12 monoclonal antibodies against smooth Brucella lipopolysaccharide. One monoclonal antibody 3 F9, presented C epitope specificity, was used to develop a competitive ELISA for the serological detection of bovine brucellosis. The competitive ELISA, a commercial competitive ELISA kit, the rose-bengal plate agglutination test, and a microplate agglutination test were all used in the detection of 6 hyperimmune antisera against other commonly cross-reacted bacterial pathogens and 110 clinical bovine serum samples. The results of the test comparisons indicated that the competitive ELISA had higher specificity than the commercial competitive ELISA kit and RBT, and comparable sensitivity with the commercial ELISA kit. This study provided a valuable detection tool with high specificity and good sensitivity, which prevent the wrong-culling of bovines in the eradication campaigns of bovine brucellosis.

  6. De novo hepatitis B virus infection developing after liver transplantation using a graft positive for hepatitis B core antibody.

    Science.gov (United States)

    Han, Jae Hyun; Kim, Dong Goo; Na, Gun Hyung; Kim, Eun Young; Lee, Soo Ho; Hong, Tae Ho; You, Young Kyoung; Choi, Jong Young; Yoon, Seung Kew

    2015-09-01

    The use of hepatitis B core antibody (HBcAb)-positive grafts is increasing, especially where hepatitis B is endemic. However, this remains controversial because of the risk of development of de novo HBV infection. We collected information obtained between January 2000 and December 2012 and retrospectively analyzed data on 187 HBsAg-negative donors and recipients were analyzed retrospectively. De novo HBV infection was defined as development of HBsAg positivity with or without detection of HBV DNA. Forty patients (21.4%) received HBcAb-positive grafts. Survival rate did not differ by donor HBcAb status (P = 0.466). De novo HBV infection occurred in five patients (12.5%) who were not treated with anti-HBV prophylaxis, and was significantly more prevalent in hepatitis B surface antibody (HBsAb)- and HBcAb-negative than HBsAb- and HBcAb-positive recipients (50% vs. 4.2%, P = 0.049). All patients except one were treated with entecavir with/without antihepatitis B immunoglobulin and four were negative in terms of HBV DNA seroconversion. No patient died. HBcAb-positive grafts are safe without survival difference. However, the risk of de novo hepatitis B virus infection was significantly increased in HBsAb- and HBcAb-negative recipients. All patients were successfully treated even after recurrence.

  7. Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker

    Science.gov (United States)

    Dorňáková, Veronika; Salazar-Sanchez, Renzo; Borrini-Mayori, Katty; Carrion-Navarro, Oscar; Levy, Michael Z.; Schaub, Günter A.; Schwarz, Alexandra

    2014-01-01

    Background Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure. Methodology and Principal Findings In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs. Conclusion Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted

  8. Development of a single dilution ELISA to detect antibody to Dermatophilus congolensis in goat and cattle sera.

    Science.gov (United States)

    Martinez, D; Mari, B; Aumont, G; Vidalenc, T

    1993-01-01

    A solid phase immunosorbent assay to detect antibodies to Dermatophilus congolensis in ruminant sera was developed to be used as a single dilution ELISA in large epidemiological surveys. Optimal conditions for the test are described. The use of blocking proteins to reduce non specific binding was necessary. Non fat dry cow milk and fetal calf serum were the only two efficient blocking agents out of six tested. Comparison of 4 antigenic fractions obtained after sonication and differential centrifugations of D. congolensis cultures showed that cell-wall (CW) or membrane (M) enriched preparations were more specific than a crude extract (CR) or a soluble (S) antigen. Whole spores and filaments performed poorly as antigens. The best sensitivity and specificity of the ELISA were obtained when the cut-off point of positivity was fixed at mean absorbance of negative sera + 2.58 sd. The specificity was then 97.6% either with M or CR antigen. The sensitivity was improved from 93.4% with CR to 98.2% with M antigen. Threshold values for a positive test varied between the 3 geographical areas tested. CW and M were also the most efficient antigens for discerning between serotypes of D. congolensis. The precision of the test was evaluated with CR antigen and expressed in residual expressed in residual coefficient of variation (CV). The precision was CV = 5.1% when each serum was titrated in duplicate and the antibody levels were expressed in absorbances. The expression of antibody levels in arbitrary standard units estimated from calibration curves reduced the precision (CV = 13.8%). Several methods were tested to decrease between plate variability but these did not greatly improve the reproducibility since it was shown that the main source of variation was within the plate.

  9. Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

    Science.gov (United States)

    Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

    2018-01-23

    The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

  10. Characterization of guinea pig antibody responses to salivary proteins of Triatoma infestans for the development of a triatomine exposure marker.

    Directory of Open Access Journals (Sweden)

    Veronika Dorňáková

    2014-04-01

    Full Text Available Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential

  11. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi

    2014-01-01

    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  12. Development of a immunochromatographic test with avidin-biotin for the detection of antibodies against antigen e of hepatitis B in human plasma

    International Nuclear Information System (INIS)

    Mainet Gonzalez, Damian; Palenzuela Gardon, Daniel O; Diaz Argudin, Tamara

    2007-01-01

    The disappearance of antigen e of hepatitis B in the presence of the plasmatic antibodies against antigen e may indicate a satisfactory therapeutic response in patients with chronic hepatitis B. The immuno-chromatographic test carried out in the diagnosis of diseases use different antibody combinations and may employ the avidin or streptavidin-biotin technology to develop a rapid immuno-chromatographic test for the detection of antibodies anti-antigen e in the plasma. They were detected in the laboratory by means of two fast immuno-chromatographic tests when using in one of them the avidin-biotin technology. These tests are carried out with a one-step competitive inhibition format and amplified or not with avidin-biotin. Monoclonal antibodies against antigen e obtained by cellular hybridization were used. Forty-six plasmatic samples classified as positive and negative to the anti-antigen antibodies were evaluated with a reference immunochromatographic test Advanced QualityTM. The possible expiry time of the biological reagents forming part of these tests were studied with accelerated thermal-stability experiments. The possible interference in the plasma of some of the biochemical compounds used in these trials was analyzed. Four murine monoclonal antibodies anti-antigen e were obtained and only one of them was used in these immunochromatographic tests with an anti-antigen polyclonal antibody conjugated with gold. Both tests and their stable biological reagents discriminated the positive and negative samples to the antibodies anti-antigen e, as well as the commercial test. There was no interference in the biochemical compounds studied in these tests. Both immuno-chromatographic tests made in the laboratory are useful to detect antibodies anti-antigen e in the plasma. The avidin-biotin increased the analytical sensitivity of this type of fast immuno-chromatographic test without altering its performance features. (Author)

  13. Development and validation of an indirect enzyme immunoassay for detection of antibody to Brucella abortus in milk.

    Science.gov (United States)

    Nielsen, K; Smith, P; Gall, D; Perez, B; Cosma, C; Mueller, P; Trottier, J; Cote, G; Boag, L; Bosse, J

    1996-09-01

    An indirect enzyme immunoassay for detection of antibody to Brucella abortus in bovine milk was developed and validated using 6238 milk samples from Canadian herds (brucellosis free) and 202 samples from herds infected with B. abortus (from Argentina and Chile). The assay utilized lipopolysaccharide as the antigen, immobilized on the polystyrene matrix, whole milk to test and a mouse monoclonal antibody, specific for an epitope of bovine IgG1, conjugated with horseradish peroxidase. The sensitivity of the assay was 95.2% +/- 3.7% at a confidence limit of 95% for samples from B. abortus infected herds obtained from chile and 98.7% +/- 0.3% at a confidence limit of 95% for samples from similar herds in Argentina. Of the negative milk samples tested, 77 gave a result above the threshold value of 0.200 optical density units. When the 77 false positive samples were retested using 7.5 mM (final concentration) of EDTA and ethyleneglycol-bis-aminoether-N,N,N', N'-tetraacetic acid (EGTA), the number of false positive reactions was reduced to 3, giving a diagnostic specificity of 99.95%. The divalent cation chelating agents did not affect positive reactions and the sensitivity remained the same. Based on control samples included with each assay, the performance of the assay was consistent.

  14. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  15. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity.

    Science.gov (United States)

    Tustian, Andrew D; Endicott, Christine; Adams, Benjamin; Mattila, John; Bak, Hanne

    2016-01-01

    There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.

  16. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    Science.gov (United States)

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  17. The Correlation between Sexual Practices and the Development of Antisperm Antibodies

    Directory of Open Access Journals (Sweden)

    Reza Salman Yazdi

    2009-01-01

    Full Text Available Background: Infertility is one of the most common and important subjects in today’s obstetricsand gynecology. Immunological factors such as the presence of antisperm antibodies (ASA arechallenging etiologies for infertility. This study was performed to determine the correlation betweenthe type of sexual practices (oral‚ anal and vaginal during menstruation and the ASA levels insemen and in the sexual partners’ serum.Materials and Methods: In this analytic cross sectional study which was performed in RoyanInstitute between 2005-2007‚ the type of sexual behaviours was determined in 51 couples withprimary or secondary infertility. The ASA level was determined in both sexual partners’ bloodserum and in the semen‚ using the Sperm Mar Test kit.Results: Using statistical analyses‚ there was no significant difference between the types of sexualpractices (anal‚ oral‚ vaginal during menstruation and the prevalence and level of ASA.Conclusion: Based on the results of this study, the prevalence and level of ASA has no significantcorrelation with the types of sexual behaviours (anal‚ oral‚ vaginal during menstruation.

  18. Development of a rapid agglutination latex test for diagnosis of enteropathogenic and enterohemorrhagic Escherichia coli infection in developing world: defining the biomarker, antibody and method.

    Directory of Open Access Journals (Sweden)

    Letícia B Rocha

    2014-09-01

    Full Text Available Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco's modified Eagle's medium (DMEM or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT was developed and tested with the same collection of bacterial isolates.EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.

  19. Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

    DEFF Research Database (Denmark)

    Claesson, M H; Endel, B; Ulrik, J

    1994-01-01

    binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2b, H-2d, as well as H-2k cells by FACS analysis. The MoAbs stained...... to two beta 2m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous beta 2m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human......-S cells were cultured at 37 degrees C. In total, these observations suggest that the MoAbs recognize conformational, presumably beta 2m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas...

  20. Generation of polyclonal antibody with high avidity to rosuvastatin and its use in development of highly sensitive ELISA for determination of rosuvastatin in plasma

    Directory of Open Access Journals (Sweden)

    Al-Malaq Hamoud A

    2011-07-01

    Full Text Available Abstract In this study, a polyclonal antibody with high avidity and specificity to the potent hypocholesterolaemic agent rosuvastatin (ROS has been prepared and used in the development of highly sensitive enzyme-linked immunosorbent assay (ELISA for determination of ROS in plasma. ROS was coupled to keyhole limpt hemocyanin (KLH and bovine serum albumin (BSA using carbodiimide reagent. ROS-KLH conjugate was used for immunization of female 8-weeks old New Zealand white rabbits. The immune response of the rabbits was monitored by direct ELISA using ROS-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and avidity to ROS was scarified and its sera were collected. The IgG fraction was isolated and purified by avidity chromatography on protein A column. The purified antibody showed high avidity to ROS; IC50 = 0.4 ng/ml. The specificity of the antibody for ROS was evaluated by indirect ELISA using various competitors from the ROS-structural analogues and the therapeutic agents used with ROS in a combination therapy. The proposed ELISA involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG and 3,3',5,5'-tetramethylbenzidine (TMB as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the color intensity in the assay wells. The assay enabled the determination of ROS in plasma at concentrations as low as 40 pg/ml.

  1. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna *

    Science.gov (United States)

    Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

    2016-01-01

    To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

  2. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

    Science.gov (United States)

    Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

    2016-02-01

    To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

  3. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  4. Basic and clinical evaluation of CA 130 RIA kit (D-7111) using two newly developed monoclonal antibodies

    International Nuclear Information System (INIS)

    Saga, Tsuneo; Endo, Keigo; Nakajima, Tetsuo

    1988-01-01

    The CA 130 RIA kit was developed with the use of two monoclonal antibodies, 130 - 22 and 145 - 9. Laboratory performance was satisfactory for precision, reproducibility, recovery, and dilution. Measurement values with CA 130 kit were almost consistent with those with CA 125 kit. Favorable standard curves were attained with smaller concentrations and shorter incubation time of CA 130 kit than those with CA 125 kit. There was less prozone phenomenon. When defining a cut-off serum level of CA 130 as 35 U/ml, false-positive rate was 0 % for healthy men and 4 % for healthy women, suggesting the involvement of menstrual cycle. Positive rate for CA 130 was 65 % for malignant ovarian tumor, 48 % for lung cancer, and 47 % for endometriosis. (Namekawa, K.)

  5. Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow.

    Science.gov (United States)

    Yamashita, Hiromichi; Kamada, Daichi; Shirasuna, Koumei; Matsui, Motozumi; Shimizu, Takashi; Kida, Katsuya; Berisha, Bajram; Schams, Dieter; Miyamoto, Akio

    2008-09-01

    Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.

  6. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Science.gov (United States)

    2012-02-17

    ... hematological malignancies such as hairy cell leukemia, chronic lymphocytic leukemia and pediatric acute lymphoblastic leukemia, and autoimmune disease such as lupus and Sjogren's syndrome. The specific antibodies...

  7. Development of a virus neutralisation test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds.

    Science.gov (United States)

    Loeffen, Willie; Quak, Sjaak; de Boer-Luijtze, Els; Hulst, Marcel; van der Poel, Wim; Bouwstra, Ruth; Maas, Riks

    2012-08-07

    At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed. A virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA's) and for (relative) quantification of antibodies.Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual

  8. Development of a virus neutralisation test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds

    Directory of Open Access Journals (Sweden)

    Loeffen Willie

    2012-08-01

    Full Text Available Abstract Background At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV, was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR on brain tissue. For epidemiological purposes, a serological assay was, however, needed. Results A virus neutralisation test (VNT was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201 of the ewes and 99% (145 out of 146 of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR, more than 90% (231 out of 255 of the ewes and 95% (795 out of 834 of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. Conclusions Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s and for (relative quantification of antibodies. Suspicions of SBV infections that were confirmed by RT-PCR were almost

  9. Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody.

    Science.gov (United States)

    Puttharugsa, Chokchai; Wangkam, Thidarat; Huangkamhang, Nongluck; Gajanandana, Oraprapai; Himananto, Orawan; Sutapun, Boonsong; Amarit, Ratthasart; Somboonkaew, Armote; Srikhirin, Toemsak

    2011-01-15

    An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Human leukocyte antigen genes and interferon beta preparations influence risk of developing neutralizing anti-drug antibodies in multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Jenny Link

    Full Text Available A significant proportion of patients with multiple sclerosis who receive interferon beta (IFNβ therapy develop neutralizing antibodies (NAbs that reduce drug efficacy. To investigate if HLA class I and II alleles are associated with development of NAbs against IFNβ we analyzed whether NAb status and development of NAb titers high enough to be biologically relevant (>150 tenfold reduction units/ml correlated with the HLA allele group carriage in a cohort of 903 Swedish patients with multiple sclerosis treated with either intramuscular IFNβ-1a, subcutaneous IFNβ-1a or subcutaneous IFNβ-1b. Carriage of HLA-DRB1*15 was associated with increased risk of developing NAbs and high NAb titers. After stratification based on type of IFNβ preparation, HLA-DRB1*15 carriage was observed to increase the risk of developing NAbs as well as high NAb titers against both subcutaneous and intramuscular IFNβ-1a. Furthermore, in patients receiving subcutaneous IFNβ-1a carriage of HLA-DQA1*05 decreased the risk for high NAb titers. In IFNβ-1b treated patients, HLA-DRB1*04 increased the risk of developing high NAb titers, and in a subgroup analysis of DRB1*04 alleles the risk for NAbs was increased in DRB1*04:01 carriers. In conclusion, there is a preparation-specific genetically determined risk to develop NAbs against IFNβ high enough to be clinically relevant in treatment decisions for patients with multiple sclerosis if confirmed in future studies. However, choice of IFNβ preparation still remains the single most significant determinant for the risk of developing NAbs.

  11. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.

    Science.gov (United States)

    Rea, Jennifer C; Lou, Yun; Cuzzi, Joel; Hu, Yuhua; de Jong, Isabella; Wang, Yajun Jennifer; Farnan, Dell

    2012-12-28

    Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 μL HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The role of antiendothelial cell antibodies in the development and follow-up of coronary and peripheral arterial diseases.

    Science.gov (United States)

    Aslim, Erdal; Hakki Akay, Tankut; Bastürk, Bilkay; Ozkan, Suleyman; Gültekin, Bahadir; Ozcobanoglu, Salih; Sirvan, Sale; Aslamaci, Sait

    2008-01-01

    Occlusive lesions in the arterial endothelium are often caused by formation of intimal hyperplasia and fibrinoid necrosis. The objective of this study was to investigate the association between antiendothelial cell antibodies (AECAs) and the development of coronary artery disease (CAD) and peripheral artery disease (PAD). In this study, 94 patients with CAD or PAD and 94 healthy volunteers serving as control subjects were examined. Frozen sections of human umbilical vein endothelial cells and primate smooth muscle cells were used to detect the presence of AECAs, which were found in 52 of 94 patients (55%) and in 15 of 94 controls (16%) (P < .001). Endothelial structure tissue damage is a major factor in arterial diseases. In the present study, a statistically significant relationship was found between AECAs and the development of CAD and PAD. The presence of AECAs has been identified as a risk factor for these diseases. According to this study, AECAs are reliable prognosticators for the development of CAD and PAD. Further studies with large numbers of serum samples are under way.

  13. The value of non-human primates in the development of therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Van Meer, P.J.K.|info:eu-repo/dai/nl/34153790X; Kooijman, M.|info:eu-repo/dai/nl/322905788; Van Der Laan, J.W.|info:eu-repo/dai/nl/374879966; Moors, E.H.M.|info:eu-repo/dai/nl/20241664X; Schellekens, H.|info:eu-repo/dai/nl/068406762

    2011-01-01

    The pharmaceutical industry is increasingly focusing on the development of biological therapeutics. These molecules generally cause no off-target toxicity and are highly species specific. Therefore, non-human primates (NHPs) are often the only relevant species in which to conduct regulatory safety

  14. Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1.

    Science.gov (United States)

    Hu, Xiaomei; Cheng, Shiliang; Liu, Xinfeng; Li, Jie; Zheng, Wen; Lu, Gang; Zhang, Jun; Zheng, Jian; Zhang, Juan

    2016-03-01

    Alanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. The complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni(2+) affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. ALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. The ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for

  15. The development of strategy for obtaining single-chain recombinant antibodies against cell-surface biomarkers on the example of human CD34

    Directory of Open Access Journals (Sweden)

    Tsapenko M. V.

    2010-10-01

    Full Text Available Antibodies against cell-surface proteins play an important role in cell detection, separation and determination of differentiation stage. The globular structure of extracellular region of cell-surface antigens is frequently characterized by heavily glycosilation and/or is stabilized by disulphide bonds. In this case the obtaining of antibodies against such proteins is a substantive problem. Aim. The development of strategy for obtaining recombinant antibodies against cell-surface biomarkers. Methods. The research strategy is based on the construction of cDNA library of VH and VL genes of animals, immunized with recombinant antigen, and subsequent cell-based biopanning for the library enrichment with desired phage clones. High-throughput automated systems were used for the detection and isolation of antigen-specific clones. Results. We have obtained a panel of five antibodies that recognize an antigen on CD34+ cell surface using the methods of immunocytochemistry and flow cytometry. Conclusions. The proposed strategy may be used for obtaining antibodies against cell-surface antigens

  16. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Science.gov (United States)

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

    2016-07-01

    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  17. Development of an immunochromatographic strip incorporating anti-mitragynine monoclonal antibody conjugated to colloidal gold for kratom alkaloids detection.

    Science.gov (United States)

    Limsuwanchote, Supattra; Putalun, Waraporn; Tanaka, Hiroyuki; Morimoto, Satoshi; Keawpradub, Niwat; Wungsintaweekul, Juraithip

    2017-12-29

    A lateral flow-based immunochromatographic strip was developed for the rapid detection of mitragynine (MG), a dominant alkaloid found in the leaves of kratom. Monoclonal antibody (mAb) against MG (anti-MG mAb) was conjugated to colloidal gold and used as a recognition probe. MG-ovalbumin conjugate (MG-OVA) and goat anti-mouse IgG were immobilized on the strip to produce a test zone and control zone, respectively. Based on the principle of a competitive assay, MG in a test sample competed with MG-OVA resident in the test zone to bind with colloidal gold-anti-MG mAb, resulting in an inverse relation of color intensity at the test zone and MG amount. The limit of detection (LOD) of the immunochromatographic strip is determined at 1 mg/mL of MG by visual assessment and 0.60 mg/mL by Image J analysis. The developed immunochromatographic strip can determine MG in kratom cocktails and kratom leaf samples. It could serve as a rapid and simple diagnostic kit for the detection of MG in kratom samples. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Development of instant kits 99Tcm-labelling of anti-CEA antibody and hIgG for scintigraphy

    International Nuclear Information System (INIS)

    Boonkittcharoen, V.

    1998-01-01

    99 Tc m -labelled monoclonal antibodies (MAbs) and human immunoglobulins (hIgG) have recently emerged as a new class of site specific radiopharmaceuticals. The role of 99 Tc m -labelled MAbs particularly anti-CEA IgG in tumour imaging has essentially established for early revealing of occult lesions in patients. Superiority of the method over X ray CT has been addressed for its capability in differentiating post-operation fibrosis from viable tumour. At present data, instant kits for preparation of this particular class of radiopharmaceuticals can be obtained from commercial sources but unfortunately at high prices. This prohibits the use of these promising diagnostic agents in developing country like Thailand. Under the assistance from IAEA through a research coordinating program, we worked on the 99 Tc m -radiochemistry in immunoglobulin labelling to establish the knowledge and acquire the know how in the development of in-house instant kits at low cost to serve the local nuclear medicine clinics in diagnosis of infectious and neoplastic diseases

  19. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    DEFF Research Database (Denmark)

    Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

    2017-01-01

    OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minim...

  20. Auto-antibodies and their association with clinical findings in women diagnosed with microscopic colitis.

    Directory of Open Access Journals (Sweden)

    Bodil Roth

    Full Text Available BACKGROUND: Microscopic colitis (MC is a disease manifested by diarrhoea and is divided into collagenous and lymphocytic colitis. The aetiology is unknown, but auto-immunity is suggested. Auto-antibodies have been only rarely examined in this entity. The aim of the study was to examine the prevalence of auto-antibodies, and to examine associations between the presence of antibodies and clinical findings. METHODS AND FINDINGS: Women with MC verified by biopsy and younger than 73 years, at any Department of Gastroenterology, in the district of Skåne, between 2002 and 2010 were invited to participate in this study. The patients were asked to complete both a questionnaire describing their medical history and the Gastrointestinal Symptom Rating Scale (GSRS. Blood samples were collected. Anti-nuclear antibodies (ANA, anti-neutrophil cytoplasmic antibodies (ANCA, anti-Saccharomyces cerevisiae antibodies (ASCA, and antibodies against glutamic acid decarboxylase (anti-GAD, islet antigens-like insulin 2 (anti-IA2, thyroid peroxidase (anti-TPO, and thyrotropin receptor (TRAK were analysed. Of 240 women identified, 133 were finally included in the study, median age 63 (59-67 years. Apart from the MC diagnosis, 52% also suffered from irritable bowel syndrome, 31% from hypertension and 31% from allergy. The prevalence of ANA (14%, ASCA IgG (13%, and anti-TPO antibodies (14% for these patients was slightly higher than for the general population, and were found together with other concomitant diseases. Patients had more of all gastrointestinal symptoms compared with norm values, irrespective of antibody expression. CONCLUSIONS: Women with MC have a slightly increased prevalence of some auto-antibodies. These antibodies are not associated with symptoms, but are expressed in patients with concomitant diseases, obscuring the pathophysiology and clinical picture of MC.

  1. Development of a highly sensitive monoclonal antibody based ELISA for detection of benzo[a]pyrene in potable water.

    Science.gov (United States)

    Matschulat, Diana; Deng, Anping; Niessner, Reinhard; Knopp, Dietmar

    2005-07-01

    In Europe, a limit value of 10 ng L(-1) was set by the European Commission for benzo[a]pyrene (B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore, sensitive and reliable methods are needed to evaluate its presence. We report here on the development of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potable water. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. The immunoassay with the least interference and the best sensitivity was optimized and characterized. As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]P solubilization for use with the developed ELISA. With the purified antibody (clone 22F12) the average IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was 65 ng L(-1) and 24 ng L(-1), respectively. From the 16 EPA-designated PAHs, only chrysene, indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than 20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthracene and benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electric conductivity 1 microS cm(-1)-2.5 mS cm(-1)), and inorganic ions (sodium, copper, iron, aluminium, manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to the Council Directive) on both signal and sensitivity of the ELISA was studied. No significant influence of these parameters on the ELISA competition curve was found. We suggest that the optimized ELISA can be used to monitor potable water samples without previous extraction from the samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levels fall close to the limit value of the new drinking water directive.

  2. Development of anti-hepatitis B surface (HBs) antibodies after HBs antigen loss in HIV-hepatitis B virus co-infected patients.

    Science.gov (United States)

    Boyd, Anders; Canini, Laetitia; Gozlan, Joël; Lascoux-Combe, Caroline; Miailhes, Patrick; Fonquernie, Laurent; Girard, Pierre-Marie; Lacombe, Karine

    2017-10-01

    Hepatitis B surface antigen (HBsAg)-seroconversion, or loss of HBsAg and acquisition of anti-hepatitis B surface (HBs) antibodies, defines functional cure of chronic hepatitis B virus (HBV) infection. After HBsAg-loss, little is known regarding the development of anti-HBs antibodies and even less so in individuals co-infected with HIV. To determine anti-HBs antibody kinetics after HBsAg-loss and explore determinants of HBsAg-seroconversion in HIV-HBV co-infected patients. Patients enrolled in the French HIV-HBV cohort were included if they had >1 study visit after HBsAg-loss. Individual patient kinetics of anti-HBs antibody levels were modeled over time using mixed-effect non-linear regression, whereby maximum specific growth rate and maximal level of antibody production were estimated from a Gompertz growth equation. Fourteen (4.6%) of 308 co-infected patients followed in the cohort exhibited HBsAg-loss, all of whom were undergoing antiretroviral therapy. Nine (64.3%) of these patients achieved HBsAg-seroconversion during a median 3.0 years (IQR=1.1-5.1) after HBsAg-loss. Across individuals with HBsAg-seroconversion, the fastest rates of antibody growth ranged between 0.57-1.93year -1 (population maximum growth rate=1.02) and antibody production plateaued between 2.09-3.66 log 10 mIU/mL at the end of follow-up (population maximal antibody levels=2.66). Patients with HBsAg-seroconversion had substantial decreases in HBV DNA viral loads (P=0.03) and proportion with elevated ALT levels (P=0.02) and HBeAg-positive serology (P=0.08). No such differences were observed in those without HBsAg-seroconversion. Most co-infected patients with HBsAg-seroconversion produced and maintained stable antibody levels, yet kinetics of anti-HBs production were much slower compared to those observed post-vaccination or after clearance of acute HBV-infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. 90Nb: potential radionuclide for application in immuno-PET. Development of appropriate production strategy and first in vivo evaluation of 90Nb-labeled monoclonal antibody

    International Nuclear Information System (INIS)

    Radchenko, Valery

    2013-01-01

    Nuclear medicine is a modern and highly effective tool for the detection and treatment of oncological disease. Molecular imaging based on radiotracers includes single photon emission tomography (SPECT) and positron emission tomography (PET), which provide non-invasive tumor visualization on nano- and picomolar level, respectively. Currently, many novel tracers for more precise discovery of small tumors and metastases have been introduced and are under investigation. Many of them are protein-based biomolecules which nature herself produces as antigens for the eradication of tumor cells. Antibodies and antibody fragments play an important role in tumor diagnostics and treatment. PET imaging with antibodies and antibody fragments is called immuno-PET. The main issue that needs to be addressed is that appropriate radiotracers with half-lives related to the half-lives of biomolecules are needed. The development of novel radiotracers is a multistep, complicated task. This task includes the evaluation of production, separation and labeling strategy for chosen radionuclide. Finally, the biomolecule-radionuclide complex should be stable in time. An equally important factor is the economic suitability of the production strategy, which will lead to a key decision for future application of the developed radionuclide. In recent work, 90 Nb has been proposed as a potential candidate for application in immuno-PET. Its half-life of 14.6 hours is suitable for application with antibody fragments and some intact antibodies. 90 Nb has a relatively high positron branching of 53% and an optimal energy of β + emission of 0.35 MeV that can provide high quality of imaging with low dose of used radionuclide. First proof-of-principle studies have shown that 90 Nb: (i) can be produced in sufficient amount and purity by proton bombardment of natural zirconium target (ii) can be isolated from target material with appropriate radiochemical purity (iii) may be used for labeling of monoclonal

  4. Impact of maternal allergy and use of probiotics during pregnancy on breast milk cytokines and food antibodies and development of allergy in children until 5 years.

    Science.gov (United States)

    Kuitunen, Mikael; Kukkonen, Anna Kaarina; Savilahti, Erkki

    2012-01-01

    Whether breast milk (BM) can protect against allergy has been studied extensively, with conflicting results. Variations in mothers' BM composition may explain some of the conflicting results. Our aim was to assess the impact of maternal allergy and probiotic intervention on BM food antibodies, transforming growth factor (TGF)-β(2) and interleukin (IL)-10 and their impact on allergy development in children until the ages of 2 and 5. We measured total IgA, IgA antibodies to cow's milk (CM), casein, β-lactoglobulin and ovalbumin (OVA), TGF-β(2) and IL-10 in 364 colostrum samples and 321 BM samples taken at 3 months from mothers participating in a prospective study evaluating the allergy-preventive effect of probiotics in a cohort with an increased risk for allergy. CM, casein and OVA antibodies, TGF-β(2) and IL-10 were detectable in most samples. Maternal allergy was associated with raised levels of IgA to casein (p = 0.04) and lower levels of TGF-β(2) (p = 0.006) in mature BM. Probiotic supplementation was associated with increased IL-10 (p = 0.046) and decreased casein IgA antibodies (p = 0.027) in mature BM. High OVA IgA antibodies in colostrum were associated with the development of atopy by the age of 2, while low levels in mature BM were a significant risk factor for the development of eczema by the age of 2. TGF-β(2) levels in BM constituted a risk for development of allergy by the age of 2. The immunologic composition of BM was only slightly affected by maternal atopy and could be altered by probiotic supplementation. Small effects of BM components on allergy development in children were evident. Copyright © 2012 S. Karger AG, Basel.

  5. Development and evaluation of drug-antibody conjugates for the treatment of human myelogenous leukemia

    International Nuclear Information System (INIS)

    Latif, Z.A.

    1978-01-01

    An immune serum to the Ph 1+ human myelogenous leukemia cell line, K-562, was developed in goats. Following exhaustive absorptions, the antiserum and its immunoglobulin (Ig) fraction were highly cytotoxic for the homologenous cells in vitro in the presence of complement. In a nude mouse-human myelogenous leukemia model system, the Ig inhibits the growth and proliferation of myelosarcomas made up of K-562 cells. At the concentration of 6 mg or more, and beginning at 7 days after transplantation of myelosarcomas in nude mice, the administration of immunoglobulins resulted in the total suppression and subsequent elimination of the tumors. The dose-response relationship between the amount of Ig injected and the growth of myelosarcomas was demonstrated to be linear i.e., the extent of inhibition of tumor growth was directly dependent upon the dose of Ig given. Also, the uptake of 125 I-labelled immunoglobulins by the K-562 myelosarcomas was at least 6-fold higher than that of the corresponding preimmune globulins

  6. Comparative Evaluation of Native Antigens for the Development of Brucellosis Antibody Detection System

    Directory of Open Access Journals (Sweden)

    Yasmin Bano

    2015-09-01

    Full Text Available Brucellosis is a highly infectious zoonotic disease and an economically important infection of humans and livestock with a worldwide distribution. The main mode of transmission of this disease to humans is through the consumption of infected milk, milk products, and uncooked or raw meat. The present study was designed to prepare few native antigens, that is, sonicated antigen (SA, cell envelope (CE antigen, and freeze and thaw (FT antigen from Brucella abortus S99 culture and to test them in a highly sensitive and specific indirect enzyme-linked immunosorbent assay (I-ELISA in both a microtiter plate and a dot-blot format for the development of field-based diagnosis. All 50 suspected bovine samples were tested by plate as well as in dot ELISA formats for all the three antigens prepared. The CE antigen was found to be more suitable as it had the maximum agreement with the Rose Bengal plate agglutination test results followed by the SA and the least agreement was found with that of the FT antigen. This detection system in microtiter plates and a dot-blot format will be useful for the rapid screening of samples for the disease surveillance and routine diagnosis.

  7. Development of novel mouse hybridomas producing monoclonal antibodies specific to human and mouse nucleolar protein SURF-6.

    Science.gov (United States)

    Polzikov, Mikhail A; Kordyukova, Maria Yu; Zavalishina, Larisa E; Magoulas, Charalambos; Zatsepina, Olga V

    2012-02-01

    SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.

  8. Correlation between HLA haplotypes and the development of antidrug antibodies in a cohort of patients with rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Benucci M

    2018-01-01

    Full Text Available Maurizio Benucci,1 Arianna Damiani,1 Francesca Li Gobbi,1 Francesca Bandinelli,1 Maria Infantino,2 Valentina Grossi,2 Mariangela Manfredi,2 Guillaume Noguier,3 Francesca Meacci2 1Rheumatology Unit, 2Immunology and Allergology Laboratory Unit, USL-Toscana Centro, Hospital S. Giovanni di Dio, Florence, Italy; 3Theradiag, Croissy Beaubourg, France Introduction: The aim of this study was to investigate the correlation between human leukocyte antigen (HLA haplotypes and the development of antidrug antibodies (ADAs in a cohort of patients with rheumatic diseases.Patients and methods: We evaluated the presence of ADAs in 248 patients with inflammatory rheumatic diseases after 6 months of treatment with anti-TNF drugs: 26 patients were treated with infliximab (IFX; three with rheumatoid arthritis [RA], 13 with ankylosing spondylitis [AS], 10 with psoriatic arthritis [PsA]; 83 treated with adalimumab (ADA; 24 with RA, 36 with AS, 23 with PsA; 88 treated with etanercept (ETA; 35 with RA, 27 with AS, 26 with PsA; 32 treated with certolizumab (CERT; 25 with RA, two with AS, five with PsA; and 19 treated with golimumab (GOL; three with RA, seven with AS, nine with PsA. Serum drug and ADA levels were determined using Lisa-Tracker Duo, the ADA-positive samples underwent an inhibition test, and the true-positive samples underwent genetic HLA typing. To have a homogeneous control population, we also performed genetic HLA typing of 11 ADA-negative patients.Results: After inhibition test, the frequency of ADAs was 2/26 patients treated with IFX (7.69%, 4/83 treated with ADA (4.81%, 0/88 treated with ETA (0%, 4/32 treated with CERT (12.5%, and 1/19 treated with GOL (5.26%. The frequency of HLA alleles in the examined patients was HLA-DRβ-11 0.636, HLA-DQ-03 0.636, and HLA-DQ-05 0.727. The estimated relative risks between the ADA-positive patients and the ADA-negative patients were HLA-DRβ-11 2.528 (95% CI 0.336–19.036, HLA-DQ-03 1.750 (95% CI 0.289–10

  9. The Development of Bispecific Hexavalent Antibodies as a Novel Class of DOCK-AND-LOCKTM (DNLTM Complexes

    Directory of Open Access Journals (Sweden)

    Chien-Hsing Chang

    2013-05-01

    Full Text Available The DOCK-AND-LOCKTM (DNLTM method provides a modular approach to develop multivalent, multifunctional complexes of defined structures, of which bispecific hexavalent antibodies (bsHexAbs are prominent examples with potential applications in targeted therapy for malignant, autoimmune, and infectious diseases. Currently, bsHexAbs are constructed by derivatizing a divalent IgG, at the carboxyl termini of either the heavy chain (the CH3-format or the light chain (the Ck-format, to contain two stabilized dimers of Fab having a different specificity from the IgG. In this review, we briefly outline the features of the DNLTM method and describe key aspects of bsHexAbs examined with diverse preclinical studies, which include binding affinity to target cells, induction of signaling pathways, effector functions, serum stability, pharmacokinetics, and antitumor activity in human tumor xenograft models. Our findings favor the selection of the CK- over the CH3-format for further exploration of bsHexAbs in clinical trials.

  10. Effects of ICOS+ T cell depletion via afucosylated monoclonal antibody MEDI-570 on pregnant cynomolgus monkeys and the developing offspring.

    Science.gov (United States)

    Nicholson, Simone M; Carlesso, Gianluca; Cheng, Lily I; Cook, Halie; DaCosta, Karma; Leininger, Joel; McKeever, Kathleen; Scott, Stephen Weasel; Taylor, Devon; Streicher, Katie; Eck, Steve; Reed, Molly; Faggioni, Raffaella; Herbst, Ronald; Dixit, Rakesh; Ryan, Patricia C

    2017-12-01

    MEDI-570 is a fully human afucosylated monoclonal antibody (MAb) against Inducible T-cell costimulator (ICOS), highly expressed on CD4+ T follicular helper (T FH ) cells. Effects of MEDI-570 were evaluated in an enhanced pre-postnatal development toxicity (ePPND) study in cynomolgus monkeys. Administration to pregnant monkeys did not cause any abortifacient effects. Changes in hematology and peripheral blood T lymphocyte subsets in maternal animals and infants and the attenuated infant IgG immune response to keyhole limpet hemocyanin (KLH) were attributed to MEDI-570 pharmacology. Adverse findings included aggressive fibromatosis in one dam and two infant losses in the high dose group with anatomic pathology findings suggestive of atypical lymphoid hyperplasia. The margin of safety relative to the no observed adverse effect level (NOAEL) for the highest planned clinical dose in the Phase 1a study was 7. This study suggests that women of child bearing potential employ effective methods of contraception while being treated with MEDI-570. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Neonatal exposure to anti-nerve growth factor antibodies affects exploratory behavior of developing mice in the hole board.

    Science.gov (United States)

    Calamandrei, G; Pennazza, S; Ricceri, L; Valanzano, A

    1996-01-01

    The aim of this study was to assess in developing mice whether the neutralization of endogenous NGF following ICV administration of anti-NGF antibodies (50 micrograms/2 microliters) on postnatal days 3, 6, 9, and 12 affected locomotor activity, exploratory behavior, and response to the cholinergic blocker scopolamine. In Experiments 1 and 2 activity and age-typical scopolamine effects were evaluated on PND 13 or 17 in an automated apparatus. No significant main effect of anti-NGF treatment was found at either age. On day 13 scopolamine (0.2, 1, or 2 mg/kg) decreased locomotion in both anti-NGF and control animals. In Experiment 3, locomotion and exploratory behavior were analyzed in an open field arena or in a hole board apparatus on PND 16. No significant effects of anti-NGF treatment on general motor activity and investigation of a novel object in the open field was found, though anti-NGF animals tended to be less active than controls. In the hole board anti-NGF pups showed a different pattern of head dipping behavior from controls, exploring mainly the holes located in the periphery of the apparatus.

  12. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

    Directory of Open Access Journals (Sweden)

    Gortázar Christian

    2008-11-01

    Full Text Available Abstract Background Bovine tuberculosis (bTB remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. Results An enzyme-linked immunosorbent assay (ELISA to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Conclusion Although some negative group animals showed an ELISA positive reaction (

  13. Development of a double-antibody radioimmunoassay for detecting ovarian tumor-associated antigen fraction OCA in plasma

    International Nuclear Information System (INIS)

    Knauf, S.; Urbach, G.I.

    1978-01-01

    Ovarian tumor-associated antigen isolated from human tumor tissue was shown to have a different mobility from that of carcinoembryonic antigen (CEA) in both acrylamide gel electrophoresis and immunoelectrophoresis in agarose. The ovarian tumor antigen is composed of six species with different electrophoretic mobility in acrylamide gel electrophoresis. Three of these species were detected in Sephadex G-100 ovarian fraction OCA (from the void volume peak) and the other three species of lower apparent molecular weight were detected in fraction OCD (from the second peak). Fractions OCA and OCD did not share common antigenic determinants as determined by immunodiffusion. CEA was shown to share antigenic determinants with both OCA and OCD. A double-antibody radioimmunoassay capable of detecting nanogram quantities of plasma OCA was developed. In a preliminary study of ovarian cancer patients, OCA appeared to be a more sensitive marker for ovarian cancer than CEA. There was virtually no correlation (r 2 = 0.1) between OCA and CEA levels in these patients, as determined by radioimmunoassay

  14. An integrated precipitation and ion-exchange chromatography process for antibody manufacturing: Process development strategy and continuous chromatography exploration.

    Science.gov (United States)

    Großhans, Steffen; Wang, Gang; Fischer, Christian; Hubbuch, Jürgen

    2018-01-19

    In the past decades, research was carried out to find cost-efficient alternatives to Protein A chromatography as a capture step in monoclonal antibody (mAb) purification processes. In this work, polyethylene glycol (PEG) precipitation has shown promising results in the case of mAb yield and purity. Especially with respect to continuous processing, PEG precipitation has many advantages, like low cost of goods, simple setup, easy scalability, and the option to handle perfusion reactors. Nevertheless, replacing Protein A has the disadvantage of renouncing a platform unit operation as well. Furthermore, PEG precipitation is not capable of reducing high molecular weight impurities (HMW) like aggregates or DNA. To overcome these challenges, an integrated process strategy combining PEG precipitation with cation-exchange chromatography (CEX) for purification of a mAb is presented. This work discusses the process strategy as well as the associated fast, easy, and material-saving process development platform. These were implemented through the combination of high-throughput methods with empirical and mechanistic modeling. The strategy allows the development of a common batch process. Additionally, it is feasible to develop a continuous process. In the presented case study, a mAb provided from cell culture fluid (HCCF) was purified. The precipitation and resolubilization conditions as well as the chromatography method were optimized, and the mutual influence of all steps was investigated. A mAb yield of over 95.0% and a host cell protein (HCP) reduction of over 99.0% could be shown. At the same time, the aggregate level was reduced from 3.12% to 1.20% and the DNA level was reduced by five orders of magnitude. Furthermore, the mAb was concentrated three times to a final concentration of 11.9mg/mL. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Amino acid-based advanced liquid formulation development for highly concentrated therapeutic antibodies balances physical and chemical stability and low viscosity.

    Science.gov (United States)

    Kemter, Kristina; Altrichter, Jens; Derwand, Roland; Kriehuber, Thomas; Reinauer, Eva; Scholz, Martin

    2018-04-16

    To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study was to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25 - >200 mg/mL) in liquid formulation was used. Pre-testing of formulations based on a stabilizing and protecting solutions (SPS®) platform was conducted in a thermal unfolding model using Differential Scanning Fluorimetry (DSF) and accelerated aging at 37 °C and 45 °C. Pre-selected amino acid combinations were further iteratively adjusted to obtain stable highly concentrated antibody formulations with low viscosity. Size Exclusion Chromatography (SE-HPLC) revealed significantly lower aggregation and fragmentation at specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa∗s of highly concentrated trastuzumab (≥200 mg/mL) were measured by falling ball viscosimetry. Moreover, less chemical degradation was found by Cationic Exchange Chromatography (CEX -HPLC) even after six months liquid storage at 25 °C. In conclusion, specifically tailored and advanced amino acid-based liquid formulations avoid aggregation and enable the development of stable and low viscous highly concentrated biopharmaceuticals. This article is protected by copyright. All rights reserved.

  16. Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

    International Nuclear Information System (INIS)

    Nagai, Kentaro; Nakahata, Shingo; Shimosaki, Shunsuke; Tamura, Tomohiro; Kondo, Yuudai; Baba, Takashi; Taki, Tomohiko; Taniwaki, Masafumi; Kurosawa, Gene; Sudo, Yukio; Okada, Seiji; Sakoda, Sumio; Morishita, Kazuhiro

    2014-01-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC

  17. Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

    Science.gov (United States)

    Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

    2015-10-05

    Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural

  18. A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

    Science.gov (United States)

    Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

    2014-01-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  19. Evaluation of capture ELISA for detection of antineutrophil cytoplasmic antibodies directed against proteinase 3 in Wegener's granulomatosis : first results from a multicentre study

    NARCIS (Netherlands)

    Csernok, E; Holle, J; Hellmich, B; Willem, J; Tervaert, C; Kallenberg, CGM; Limburg, PC; Niles, J; Pan, GL; Specks, U; Westman, K; Wieslander, J; Gross, WL

    Objective: To evaluate the performance characteristics of direct and capture ELISA for the detection of PR3-ANCA in Wegener's granulomatosis (WG) in international ANCA reference laboratories. Methods: Serum samples were derived from patients with histological and clinical diagnosis of WG (n = 60),

  20. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  1. Development of antibodies against growth hormone (GH) during rhGH therapy in a girl with idiopathic GH deficiency: a case report.

    Science.gov (United States)

    Meazza, Cristina; Schaab, Michael; Pagani, Sara; Calcaterra, Valeria; Bozzola, Elena; Kratzsch, Juergen; Bozzola, Mauro

    2013-01-01

    A 12.5-year-old Italian girl was referred to our institute for progressive growth failure from the age of 6 years, with a height of 128.2 cm (-3.37 SDS) and a bone age of 9 years. Endocrinological evaluation revealed a partial growth hormone deficiency (GHD) and GH therapy was started at a dosage of 0.25 mg/kg/week. During the first 3 years, she showed an increase in growth rate and experienced pubertal development onset. Then a poor growth rate (2 cm/year=0.43 SDS) was observed, notwithstanding an increase in GH dosage (0.35 mg/kg/week) and good compliance. We found a positive anti-GH antibody titre (1:1850, cutoff 1/100), confirmed 6 months later (1:2035); the antibodies had low binding capacity (0.63 μg/mL) and were only partially capable of inhibiting the GH effect. However, GH treatment was discontinued, and after 3 months the antibody titre decreased (1:950). In conclusion, we suggest evaluation of anti-GH antibodies in GH-treated idiopathic GHD children in whom growth response decreases after some years of therapy.

  2. Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2016-01-29

    The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

  3. Approach to a case of multiple irregular red cell antibodies in a liver transplant recipient: Need for developing competence.

    Science.gov (United States)

    Dara, Ravi C; Tiwari, Aseem K; Pandey, Prashant; Arora, Dinesh

    2015-01-01

    Liver transplant procedure acts as a challenge for transfusion services in terms of specialized blood components, serologic problems, and immunologic effects of transfusion. Red cell alloimmunization in patients awaiting a liver transplant complicate the process by undue delay or unavailability of compatible red blood cell units. Compatible blood units can be provided by well-equipped immunohematology laboratory, which has expertise in resolving these serological problems. This report illustrates resolution of a case with multiple alloantibodies using standard techniques, particularly rare antisera. Our case re-emphasizes the need for universal antibody screening in all patients as part of pretransfusion testing, which helps to identify atypical antibodies and plan for appropriate transfusion support well in time. We recommend that the centers, especially the ones that perform complex procedures like solid organ transplants and hematological transplants should have the necessary immunohematological reagents including rare antisera to resolve complex cases of multiple antibodies as illustrated in this case.

  4. Approach to a case of multiple irregular red cell antibodies in a liver transplant recipient: Need for developing competence

    Directory of Open Access Journals (Sweden)

    Ravi C Dara

    2015-01-01

    Full Text Available Liver transplant procedure acts as a challenge for transfusion services in terms of specialized blood components, serologic problems, and immunologic effects of transfusion. Red cell alloimmunization in patients awaiting a liver transplant complicate the process by undue delay or unavailability of compatible red blood cell units. Compatible blood units can be provided by well-equipped immunohematology laboratory, which has expertise in resolving these serological problems. This report illustrates resolution of a case with multiple alloantibodies using standard techniques, particularly rare antisera. Our case re-emphasizes the need for universal antibody screening in all patients as part of pretransfusion testing, which helps to identify atypical antibodies and plan for appropriate transfusion support well in time. We recommend that the centers, especially the ones that perform complex procedures like solid organ transplants and hematological transplants should have the necessary immunohematological reagents including rare antisera to resolve complex cases of multiple antibodies as illustrated in this case.

  5. Frequent antibody production against RARalpha in both APL mice and patients.

    Science.gov (United States)

    Robin, Marie; Andreu-Gallien, Juliette; Schlageter, Marie-Helene; Bengoufa, Djaouida; Guillemot, Isabelle; Pokorna, Katerina; Robert, Carine; Larghero, Jerome; Rousselot, Philippe; Raffoux, Emmanuel; Dombret, Herve; Fenaux, Pierre; Pla, Marika; Charron, Dominique; Padua, Rose-Ann; Chomienne, Christine

    2006-09-15

    In an acute promyelocytic leukemia (APL)-transplantable mouse model, we previously reported the presence of antibodies recognizing PML-RARalpha and RARalpha in the sera of ATRA-treated mice. To evaluate this immune response, we determined the prevalence of anti-RARalpha antibodies in a cohort of 48 APL mice, treated by ATRA (n = 24) or by placebo pellets (n = 24), and in a preliminary subset of 9 patients with APL using a specific enzyme-linked immunosorbent assay (ELISA). In APL mice, significantly higher antibody levels were observed at the latest time points (day 48 to 58 levels superior to day 15 to 18 or day 28 to 38 levels). Antibody levels were higher in ATRA-treated mice than in placebo-treated mice and were also predictive of better survival. In the patients with APL, anti-RARalpha antibodies were detected at diagnosis and after maintenance therapy, reminiscent of the ATRA-treated APL mice. Antinuclear or antineutrophil cytoplasmic autoantibodies were also detected. These data reveal for the first time that in patients with APL an immune response may be detected at diagnosis and enhanced after maintenance therapy.

  6. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  7. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  8. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  9. Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma

    Science.gov (United States)

    May, RD; Monk, PD; Cohen, ES; Manuel, D; Dempsey, F; Davis, NHE; Dodd, AJ; Corkill, DJ; Woods, J; Joberty-Candotti, C; Conroy, LA; Koentgen, F; Martin, EC; Wilson, R; Brennan, N; Powell, J; Anderson, IK

    2012-01-01

    BACKGROUND AND PURPOSE IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma. PMID:21895629

  10. Progression of Mycoplasma hyosynoviae infection in three pig herds. Development of tonsillar carrier state, arthritis and antibodies in serum and synovial fluid in pigs from birth to slaughter

    DEFF Research Database (Denmark)

    Hagedorn-Olsen, T.; Nielsen, N.C.; Friis, N.F.

    1999-01-01

    In this investigation, natural infection with Mycoplasma hyosynoviae was followed in groups of individual pigs in three different herds with regard to occurrence of tonsillar carrier state, clinical arthritis and development of antibodies in serum and in synovial fluid. Antibodies were detected...... by a polyclonal enzyme-linked immunosorbent assay (ELISA) developed for experimental use. The infection with M hyosynoviae progressed very differently in the three herds investigated. In one herd, the infection was apparently limited to adult Figs. In a second herd, all pigs became tonsillar carriers of M....... hyosynoviae, but no mycoplasma-related arthritis nor any serological response was demonstrated within the growing-finishing period. In the third herd investigated, tonsillar infection was detected in all pigs, clinical cases of M. hyosynoviae arthritis followed and a moderate serological response was observed...

  11. Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model.

    Science.gov (United States)

    Eksteen, Mariana; Heide, Gøril; Tiller, Heidi; Zhou, Yan; Nedberg, Nora Hersoug; Martinez-Zubiaurre, Inigo; Husebekk, Anne; Skogen, Bjørn R; Stuge, Tor B; Kjær, Mette

    2017-04-21

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin β3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin β3 is also associated with integrin αV forming the αVβ3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells. An in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells. We found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells. Our findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast

  12. Development of monoclonal antibodies bearing the internal image of the gizzerosine epitope and application in a competitive ELISA for fish meal.

    Science.gov (United States)

    Manosalva, Headdy; De Ioannes, Alfredo E; Becker, María Inés

    2004-02-01

    Gizzerosine (GZ), a derivative of histamine, is a biogenic amine found in fish meal, and one of the causative agents of black vomit, a poultry disease. We describe here the preparation of anti-idiotype antibodies to the anti-GZ monoclonal antibody (anti-GZ 3H4) and their possible application to an immunoassay. BALB-c mice were immunized with anti-GZ 3H4 antibody coupled to hemocyanin from Concholepas concholepas. Using somatic cell fusion between NSO/2 cells and splenic lymphocytes from the immunized mice, we obtained 34 potential anti-idiotype antibodies. They were characterized by passive agglutination with supernatants from hybridoma cultures and latex particles conjugated to the idiotype. Anti-idiotype antibodies were analyzed by a competitive RIA, to determine their ability to dissociate the interaction between (125)I-GZ and the anti-GZ 3H4-idiotype antibody. They were also characterized by GZ inhibition of latex passive agglutination assay. Three anti-idiotypes named 2D11, 2H6, and 3A12, all of the IgG isotype, were obtained. They were evaluated by a competitive ELISA, in which GZ competes with the tracer (HRP-idiotype). All presented sensitivity in the range of 0.1-10 microg/mL of GZ; and the 3A12 anti-idiotype antibody showed the best performance. An ELISA was developed using the idiotype bound to the solid phase and the anti-idiotype 3A12-HRP as the tracer. The assay showed a similar sensitivity and cross-reactivity with histamine was only observed at concentrations over 10 microg/mL. Lysine and histidine did not interfere with the assay up to 500 microg/mL. An experiment was conducted with fish meal contaminated with synthetic GZ. The results are promising, and showed that no other compounds of the fish meal interfere with the ELISA system; however the extraction procedure of the sample needs to be improved. From the results presented here, we conclude that the idiotype anti-idiotype ELISA would be an appropriate method to determine GZ in fish meal.

  13. BACE1 elevation is involved in amyloid plaque development in the triple transgenic model of Alzheimer's disease: differential Aβ antibody labeling of early-onset axon terminal pathology.

    Science.gov (United States)

    Cai, Yan; Zhang, Xue-Mei; Macklin, Lauren N; Cai, Huaibin; Luo, Xue-Gang; Oddo, Salvatore; Laferla, Frank M; Struble, Robert G; Rose, Gregory M; Patrylo, Peter R; Yan, Xiao-Xin

    2012-02-01

    β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer's disease (FAD). Some FAD-based mouse models produce amyloid plaques, others do not. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.

  14. [Development of an antigen 'sandwich' enzyme immunoassay for the detection of antibodies against HIV-2 by using a biotinylated synthetic peptide of gp36 protein].

    Science.gov (United States)

    Delahanty-Fernández, Aurora; Bequer-Ariza, Dunia Clara; Hernández-Marín, Milenen; Zulueta-Rodríguez, Orlando; Pozo-Peña, Lilliam; Hernández-Spengler, Idialis; Ramos-Martínez, Grisell; Valdespino-Díaz, Marcos Antonio; Ventura-Paz, Julio

    2015-01-01

    Among the several existing methods for the detection of antibodies to HIV, the 'sandwich' ELISA is currently the most used. This study aims to assess a biotinylated monomeric synthetic peptide of the glycoprotein trans-membrane gp36 from HIV-2, in a sandwich assay, for the detection of antibodies against this HIV-2 protein. To perform the assay, plates coated with recombinant protein gp36 at 0.5μg/mL and synthetic peptide gp36(5) at 1μg/mL were used. The concentration of the biotinylated synthetic peptide (gp36(5)-B) used was 0.1μg/mL prepared with a Tris-BSA-NaCl buffer solution and the Streptavidin- Alkaline Phosphatase conjugate diluted 1:30000 prepared with a PBS-Sucrose-BSA solution. Positive serum samples to antibodies against HIV-1 and HIV-2 viruses (88 and 34, respectively) were tested, with 483 negative samples from blood donors and 96 serum samples to assess the analytical specificity. All the samples were tested using the UMELISA HIV 1+2 RECOMBINANT assay, and all positives were confirmed using a DAHIV-BLOT assay. Thirty four samples with antibodies against HIV-2 were assessed as positive for both coating variants. The highest specificity was obtained with the variant using the synthetic peptide gp36(5) in its coating. The antigen 'sandwich' assay developed by using gp36(5)-B enables the detection of antibodies against gp36 protein of HIV-2. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  15. Expression of the rubber-like protein, resilin, in developing and functional insect cuticle determined using a Drosophila anti-Rec 1 resilin antibody.

    Science.gov (United States)

    Wong, Darren C C; Pearson, Roger D; Elvin, Christopher M; Merritt, David J

    2012-02-01

    The natural elastomeric protein, insect resilin, is the most efficient elastic material known, used to store energy for jumping and flight in a variety of insects. Here, an antibody to recombinant Drosophila melanogaster pro-resilin is used to examine resilin expression in Drosophila and a wider range of insects. Immunostaining of Drosophila embryos reveals anti-resilin reactivity in epidermal patches that exhibit a dynamic spatial and temporal expression through late embryogenesis. Resilin is also detected in stretch receptors in the embryo. In developing adult Drosophila, resilin pads are described at the base of wings and at the base of flexible sensory hairs in pupae. Resilin is also detected in embryos of the tephritid fruitfly, Bactrocera tryoni, and two well-known concentrations of insect resilin: the flight muscle tendon of the dragonfly and the pleural arch of the flea. The anti-Rec1 antibody antibody developed using Drosophila pro-resilin as antigen is cross-reactive and is useful for detection of resilin in diverse insects. For the first time, resilin expression has been detected during embryogenesis, revealing segmental patches of resilin in the developing epidermis of Drosophila. Copyright © 2011 Wiley Periodicals, Inc.

  16. Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

    Directory of Open Access Journals (Sweden)

    Nadya V. Ilicheva

    2018-03-01

    Full Text Available Background: TRF2 (telomeric repeat binding factor 2 is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2 between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3. The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina. Keywords: Chromosomes, Molecular cloning, Nuclear lamina, Nucleoprotein complexes, Polyclonal antibodies, Recombinant polypeptide, Shelterin, Telomere-binding protein TRF2, Telomeres, Telomeric DNA, TTAGGG repeats

  17. The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.

    Science.gov (United States)

    Mytych, Daniel T; La, Stephon; Barger, Troy; Ferbas, John; Swanson, Steven J

    2009-02-20

    A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.

  18. Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies.

    Science.gov (United States)

    Oei, A L M; Boerman, O C; Geurts-Moespot, A; van Eerd, J E; van Tienoven, D; Courtenay-Luck, N; Thomas, C M G; Massuger, L F A G; Sweep, F C G J

    2007-01-01

    The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.

  19. Correlation between HLA haplotypes and the development of antidrug antibodies in a cohort of patients with rheumatic diseases.

    Science.gov (United States)

    Benucci, Maurizio; Damiani, Arianna; Li Gobbi, Francesca; Bandinelli, Francesca; Infantino, Maria; Grossi, Valentina; Manfredi, Mariangela; Noguier, Guillaume; Meacci, Francesca

    2018-01-01

    The aim of this study was to investigate the correlation between human leukocyte antigen (HLA) haplotypes and the development of antidrug antibodies (ADAs) in a cohort of patients with rheumatic diseases. We evaluated the presence of ADAs in 248 patients with inflammatory rheumatic diseases after 6 months of treatment with anti-TNF drugs: 26 patients were treated with infliximab (IFX; three with rheumatoid arthritis [RA], 13 with ankylosing spondylitis [AS], 10 with psoriatic arthritis [PsA]); 83 treated with adalimumab (ADA; 24 with RA, 36 with AS, 23 with PsA); 88 treated with etanercept (ETA; 35 with RA, 27 with AS, 26 with PsA); 32 treated with certolizumab (CERT; 25 with RA, two with AS, five with PsA); and 19 treated with golimumab (GOL; three with RA, seven with AS, nine with PsA). Serum drug and ADA levels were determined using Lisa-Tracker Duo, the ADA-positive samples underwent an inhibition test, and the true-positive samples underwent genetic HLA typing. To have a homogeneous control population, we also performed genetic HLA typing of 11 ADA-negative patients. After inhibition test, the frequency of ADAs was 2/26 patients treated with IFX (7.69%), 4/83 treated with ADA (4.81%), 0/88 treated with ETA (0%), 4/32 treated with CERT (12.5%), and 1/19 treated with GOL (5.26%). The frequency of HLA alleles in the examined patients was HLA-DRβ-11 0.636, HLA-DQ-03 0.636, and HLA-DQ-05 0.727. The estimated relative risks between the ADA-positive patients and the ADA-negative patients were HLA-DRβ-11 2.528 (95% CI 0.336-19.036), HLA-DQ-03 1.750 (95% CI 0.289-10.581), and HLA-DQ-05 2.424 (95% CI 0.308-15.449). This is the first study that shows an association between HLA and genetic factors associated with the occurrence of ADAs in patients with rheumatic diseases, but the number of samples is too small to draw any definite conclusion.

  20. Antithyroid microsomal antibody

    Science.gov (United States)

    ... that you have a higher chance of developing thyroid disease in the future. Antithyroid microsomal antibodies may be ... PA: Elsevier; 2016:chap 11. Weiss RE, Refetoff S. Thyroid function testing. In: Jameson JL, De Groot LJ, eds. Endocrinology: Adult and ... Lupus Read more ...

  1. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  2. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  3. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

  4. Development of gastric cancer in nonatrophic stomach with highly active inflammation identified by serum levels of pepsinogen and Helicobacter pylori antibody together with endoscopic rugal hyperplastic gastritis.

    Science.gov (United States)

    Watanabe, Mika; Kato, Jun; Inoue, Izumi; Yoshimura, Noriko; Yoshida, Takeichi; Mukoubayashi, Chizu; Deguchi, Hisanobu; Enomoto, Shotaro; Ueda, Kazuki; Maekita, Takao; Iguchi, Mikitaka; Tamai, Hideyuki; Utsunomiya, Hirotoshi; Yamamichi, Nobutake; Fujishiro, Mitsuhiro; Iwane, Masataka; Tekeshita, Tatsuya; Mohara, Osamu; Ushijima, Toshikazu; Ichinose, Masao

    2012-12-01

    This study aimed to elucidate groups at high risk of developing cancer among patients with serologically identified Helicobacter pylori infection and nonatrophic stomach. Annual endoscopy was performed for a mean of 5.4 years in 496 asymptomatic middle-aged men who were H. pylori antibody-positive and pepsinogen (PG) test-negative. Subjects were stratified according to the activity of H. pylori-associated gastritis measured by serum levels of PG and H. pylori antibody, and/or by endoscopic findings of rugal hyperplastic gastritis (RHG), and cancer development was investigated. During the study period, seven cases of cancer developed in the cohort (incidence rate, 261/100,000 person-years), with 85.7% developing in the group showing a PGI/II ratio ≤ 3.0, reflecting active inflammation-based high PGII levels. Cancer incidence was significantly higher in this group (750/100,000 person-years) than in groups with less active gastritis. Furthermore, cancer incidence for this group was significantly higher in the subgroup with high H. pylori antibody titers than in the low-titer subgroup. Meanwhile, endoscopic findings revealed that 11.7% of subjects showed RHG reflecting localized highly active inflammation, and cancer risk was significantly higher in patients with RHG than in patients without. Combining the two serum tests and endoscopic examination for RHG allowed identification of subjects with more active gastritis and higher cancer risk. No cancer development was observed in these high-risk subjects after H. pylori eradication. Subjects with highly active gastritis identified by the two serological tests and endoscopic RHG constitute a group at high risk of cancer development with H. pylori-infected nonatrophic stomach. Copyright © 2012 UICC.

  5. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  6. A novel Fc-engineered human ICAM-1/CD54 antibody with potent anti-myeloma activity developed by cellular panning of phage display libraries.

    Science.gov (United States)

    Klausz, Katja; Cieker, Michael; Kellner, Christian; Oberg, Hans-Heinrich; Kabelitz, Dieter; Valerius, Thomas; Burger, Renate; Gramatzki, Martin; Peipp, Matthias

    2017-09-29

    To identify antibodies suitable for multiple myeloma (MM) immunotherapy, a cellular screening approach was developed using plasma cell lines JK-6L and INA-6 and human synthetic single-chain fragment variable (scFv) phage libraries. Isolated phage antibodies were screened for myeloma cell surface reactivity. Due to its binding characteristics, phage PIII-15 was selected to generate the scFv-Fc fusion protein TP15-Fc with an Fc domain optimized for FcγRIIIa binding. Various MM cell lines and patient-derived CD138-positive malignant plasma cells, but not granulocytes, B or T lymphocytes from healthy donors were recognized by TP15-Fc. Human intercellular adhesion molecule-1 (ICAM-1/CD54) was identified as target antigen by using transfected Chinese hamster ovary (CHO) cells. Of note, no cross-reactivity of TP15-Fc with mouse ICAM-1 transfected cells was detected. TP15-Fc was capable to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against different human plasma cell lines and patients' myeloma cells with peripheral blood mononuclear cells (PBMC) and purified NK cells. Importantly, TP15-Fc showed potent in vivo efficacy and completely prevented growth of human INA-6.Tu1 plasma cells in a xenograft SCID/beige mouse model. Thus, the novel ADCC-optimized TP15-Fc exerts potent anti-myeloma activity and has promising characteristics to be further evaluated for MM immunotherapy.

  7. Development of a Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay Using a Synthetic Peptide Antigen for Detection of Avian Metapneumovirus Antibodies in Turkey Sera

    Science.gov (United States)

    Alvarez, Rene; Njenga, M. Kariuki; Scott, Melissa; Seal, Bruce S.

    2004-01-01

    Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes. PMID:15013970

  8. Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by northern blot and flow cytometry.

    Science.gov (United States)

    Borth, N; Strutzenberger, K; Kunert, R; Steinfellner, W; Katinger, H

    1999-01-08

    The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

  9. Development of a vaccine to mitigate greenhouse gas emissions in agriculture: vaccination of sheep with methanogen fractions induces antibodies that block methane production in vitro.

    Science.gov (United States)

    Wedlock, D N; Pedersen, G; Denis, M; Dey, D; Janssen, P H; Buddle, B M

    2010-02-01

    To develop an understanding of the immune responses of ruminants to methanogens, and to provide proof of a concept that harnessing the immune system of ruminants is a potentially viable approach to mitigate greenhouse gas emissions from agriculture. Four subcellular fractions, namely cytoplasmic, two cell-wall preparations, and cell wall-derived proteins were prepared from Methanobrevibacter ruminantium M1. Twenty sheep (10 months of age) were vaccinated with these fractions or with whole cells (n=4 per group). Sheep were re-vaccinated once after 3 weeks, and antibody responses to M. ruminantium M1 antigens in sera and saliva measured using ELISA at 2 weeks after the second vaccination. Antigens recognised by the antisera were visualised using Western blotting. The antisera were tested in vitro for their impact on M. ruminantium M1, measuring the effect on cell growth, methane production, and ability to induce agglutination. Basal levels (pre-vaccination) of antibodies against M. ruminantium M1 antigens were low. Vaccination with the antigenic fractions induced strong antibody responses in serum. Both IgG and IgA responses to methanogen antigens were detected in saliva following vaccination. Western blot analysis of the antisera indicated reactivity of antibodies, and a wide range of proteins was present in the different methanogen fractions. Antisera against the various fractions agglutinated methanogens in an in-vitro assay. In addition, these antisera decreased the growth of a pure culture of a methanogen and production of methane in vitro. Antigens from methanogens are immunogenic in ruminants, and antisera from sheep vaccinated with fractions of methanogens have a significant impact on these organisms, inducing cell agglutination, and decreasing growth of methanogens and production of methane. Only antisera to selected methanogen fractions were able to achieve these effects. The results demonstrate the feasibility of a vaccination strategy to mitigate emission

  10. BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Picceli, V F; Skare, T L; Nisihara, R M; Nass, F R; Messias-Reason, I T; Utiyama, S R R

    2016-04-01

    B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti β2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti β2GPI). There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients. © The Author(s) 2015.

  11. Cancer development based on chronic active gastritis and resulting gastric atrophy as assessed by serum levels of pepsinogen and Helicobacter pylori antibody titer.

    Science.gov (United States)

    Yoshida, Takeichi; Kato, Jun; Inoue, Izumi; Yoshimura, Noriko; Deguchi, Hisanobu; Mukoubayashi, Chizu; Oka, Masashi; Watanabe, Mika; Enomoto, Shotaro; Niwa, Toru; Maekita, Takao; Iguchi, Mikitaka; Tamai, Hideyuki; Utsunomiya, Hirotoshi; Yamamichi, Nobutake; Fujishiro, Mitsuhiro; Iwane, Masataka; Takeshita, Tatsuya; Ushijima, Toshikazu; Ichinose, Masao

    2014-03-15

    Our study investigated the relationship between gastric cancer development and activity of Helicobacter pylori-associated chronic gastritis or the resulting chronic atrophic gastritis (CAG). A cohort of 4,655 healthy asymptomatic subjects, in whom serum pepsinogen (PG) and H. pylori antibody titer had been measured to assess the activity and stage of H. pylori-associated chronic gastritis, was followed for up to 16 years, and cancer development was investigated. In subjects with a serologically diagnosed healthy stomach (H. pylori-negative/CAG-negative), cancer incidence rate was low, at 16/100,000 person-years. With the establishment of H. pylori infection and progression of chronic gastritis, significant stepwise cancer risk elevations were seen from CAG-free subjects (H. pylori-positive/CAG-negative) [hazard ratio (HR) = 8.9, 95% confidence interval (CI) = 2.7-54.7] to subjects with CAG (H. pylori-positive/CAG-positive) (HR = 17.7, 95% CI = 5.4-108.6) and finally to subjects with metaplastic gastritis (H. pylori-negative/CAG-positive) (HR = 69.7, 95% CI = 13.6-502.9). In H. pylori-infected CAG-free subjects, significantly elevated cancer risk was observed in the subgroup with active inflammation-based high PG II level or potent immune response-based high H. pylori antibody titer; the former was associated with a particularly high risk of diffuse-type cancer, and both subgroups showed high cancer incidence rates of around 250/100,000 person-years, comparable to that in subjects with CAG. No such risk elevation was observed in H. pylori-infected subjects with CAG. These results clearly indicate that gastric cancer develops mainly from the gastritis-atrophy-metaplasia-cancer sequence and partly from active inflammation-based direct carcinogenesis, and that serum levels of PG and H. pylori antibody titer provide indices of cancer development in H. pylori-infected subjects. © 2013 UICC.

  12. Antibody therapeutics - the evolving patent landscape.

    Science.gov (United States)

    Petering, Jenny; McManamny, Patrick; Honeyman, Jane

    2011-09-01

    The antibody patent landscape has evolved dramatically over the past 30 years, particularly in areas of technology relating to antibody modification to reduce immunogenicity in humans or improve antibody function. In some cases antibody techniques that were developed in the 1980s are still the subject of patent protection in the United States or Canada. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana

    1994-01-01

    A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody resp...... against the infection. On the other hand, immune complexes between the beta-lactamase and corresponding antibodies could play a role in the pathogenesis of bronchopulmonary injury in CF by mediating hyperimmune reactions....

  14. Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

    Science.gov (United States)

    Guo, Chun-yan; Tang, Yi-gui; Qi, Zong-li; Liu, Yang; Zhao, Xiang-rong; Huo, Xue-ping; Li, Yan; Feng, Qing; Zhao, Peng-hua; Wang, Xin; Li, Yuan; Wang, Hai-fang; Hu, Jun; Zhang, Xin-jian

    2015-08-01

    To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. No development of neutralizing antibodies against recombinant interferon-alpha in Ph-negative myeloproliferative neoplasms-a prospective study

    DEFF Research Database (Denmark)

    Ocias, Lukas Frans; Lund Hansen, Dennis; Kielsgaard Kristensen, Thomas

    2015-01-01

    neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients......: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET). The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range...

  16. [Antibody induction after intrauterine interventions].

    Science.gov (United States)

    Hoch, J; Giers, G; Bald, R; Hansmann, M; Hanfland, P

    1993-06-01

    Immunohematologic and clinical data, i.e., antibody profile, location of the placenta, mode of cordocentesis, obtained from 48 pregnant patients with irregular erythrocyte antibodies during the last 2 years have been retrospectively evaluated. All fetuses of the patients received intrauterine transfusions for the treatment of fetal erythroblastosis. In 16 (33%) patients (group I) a secondarily induced antibody was detected after the onset of intrauterine transfusion therapy. 32 (67%) patients (group II) did not further develop new antibody specificities. Group I exhibited a significantly different distribution in the location of the placenta (p pregnant women. In group I a 5-fold higher rate of anterior than posterior placenta location was found. The mode of cordocentesis differed significantly (p antibodies by invasive intrauterine interventions in our patients depended indirectly on the location of the placenta and directly on the mode of the puncture (trans- vs. paraplacental access).

  17. Development of a novel long-acting antidiabetic FGF21 mimetic by targeted conjugation to a scaffold antibody.

    Science.gov (United States)

    Huang, Jie; Ishino, Tetsuya; Chen, Gang; Rolzin, Paul; Osothprarop, Trina F; Retting, Kelsey; Li, Lingna; Jin, Ping; Matin, Marla J; Huyghe, Bernard; Talukdar, Saswata; Bradshaw, Curt W; Palanki, Moorthy; Violand, Bernard N; Woodnutt, Gary; Lappe, Rodney W; Ogilvie, Kathleen; Levin, Nancy

    2013-08-01

    Fibroblast growth factor (FGF)21 improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF21 mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF21 through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF21. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic β-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF21 protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF21 while preserving full therapeutic functionality.

  18. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

    Science.gov (United States)

    Ribotta, M J; Higgins, R; Gottschalk, M; Lallier, R

    2000-01-01

    Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.

  19. Antibody Drug Conjugates: Preclinical Considerations.

    Science.gov (United States)

    Bornstein, Gadi G

    2015-05-01

    The development path for antibody drug conjugates (ADCs) is more complex and challenging than for unmodified antibodies. While many of the preclinical considerations for both unmodified and antibody drug conjugates are shared, special considerations must be taken into account when developing an ADC. Unlike unmodified antibodies, an ADC must preferentially bind to tumor cells, internalize, and traffic to the appropriate intracellular compartment to release the payload. Parameters that can impact the pharmacological properties of this class of therapeutics include the selection of the payload, the type of linker, and the methodology for payload drug conjugation. Despite a plethora of in vitro assays and in vivo models to screen and evaluate ADCs, the challenge remains to develop improved preclinical tools that will be more predictive of clinical outcome. This review will focus on preclinical considerations for clinically validated small molecule ADCs. In addition, the lessons learned from Mylotarg®, the first in class FDA-approved ADC, are highlighted.

  20. Anti-VEGF strategies - from antibodies to tyrosine kinase inhibitors: background and clinical development in human cancer.

    LENUS (Irish Health Repository)

    Korpanty, Grzegorz

    2012-01-01

    Tumour angiogenesis (formation of new blood vessels supporting tumour growth and metastasis) is a result of complex interactions between the tumour and the surrounding microenvironment. Targeting tumours with anti-angiogenic therapy remains an exciting area of preclinical and clinical studies. Although many significant advances have been achieved and the clinical use of anti-angiogenic drugs is now well recognized in many solid malignancies, these therapies fall short of their anticipated clinical benefits and leave many unanswered questions like exact mechanism of action, patients\\' selection and monitoring response to anti-angiogenic drugs. Tumour angiogenesis is controlled by complex signaling cascades and ongoing research into molecular mechanisms of tumour angiogenesis not only helps to understand its basic mechanisms but hopefully will identify new therapeutic targets. In 2012, both monoclonal antibodies and small molecule tyrosine kinase inhibitors remain the two major clinically useful therapeutic options that interfere with tumour angiogenesis in many solid malignancies.

  1. Development and validation of novel enzyme activity methods to assess inhibition of matrix metalloproteinases (MMPs) in human serum by antibodies against enzyme therapeutics.

    Science.gov (United States)

    Edkins, Thomas J; Alhadeff, Jack A; Kwok, Vincent; Kalensky, Charles; Rock, Marie T; Vidmar, Thomas J; Del Tito, Benjamin J

    2012-11-01

    This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

    Science.gov (United States)

    Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

    2018-04-01

    Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

  3. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation.

    Science.gov (United States)

    Noble, James E; Bunk, David M; Christenson, Robert H; Cole, Kenneth D; He, Hua-Jun; Katrukha, Alexei G; Panteghini, Mauro; Porter, Robert A; Schimmel, Heinz; Tate, Jillian R; Wang, Lili

    2010-11-01

    In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.

  5. Development and evaluation of a new lateral flow assay for simultaneous detection of antibodies against African Horse Sickness and Equine Infectious Anemia viruses.

    Science.gov (United States)

    Costa, Sofia; Sastre, Patricia; Pérez, Teresa; Tapia, Istar; Barrandeguy, María; Sánchez-Vizcaíno, José M; Sánchez-Matamoros, Almudena; Wigdorovitz, Andrés; Sanz, Antonio; Rueda, Paloma

    2016-11-01

    African horse sickness (AHS) and equine infectious anemia (EIA) are both notifiable equid specific diseases that may present similar clinical signs. Considering the increased global movement of horses and equine products over the past decades, together with the socio-economic impact of previous AHS and EIA outbreaks, there is a clear demand for an early discrimination and a strict control of their transmission between enzootic and AHS/EIA-free regions. Currently, the individual control and prevention of AHS or EIA relies on a series of measures, including the restriction of animal movements, vector control, and the use of several laboratory techniques for viral identification, amongst others. Despite being widely employed in surveillance programmes and in the control of animal movements, the available serological assays can only detect AHS- or EIA-specific antibodies individually. In this work, a duplex lateral flow assay (LFA) for simultaneous detection and differentiation of specific antibodies against AHS virus (AHSV) and EIA virus (EIAV) was developed and evaluated with experimental and field serum samples. The duplex LFA was based on the AHSV-VP7 outer core protein and the EIAV-P26 major core protein. The results indicated that the duplex LFA presented a good analytical performance, detecting simultaneously and specifically antibodies against AHSV and EIAV. The initial diagnostic evaluation revealed a good agreement with results from the AHS and EIA tests prescribed by the OIE, and it highlighted the usefulness of the new AHSV/EIAV duplex LFA for an on-field and point-of-care first diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures.

    Science.gov (United States)

    Devlin, Shauna; Meneely, Julie P; Greer, Brett; Campbell, Katrina; Vasconcelos, Vitor; Elliott, Christopher T

    2014-05-01

    A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Magnetic Purification of Antibodies

    Science.gov (United States)

    Dhadge, Vijaykumar Laxman

    This work aimed at the development of magnetic nanoparticles for antibody purification and at the evaluation of their performance in Magnetic fishing and in a newly developed hybrid technology Magnetic Aqueous Two Phase Systems. Magnetic materials were produced by coprecipitation and solvothermal approaches. Natural polymers such as dextran, extracellular polysaccharide and gum Arabic were employed for coating of iron oxide magnetic supports. Polymer coated magnetic supports were then modified with synthetic antibody specific ligands,namely boronic acid, a triazine ligand (named 22/8) and an Ugi ligand (named A2C7I1). To optimize the efficacy of magnetic nanoparticles for antibody magnetic fishing, various solutions of pure and crude antibody solutions along with BSA as a non-specific binding protein were tested. The selectivity of magnetic nanoparticle for antibody, IgG, was found effective with boronic acid and ligand 22/8. Magnetic supports were then studied for their performance in high gradient magnetic separator for effective separation capability as well as higher volume handling capability. The magnetic materials were also supplemented to aqueous two phase systems, devising a new purification technology. For this purpose, magnetic particles modified with boronic acid were more effective. This alternative strategy reduced the time of operation,maximized separation capability (yield and purity), while reducing the amount of salt required. Boronic acid coated magnetic particles bound 170 +/- 10 mg hIgG/g MP and eluted 160 +/- 5 mg hIgG/g MP, while binding only 15 +/- 5 mg BSA/g MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 x 105 M-1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed/g MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely

  8. Risk factors for development of left ventricular thrombus after first acute anterior myocardial infarction-association with anticardiolipin antibodies

    Directory of Open Access Journals (Sweden)

    Okuyan Ertuğrul

    2010-09-01

    Full Text Available Abstract Background Left ventricular thrombus(LVT] formation is a frequent complication in patients with acute anterior myocardial infarction(MI. LVT is associated with increased risk of embolism and higher mortality rates after acute MI. Anticardiolipin antibodies (ACA are immunoglobulins that react with phospholipid-binding proteins interfering with the prothrombin activator complex. The effects of phospholipids on pathophysiology of cardiovascular thrombotic events are well known. In this study, we aimed to evaluate the importance of clinical and biochemical parameters including anticardiolipin antibodies on left ventricular thrombus formation after acute anterior MI. Methods and Results Seventy patients with a first anterior AMI were prospectively and consecutively enrolled. Patients with previous MI, autoimmune disease, collagen vascular disease and arterial or venous thrombosis history were excluded from this study. At the time of hospitalization, key demographic and clinical characteristics were collected including age, gender, ethanol intake and presence of traditional risk factors for atherosclerosis (hypertension, diabetes, smoking, hyperlipidemia, positive family history. Patients were evaluated for echocardiographic data, blood chemistry and ACA. Two-dimensional and Doppler echocardiographic examinations were performed in all patients within the first week and at 14 days after MI. LV thrombus was detected in 30 (42.8% patients. ACA IgM levels were significantly higher in the patient group with LV thrombus than in the group without thrombus (12.44 ±4.12 vs. 7.69 ± 4.25 mpl, p = 0,01. ACA IgG levels were also found higher in the group with LV thrombus (24.2 ± 7.5 vs.17.98 ± 6.45 gpl, p = 0.02. Multivariate analyses revealed diabetes mellitus, higher WMSI, lower MDT and higher ACA IgM and higher ACA IgG levels as independent predictors of left ventricular thrombus formation. Conclusions Our data demonstrate that beside the low

  9. Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples.

    Science.gov (United States)

    Xing, Yue; Meng, Meng; Xue, Huyin; Zhang, Taichang; Yin, Yongmei; Xi, Rimo

    2012-09-15

    Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC(50) value of 0.52 ng mL(-1) and detection limit of 25 pg mL(-1) (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%-106%), intra-assay (foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Optimization and Validation of a Plaque Reduction Neutralization Test for the Detection of Neutralizing Antibodies to Four Serotypes of Dengue Virus Used in Support of Dengue Vaccine Development

    Science.gov (United States)

    Timiryasova, Tatyana M.; Bonaparte, Matthew I.; Luo, Ping; Zedar, Rebecca; Hu, Branda T.; Hildreth, Stephen W.

    2013-01-01

    A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype–specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT50 for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT50 was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10. PMID:23458954

  11. Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

    Science.gov (United States)

    Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

    2017-11-22

    Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

  12. Development of novel anti-Kv 11.1 antibody-conjugated PEG-TiO2 nanoparticles for targeting pancreatic ductal adenocarcinoma cells

    Science.gov (United States)

    Sette, Angelica; Spadavecchia, Jolanda; Landoulsi, Jessem; Casale, Sandra; Haye, Bernard; Crociani, Olivia; Arcangeli, Annarosa

    2013-12-01

    Titanium dioxide (TiO2) has been widely used in many nanotechnology areas including nanomedicine, where it could be proposed for the photodynamic and sonodynamic cancer therapies. However, TiO2 nanoformulations have been shown to be toxic for living cells. In this article, we report the development of a new delivery system, based on nontoxic TiO2 nanoparticles, further conjugated with a monoclonal antibody against a novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel. We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG TiO2 nanocrystals (PEG-TiO2 NPs). Anti-Kv 11.1 monoclonal antibodies (Kv 11.1-Mab) were further linked to the terminal carboxylic acid groups. Proper conjugation was confirmed by X-ray photoelectron spectroscopy analysis. Kv 11.1-Mab-PEG-TiO2 NPs efficiently recognized the specific Kv 11.1 antigen, both in vitro and in pancreatic ductal adenocarcinoma (PDAC) cells, which express the Kv 11.1 channel onto the plasma membrane. Both PEG TiO2 and Kv 11.1-Mab-PEG-TiO2 NPs were not cytotoxic, but only Kv 11.1-Mab-PEG-TiO2 NPs were efficiently internalized into PDAC cells. Data gathered from this study may have further applications for the chemical design of nanostructures to be applied for therapeutic purposes in pancreatic cancer.

  13. Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

    Science.gov (United States)

    Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

    2015-01-01

    The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

  14. Anti-hLAMP2-antibodies and dual positivity for anti-GBM and MPO-ANCA in a patient with relapsing pulmonary-renal syndrome

    Directory of Open Access Journals (Sweden)

    Kistler Thomas

    2011-06-01

    Full Text Available Abstract Background Pulmonary-renal syndrome associated with anti-glomerular basement membrane (GBM antibodies, also known as Goodpasture's syndrome, is a rare but acute and life-threatening condition. One third of patients presenting as anti-GBM antibody positive pulmonary-renal syndrome or rapidly progressive glomerulonephritis are also tested positive for anti-neutrophil cytoplasmic antibodies (ANCA. Whilst anti-GBM disease is considered a non-relapsing condition, the long-term course of double-positive patients is less predictable. Case Presentation We report a patient with such dual positivity, who presented with pulmonary hemorrhage, crescentic glomerulonephritis and membranous nephropathy. Plasmapheresis in combination with immunosuppresive therapy led to a rapid remission but the disease relapsed after two years. The serum of the patient was tested positive for antibodies to human lysosomal membrane protein 2 (hLAMP2, a novel autoantigen in patients with active small-vessel vasculitis (SVV. The anti-hLAMP2 antibody levels correlated positively with clinical disease activity in this patient. Conclusion We hypothesize that this antibody may indicate a clinical course similar to ANCA-associated vasculitis in double-positive patients. However, this needs to be confirmed on comprehensive patient cohorts.

  15. Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics

    OpenAIRE

    Zhou, Lei; Hoofring, Sarah A.; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J.; Chirmule, Narendra; Starcevic, Marta

    2012-01-01

    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic....

  16. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Directory of Open Access Journals (Sweden)

    Morten A Nielsen

    Full Text Available The disease caused by Plasmodium falciparum (Pf involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM is one such manifestation in which Pf infected erythrocytes (IE bind to chondroitin sulphate A (CSA through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2 expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens

  17. Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody-associated glomerulonephritis.

    Science.gov (United States)

    Yang, Jia Jin; Pendergraft, William F; Alcorta, David A; Nachman, Patrick H; Hogan, Susan L; Thomas, Robin P; Sullivan, Pamela; Jennette, J Charles; Falk, Ronald J; Preston, Gloria A

    2004-08-01

    Granulopoiesis-related genes are distinctively upregulated in peripheral leukocytes of patients with antineutrophil cytoplasmic autoantibodies (ANCA)-associated glomerulonephritis. Affymetrix microarrays identified the upregulation of nine neutrophilic primary granule genes, including myeloperoxidase (MPO) and proteinase 3 (PR3), plus five secondary granule genes. Coordinate expression of granulocyte maturation marker CD35, measured by TaqMan PCR, and positive in situ staining for PR3 transcripts in polymorphic neutrophils and monocytes indicate that these genes are expressed in "mature" cells. Increased transcripts correlated with disease activity and absolute neutrophil values but not with "left shift," drug regimen, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender, or age. Upregulation of PR3 and MPO transcripts was specifically associated with ANCA disease (n = 56) as these changes were not detected in patients with ESRD (n = 25) or systemic lupus erythematosus (n = 17), as determined by TaqMan PCR. This is the first report of this phenomenon in nonneoplastic cells. The data raise the hypothesis that, in addition to the presence of anti-MPO or anti-PR3 autoantibodies, a second critical component in the cause of this disease is the reactivation of once-silenced genes leading to increased antigen availability.

  18. Generation of monoclonal antibodies and development of an immunofluorometric assay for the detection of CUZD1 in tissues and biological fluids.

    Science.gov (United States)

    Farkona, Sofia; Soosaipillai, Antoninus; Filippou, Panagiota; Korbakis, Dimitrios; Serra, Stefano; Rückert, Felix; Diamandis, Eleftherios P; Blasutig, Ivan M

    2017-12-01

    CUB and zona pellucida-like domain-containing protein 1 (CUZD1) was identified as a pancreas-specific protein and was proposed as a candidate biomarker for pancreatic related disorders. CUZD1 protein levels in tissues and biological fluids have not been extensively examined. The purpose of the present study was to generate specific antibodies targeting CUZD1 to assess CUZD1 expression within tissues and biological fluids. Mouse monoclonal antibodies against CUZD1 were generated and used to perform immunohistochemical analyses and to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA). CUZD1 protein expression was assessed in various human tissue extracts and biological fluids and in gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant protein. Immunohistochemical staining of CUZD1 in pancreatic tissue showed that the protein is localized to the acinar cells and the lumen of the acini. Western blot analysis detected the protein in pancreatic tissue extract and pancreatic juice. The newly developed ELISA measured CUZD1 in high levels in pancreas and in much lower but detectable levels in several other tissues. In the biological fluids tested, CUZD1 expression was detected exclusively in pancreatic juice. The analysis of gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant CUZD1 suggested that the protein exists in high molecular weight protein complexes. This study describes the development of tools targeting CUZD1 protein, its tissue expression pattern and levels in several biological fluids. These new tools will facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  19. Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

    Science.gov (United States)

    Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

    2011-06-01

    Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

  20. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.

    Science.gov (United States)

    Sack, Markus; Rademacher, Thomas; Spiegel, Holger; Boes, Alexander; Hellwig, Stephan; Drossard, Juergen; Stoger, Eva; Fischer, Rainer

    2015-10-01

    The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Follow-up of pregnant women exposed to chicken pox: an audit of relationship between level of antibody and development of chicken pox.

    Science.gov (United States)

    Boxall, E H; Maple, P A C; Rathod, P; Smit, E

    2011-10-01

    The purpose of this study was to validate through natural exposure a cut-off level of varicella zoster IgG as protective against infection with varicella zoster virus (VZV). Laboratory testing to determine VZV immune status of pregnant women exposed to varicella is recommended. Quantitative assays are now available which are sensitive and specific. More than 200 consecutive requests for screening in pregnant patients with recent varicella contacts were followed-up by questionnaire. DiaSorin LIAISON and VZV time resolved fluorescence immuno assay (VZV TRFIA) were used to measure VZV antibody level. One hundred fifty out of 209 (72%) questionnaires were returned; 14 patients developed varicella, 129 did not and seven were not known. Patients who had been given VZIG and developed varicella on follow-up had a mean antibody level before VZIG of 28 mIU/ml and 62 mIU/ml, by LIAISON and TRFIA, respectively. The mean IgG level of those that did not develop varicella was 885 and 866 mIU/ml by LIAISON and TRFIA, respectively. Those with levels pox than those with levels >100 mIU/ml (relative risk of 10.4 for LIAISON and 8.8 for TRFIA). On the basis of the relatively small numbers in this study, quantitative assays, using a 100mIU/ml cut-off, can differentiate between those who are susceptible and those who are protected against exposure, however follow-up studies should include sampling for VZV DNA and IgM.

  2. Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

    Directory of Open Access Journals (Sweden)

    R Hoare

    Full Text Available Infectious salmon anaemia virus (ISAV is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs were developed against Segment 3 (encoding the viral nucleoprotein, NP of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA, indirect fluorescent antibody technique (IFAT and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L. smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p. injection of virus at 50% Tissue Culture Infective Dose (TCID50 = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point, was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections. To our knowledge this is the first report of the use of Luminex (Bio-Plex technology for the detection of a fish pathogen.

  3. Interferon-beta specific T cells are associated with the development of neutralizing antibodies in interferon-beta treated multiple sclerosis patients.

    Science.gov (United States)

    Kalluri, Sudhakar Reddy; Grummel, Verena; Hracsko, Zsuzsanna; Pongratz, Viola; Pernpeintner, Verena; Gasperi, Christiane; Buck, Dorothea; Hemmer, Bernhard

    2018-03-01

    Beta-interferons are still among the most commonly used drugs to treat Multiple Sclerosis (MS). The use of beta-interferons is limited by the development of anti-drug antibodies (ADA), which may abrogate the treatment effect of the drug. Although the antibody response has been well studied, little is known about the T cell response to interferon-beta (IFN-β). We investigated T cell responses in four treatment naïve MS patients and twenty-three patients treated with IFN-β who had or had not developed ADA to IFN-β. T cell responses were determined by split-well and primary proliferation assays against different IFN-β protein preparations and a set of overlapping peptides covering the full sequence of IFN-β. T cell responses to IFN-β were observed in all donors. ADA positive patients showed higher T cell responses to IFN-β protein than ADA negative patients and untreated controls. We identified two immunodominant regions; T cell responses to IFN-β 1-40 were observed in all patients independent of ADA status, while T cell responses to IFN-β 125-159 were stronger in ADA positive than ADA negative patients. IFN-β specific T cell responses were HLA class II restricted and in ADA positive patients skewed towards a Th2 phenotype. In IFN-β treated patients we observed a correlation between IFN-β specific T cell responses, serum ADA titer and loss of biological activity of IFN-β treatment. Our studies demonstrate the occurrence of an antigen specific HLA class II restricted Th2 T cell response associated with the development of ADA in IFN-β treated patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  5. Anti-Myeloperoxidase Antibodies Associate with Future Proliferative Lupus Nephritis

    Directory of Open Access Journals (Sweden)

    S. W. Olson

    2017-01-01

    Full Text Available Background. The subclinical pathophysiology of proliferative lupus nephritis (PLN has not been fully elucidated. Myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA is associated with PLN, but prediagnostic levels have not been reported. Methods. We performed a retrospective case-control Department of Defense Serum Repository (DoDSR study comparing MPO-ANCA levels in longitudinal prediagnostic serum samples for 23 biopsy confirmed proliferative lupus nephritis (PLN patients to DoDSR identified age, sex, race, and age of serum matched healthy and SLE without LN disease controls. We also compared the temporal relationship of MPO-ANCA to anti-double stranded DNA antibodies (dsDNAab. Results. A greater proportion of PLN patients had prediagnostic MPO-ANCA levels above ≥3 U/mL and ≥6 U/mL compared to SLE without LN (91% versus 43%, p<0.001; 57% versus 5%, p<0.001, resp.. In subgroup analysis, the MPO-ANCA threshold of ≥3 U/mL was significant at <1 year (88% versus 39%, p=0.007 and 1–4 years (87% versus 38%, p=0.009 prior to diagnosis. Statistically significant subclinical MPO-ANCA levels (≥3 U/mL occurred prior to statistically significant dsDNAab ≥ 3 IU/ml (89% versus 11%, p=0.003. Conclusions. Subclinical MPO-ANCA levels could distinguish future PLN from SLE without LN. MPO-ANCA manifests prior to clinical disease and subclinical dsDNAab to suggest that it may contribute directly to PLN pathogenicity.

  6. Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

    Science.gov (United States)

    Wheaton, Catharine J; Mylniczenko, Natalie D; Rimoldi, John M; Gadepalli, Rama S V S; Hart, R; O'Hara, Bobbi R; Evans, Andrew N

    2018-01-31

    Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays

  7. Development of a method to measure kinetics of radiolabelled monoclonal antibody in human tumour with applications to microdosimetry: positron emission tomography studies of iodine-124 labelled 3F8 monoclonal antibody in glioma

    International Nuclear Information System (INIS)

    Daghighian, F.; Pentlow, K.S.; Graham, M.C.; Finn, R.D.; Larson, S.M.; Yeh, S.D.J.; Macapinlac, H.; DiResta, G.R.; Arbit, E.; Cheung, N.V.

    1993-01-01

    We present a method to assess quantitatively the immuhological characteristics of tumours using radiolabelled monoclonal antibody and positron emission tomography (PET) to improve dosimetry for radioimmunotherapy. This method is illustrated with a glioma patient who injected with 96.2 MPq of iodine-124 labelled 3FB, a murine antibody (IgG3) specific against the ganglioside G D2 . Serial PET scans and plasma samples were taken over 11 days. A three-compartment model was used to estimate the plasma to tumour transfer constant (K 1 ), the tumour to plasma transfer constant k 2 , the association and dissociation constants (k 3 , k 4 ) of antibody binding, and the binding potential. Tumour radioactivity peaked at 18 h at 0.0045% ID/g. The kinetic parameters were estimated to be: K 1 =0.048 ml h -1 g -1 , k 2 =0.16 h -1 , k 3 =0.03 h -1 , k 4 =0.015 h -1 and BP=2.25. Based on these kinetic parameters, the amount of tumour-bound radiolabelled monoclonal antibody was calculated. This method permits estimates of both macrodosimetry and microdosimetry at the cellular level based on in vivo non-invasive measurement. (orig.)

  8. Antibodies against Pseudomonas aeruginosa chromosomal beta-lactamase inpatients with cystic fibrosis are markers of the development of resistance of P. aeruginosa to beta-lactams

    DEFF Research Database (Denmark)

    Ciofu, O; Giwercman, B; Walter-Rasmussen, J

    1995-01-01

    Chromosomal beta-lactamase production is considered to be the most important resistance mechanism of Pseudomonas aeruginosa against beta-lactams. Recently we have detected serum and sputum antibodies against P. aeruginosa chromosomal beta-lactamase (a beta ab), using immunoblotting techniques....... In this study we have developed an enzyme-linked-immunosorbent assay to measure serum a beta ab response in 124 cystic fibrosis patients in a cross-sectional study and in 54 cystic fibrosis patients in a longitudinal study. The a beta ab response occurred after a median of 3 years following onset of chronic...... infection and was significantly higher (P beta ab levels correlated (r = 0.51, P = 0.0001) with the number of beta-lactam courses. A 14 fold increase in a beta ab...

  9. Viral RNA in the influenza vaccine may have contributed to the development of ANCA-associated vasculitis in a patient following immunisation.

    Science.gov (United States)

    Jeffs, Lisa S; Nitschke, Jodie; Tervaert, Jan Willem Cohen; Peh, Chen Au; Hurtado, Plinio R

    2016-04-01

    A number of large studies have demonstrated influenza vaccinations to be safe and effective. However, there have been some sporadic case reports, describing a temporal association of influenza vaccination with onset or relapse of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The nature of this association, beyond time of occurrence, remains unknown. The presentation of a previously healthy patient who developed ANCA-associated vasculitis (AAV) shortly after influenza vaccination provided us with the rare opportunity to study the possible mechanisms behind this observation. We tested the ability of different types and batches of influenza vaccines to stimulate proteinase-3 ANCA (PR3-ANCA) production in vitro. We found that only some influenza vaccines stimulated PR3-ANCA production in this patient. We demonstrated that this unusual response was associated with those vaccines that contained viral ribonucleic acid (RNA), the natural ligand for Toll-like receptor-7. Exome sequencing of the patient's DNA did not show any mutation in any of the molecules associated with Toll-like receptor signalling. We propose that hyper-reaction to viral RNA in the influenza vaccine may have contributed to the development of AAV following influenza vaccination in this patient.

  10. Primary alveolar echinococcosis: course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis.

    Science.gov (United States)

    Matsumoto, Jun; Kouguchi, Hirokazu; Oku, Yuzaburo; Yagi, Kinpei

    2010-09-01

    We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host-parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.

  11. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  12. Autophagy facilitates the development of breast cancer resistance to the anti-HER2 monoclonal antibody trastuzumab.

    Directory of Open Access Journals (Sweden)

    Alejandro Vazquez-Martin

    Full Text Available Autophagy has been emerging as a novel cytoprotective mechanism to increase tumor cell survival under conditions of metabolic stress and hypoxia as well as to escape chemotherapy-induced cell death. To elucidate whether autophagy might also protect cancer cells from the growth inhibitory effects of targeted therapies, we evaluated the autophagic status of preclinical breast cancer models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody trastuzumab (Tzb. We first examined the basal autophagic levels in Tzb-naive SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR, which optimally grow in the presence of Tzb doses as high as 200 microg/ml Tzb. Fluorescence microscopic analyses revealed that the number of punctate LC3 structures -a hallmark of autophagy- was drastically higher in Tzb-refractory cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses confirmed that the lipidation product of the autophagic conversion of LC3 was accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein expression, a phenomenon characterizing the occurrence of increased autophagic flux. Moreover, increased autophagy was actively used to survive Tzb therapy as TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal formation/function. Knockdown of LC3 expression via siRNA similarly resulted in reduced TzbR cell proliferation and supra-additively interacted with Tzb to re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells accumulated LC3 punctate structures and decreased p62 expression after treatment with high-dose Tzb, likely promoting their own resistance. This is the first report showing that HER2-overexpressing breast cancer cells chronically exposed to Tzb exhibit a bona fide up-regulation of the autophagic activity that efficiently works to protect breast cancer cells

  13. [In vitro immunization for the production of antibodies to tetanus toxin and toxoid. 1. Systems for the detection of in vitro synthetized specific immunoglobulins. Strategies of test development].

    Science.gov (United States)

    Kiessig, S T; Jahn, S; Porstmann, T; von Baehr, R

    1987-01-01

    By means of semipurified tetanus toxin for solid phase coating in an enzyme immunoassay (ELISA) for detection of specific IgG and IgM antibodies a detection limit of 0.02 IU per litre was achieved. The addition of serum from animals like horses or goats as inert protein to the dilution medium was omitted to prevent a displacement of human antibodies by antitetanus antibodies present in the animals sera. The specificity of the ELISA was demonstrated by inhibition experiments with soluble antigen and in an ELISA for detection of anti-tetanus toxin antibodies from mice immunized with the toxoid from the different purification steps.

  14. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Okada, H.

    1990-05-01

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

  15. Antimitochondrial antibody

    Science.gov (United States)

    ... from a vein. The procedure is called a venipuncture . How to Prepare for the Test Your health ... Guidelines Viewers & Players MedlinePlus Connect for EHRs For Developers U.S. National Library of Medicine 8600 Rockville Pike, ...

  16. Development of Insulin Detemir/Insulin Aspart Cross-Reacting Antibodies Following Treatment with Insulin Detemir: 104-week Study in Children and Adolescents with Type 1 Diabetes Aged 2-16 Years.

    Science.gov (United States)

    Thalange, Nandu; Bereket, Abdullah; Jensen, Lisbeth Bjerring; Hiort, Line Conradsen; Peterkova, Valentina

    2016-12-01

    To study the long-term development (104 weeks) of insulin antibodies during treatment with insulin detemir (IDet) and insulin aspart (IAsp) in children with type 1 diabetes aged 2-16 years. A 52-week, two-arm, randomized trial comparing IDet and neutral protamine Hagedorn insulin, both in combination with IAsp, was followed by a one-arm, 52-week extension trial of the IDet + IAsp arm. The present analysis was conducted in children who completed the randomized trial and entered into the extension trial. Of the 177 children randomized to IDet treatment, 146 entered the extension trial. IDet-IAsp cross-reacting antibodies peaked within the first 39 weeks of treatment before gradually declining. A similar pattern was seen for IDet-specific and IAsp-specific antibodies. At end of trial (EOT), no correlation was observed between the level of IDet-specific or IAsp-specific antibodies or IDet-IAsp cross-reacting antibodies and either glycated hemoglobin (HbA1c) or basal insulin dose. Mean HbA1c was stable during the treatment period, with a slight increase over time from 8.41% (68.4 mmol/mol) at baseline to 8.74% (72 mmol/mol) at EOT. Mean IDet dose increased from 0.43 U/kg at baseline to 0.66 U/kg at EOT. Mean IAsp dose increased from 0.46 U/kg to 0.51 U/kg at EOT. Although treatment with IDet and IAsp is associated with development of specific and cross-reacting antibodies, no correlation between insulin antibodies and basal insulin dose or HbA1c was found. Novo Nordisk A/S. ClinicalTrials.gov identifiers: NCT00435019 and NCT00623194.

  17. The value of pattern capillary changes and antibodies to predict the development of systemic sclerosis in patients with primary Raynaud's phenomenon.

    Science.gov (United States)

    Pavlov-Dolijanovic, Slavica R; Damjanov, Nemanja S; Vujasinovic Stupar, Nada Z; Baltic, Snezana; Babic, Dragan D

    2013-12-01

    The aim of this study is to assess the prognostic value of major provisional criteria for the development of systemic sclerosis (SSc) in primary Raynaud's phenomenon (RP) patients. We retrospectively studied the chart of 497 patients with primary RP in whom anticentromere (ACA) and antitopoisomerase I (ATA) antibodies tests and a capillary reading were available. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratios (LHR+), negative likelihood ratios (LHR-), odds ratio (OR), and area under the receiver operating characteristics curve (AUC) of those criteria were assessed to predict the development of SSc. During the average follow-up of 2.3 ± 1.9 years, 159 (32 %) patients evolved to SSc, 245 (49.3 %) evolved to other connective tissue diseases, and 93 (18.7 %) patients did not progress. The SSc pattern predicted SSc satisfactorily (LHR+ 4.12, LHR- 0.07, OR 63, AUC 0.819; P pattern and ACA or ATA were significantly associated with the development of SSc (LHR+ 2.98, LHR- 0.70, OR 4.2, AUC 0.674; P pattern or ATA as independent risk factors, as well as following two parameters together (SSc pattern and ATA or SSc pattern and ACA) were good predictors for the development of SSc.

  18. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-10-01

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

  19. Assessing Zika virus replication and the development of Zika-specific antibodies after a mid-gestation viral challenge in guinea pigs.

    Science.gov (United States)

    Bierle, Craig J; Fernández-Alarcón, Claudia; Hernandez-Alvarado, Nelmary; Zabeli, Jason C; Janus, Bradley C; Putri, Dira S; Schleiss, Mark R

    2017-01-01

    Primary Zika virus (ZIKV) infections that occur during pregnancy can cause spontaneous abortion and profoundly disrupt fetal development. While the full range of developmental abnormalities associated with congenital Zika syndrome is not yet known, severe cases of the syndrome can present with microcephaly, extensive neurologic and ocular damage, and pronounced joint malformations. Animal models that accurately recapitulate congenital Zika syndrome are urgently needed for vaccine development and for the study of ZIKV pathogenesis. As guinea pigs have successfully been used to model transplacental infections by cytomegalovirus, syphilis, and Listeria monocytogenes, we sought to test whether ZIKV could productively infect guinea pigs and whether viral transmission with attendant fetal pathology would occur after a mid-gestation viral challenge. We found that guinea pig cells supported ZIKV replication in vitro. Experimental infection of non-pregnant animals did not result in overt disease but low-level, detectable viremia was observed. When pregnant guinea pigs were challenged with ZIKV at between 18 and 21 days gestational age, ZIKV was not detected in maternal or pup blood, plasma, or tissues and no significant differences in maternal weight gain or pup size were observed following challenge. Nonetheless, a robust antibody response against ZIKV was detected in both the pups and dams. These results suggest that, while guinea pigs can model aspects of the immune response to ZIKV infection during pregnancy, naturally circulating ZIKV strains are not pathogenic during the pregnancy of immunocompetent guinea pigs and do not interfere with normal pup development.

  20. Assessing Zika virus replication and the development of Zika-specific antibodies after a mid-gestation viral challenge in guinea pigs.

    Directory of Open Access Journals (Sweden)

    Craig J Bierle

    Full Text Available Primary Zika virus (ZIKV infections that occur during pregnancy can cause spontaneous abortion and profoundly disrupt fetal development. While the full range of developmental abnormalities associated with congenital Zika syndrome is not yet known, severe cases of the syndrome can present with microcephaly, extensive neurologic and ocular damage, and pronounced joint malformations. Animal models that accurately recapitulate congenital Zika syndrome are urgently needed for vaccine development and for the study of ZIKV pathogenesis. As guinea pigs have successfully been used to model transplacental infections by cytomegalovirus, syphilis, and Listeria monocytogenes, we sought to test whether ZIKV could productively infect guinea pigs and whether viral transmission with attendant fetal pathology would occur after a mid-gestation viral challenge. We found that guinea pig cells supported ZIKV replication in vitro. Experimental infection of non-pregnant animals did not result in overt disease but low-level, detectable viremia was observed. When pregnant guinea pigs were challenged with ZIKV at between 18 and 21 days gestational age, ZIKV was not detected in maternal or pup blood, plasma, or tissues and no significant differences in maternal weight gain or pup size were observed following challenge. Nonetheless, a robust antibody response against ZIKV was detected in both the pups and dams. These results suggest that, while guinea pigs can model aspects of the immune response to ZIKV infection during pregnancy, naturally circulating ZIKV strains are not pathogenic during the pregnancy of immunocompetent guinea pigs and do not interfere with normal pup development.

  1. Acetylcholine receptor antibody

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

  2. Platelet antibodies blood test

    Science.gov (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  3. The future of antibodies as cancer drugs.

    Science.gov (United States)

    Reichert, Janice M; Dhimolea, Eugen

    2012-09-01

    Targeted therapeutics such as monoclonal antibodies (mAbs) have proven successful as cancer drugs. To profile products that could be marketed in the future, we examined the current commercial clinical pipeline of mAb candidates for cancer. Our analysis revealed trends toward development of a variety of noncanonical mAbs, including antibody-drug conjugates (ADCs), bispecific antibodies, engineered antibodies and antibody fragments and/or domains. We found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets and mechanisms of action (MOA). Although well-validated targets, such as epidermal growth factor receptor (EGFR) and CD20, continue to provide opportunities for companies, we found notable trends toward targeting less-well-validated antigens and exploration of innovative MOA such as the generation of anticancer immune responses or recruitment of cytotoxic T cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes.

    Science.gov (United States)

    Holder, Angela L; Kennedy, Lorna J; Ollier, William E R; Catchpole, Brian

    2015-10-15

    Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n=942) compared with non-diabetic dogs (n=100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic

  5. ALPHA–2–MACROGLOBULIN COMPLEXES WITH IGG ANTIBODIES, PLASMIN, AND THEIR INTERRELATION WITH OTHER FACTORS OF HUMORAL IMMUNITY DURING THE DEVELOPMENT OF RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    V. N. Zorina

    2005-01-01

    Full Text Available Abstract. Human alpha–2–macroglobulin (α2–MG acts as a broad–spectrum cytokine and proteasebinding protein, and it represents an evolutionarily conserved arm of innate immune system. Meanwhile, previous studies have shown that rheumatoid arthritis (RA development may cause alterations in α2–MG conformation and lessen its ability to bind and utilise regulatory substances. We investigated serum contents of α2–MG–IgG and α2–MG–plasmin complexes, as well as total concentrations ofα2–MG, plasmin (Pl, IgG, IL–6, IL–1β, TNFα and Rf–IgM, in order to evaluate the levels of anti–α2–MG antibody production in sera of patients with different degrees of RA activity and some interrelations of preformed immune complex with some other substances implicated in RA development. Serum samples were obtained in acute phase of RA, before the treatment was started. We have revealed significantly increased levels of α2–MG–IgG complex in the groups with severe RA, accompanied by increase in total IgG levels, without significant changes in total α2–MG concentrations. We have also demonstrated that increased levels of Pl–α2–MG complex did correspond to the severity of disease, and showed statistically high correlation with α2–MG–IgG levels. We have found a significant increase of IL–1β, IL–6, TNFα and Rf–IgM in RA, in absence of significant correlations with α2–MG–IgG contents. The results obtained allow us to suggest that abundant accumulation of serum antibodies to α2–MG or Pl–α2–MG complex during inflammation in the people with innate α2–MG deficiency, followed by increased levels of proinflammatory cytokines, may provoke a cascade–like development of RA. Serum levels of α2–MG may serve as prognostic marker in RA. (Med. Immunol., 2005, vol.7, № 5–6, pp. 557–562

  6. Replacing reprogramming factors with antibodies selected from combinatorial antibody libraries.

    Science.gov (United States)

    Blanchard, Joel W; Xie, Jia; El-Mecharrafie, Nadja; Gross, Simon; Lee, Sohyon; Lerner, Richard A; Baldwin, Kristin K

    2017-10-01

    The reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is usually achieved by exogenous induction of transcription by factors acting in the nucleus. In contrast, during development, signaling pathways initiated at the membrane induce differentiation. The central idea of this study is to identify antibodies that can catalyze cellular de-differentiation and nuclear reprogramming by acting at the cell surface. We screen a lentiviral library encoding ∼100 million secreted and membrane-bound single-chain antibodies and identify antibodies that can replace either Sox2 and Myc (c-Myc) or Oct4 during reprogramming of mouse embryonic fibroblasts into iPSCs. We show that one Sox2-replacing antibody antagonizes the membrane-associated protein Basp1, thereby de-repressing nuclear factors WT1, Esrrb and Lin28a (Lin28) independent of Sox2. By manipulating this pathway, we identify three methods to generate iPSCs. Our results establish unbiased selection from autocrine combinatorial antibody libraries as a robust method to discover new biologics and uncover membrane-to-nucleus signaling pathways that regulate pluripotency and cell fate.

  7. Antibodies from plants for bionanomaterials.

    Science.gov (United States)

    Edgue, Gueven; Twyman, Richard M; Beiss, Veronique; Fischer, Rainer; Sack, Markus

    2017-11-01

    Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as 'green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications. WIREs Nanomed Nanobiotechnol 2017, 9:e1462. doi: 10.1002/wnan.1462 For further resources related to this article, please visit the WIREs website. © 2017 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.

  8. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  9. Development of a time-resolved fluorescence immunoassay for Epstein-Barr virus Zta IgA antibodies in human serum.

    Science.gov (United States)

    Chen, Juanjuan; Liu, Tiancai; Chen, Zhenhua; Hou, Jingyuan; Wu, Yingsong; Li, Ming

    2015-04-01

    The Epstein-Barr virus (EBV) transactivator protein (ZEBRA) is an immediate-early protein that plays an important role in the switch from latency to productive cycle in EBV virus. ZEBRA is an important marker of EBV reactivation. In order to diagnose EBV infection status correctly and timely, a novel immunoassay was developed based on an indirect time-resolved fluoroimmunoassay (TRFIA) for Zta IgA, which used recombinant Zta antigen as solid-phase antigen and Eu(3+)-labeled mouse antihuman IgA as corresponding probe. The precision, sensitivity, specificity test, and stability of the TRFIA kit were evaluated, and comparison with the traditional enzyme-linked immunosorbent assay (ELISA) was also investigated. The cutoff value for the TRFIA was 2.5. Intra- and interassay coefficients of variation for the TRFIA were 2.45-3.30% and 3.38-4.61% respectively. There was no cross-reactivity with the antibodies of cytomegalovirus (CMV) or herpes simplex virus (HSV) types 1 and 2, or other potential interferences. The established assay kit also behaved better in sensitivity and stability than the ELISA one. Additionally, the results in 382 serum samples using two analytical methods showed there was good agreement between the TRFIA and commercial ELISA kit. In the current study, the results demonstrated that the TRFIA that was developed for Zta IgA detection was more sensitive and reliable for the diagnosis of EBV infection and had potential value in automation and high-throughput screening.

  10. Development and scale-up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach.

    Science.gov (United States)

    Herzer, Sibylle; Bhangale, Atul; Barker, Gregory; Chowdhary, Isha; Conover, Matthew; O'Mara, Brian W; Tsang, Lily; Wang, Shi-Yu; Krystek, Stanley R; Yao, Yan; Rieble, Siegfried

    2015-07-01

    A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk. © 2015 Wiley Periodicals, Inc.

  11. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Directory of Open Access Journals (Sweden)

    Kathleen Ingenhoven

    2017-07-01

    Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

  12. Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk*

    Science.gov (United States)

    Huang, Bin; Yin, Yun; Lu, Lei; Ding, Hai; Wang, Lin; Yu, Ting; Zhu, Jia-jin; Zheng, Xiao-dong; Zhang, Yan-zhen

    2010-01-01

    A convenient competitive enzyme-linked immunosorbent assay (ELISA) for ciprofloxacin (CPFX) was developed by using rabbit monoclonal antibodies (RabMAbs) against a hapten-protein conjugate of CPFX-bovine serum albumin (BSA). The indirect competitive ELISA of CPFX had a concentration at 50% inhibition (IC50) of 1.47 ng/ml and a limit of detection (LOD) of 0.095 ng/ml. The mAb exhibited some cross-reactivity, however, not so high with enrofloxacin (28.8%), ofloxacin (13.1%), norfloxacin (11.0%), fleroxacin (22.6%), and pefloxacin (20.4%). And it showed almost no cross-reactivity with other antibiotics or sulfonamides evaluated in this study. The competitive ELISA kit developed here could be used as a screening tool to detect and control illegal addition of CPFX in food products. This kit had been applied to milk detection and the recovery rates from samples spiked by CPFX were in a range of 63.02%–84.60%, with coefficients of variation of less than 12.2%. PMID:20872990

  13. Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7.

    NARCIS (Netherlands)

    Purushothaman, A.; Fukuda, J.; Mizumoto, S.; Dam, G.B. ten; Kuppevelt, A.H.M.S.M. van; Kitagawa, H.; Mikami, T.; Sugahara, K.

    2007-01-01

    Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the

  14. Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

    Science.gov (United States)

    Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

    2018-01-11

    A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

  15. Study of the viability of technetium-99m labeling of whole antimyosin antibody and its fragment: development of radiopharmaceutical for cardiac survey

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro

    2007-01-01

    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled

  16. A system identification approach for developing model predictive controllers of antibody quality attributes in cell culture processes.

    Science.gov (United States)

    Downey, Brandon; Schmitt, John; Beller, Justin; Russell, Brian; Quach, Anthony; Hermann, Elizabeth; Lyon, David; Breit, Jeffrey

    2017-11-01

    As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed-batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1647-1661, 2017. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

  17. A monoclonal antibody (SJ-9A4) to P24 present on common alls, neuroblastomas and platelets - I. Characterization and development of a unique radioimmunometric assay.

    Science.gov (United States)

    Komada, Y; Peiper, S C; Melvin, S L; Metzger, D W; Tarnowski, B H; Green, A A

    1983-01-01

    We report the development and characterization of SJ-9A4, a monoclonal antibody (MoAb) produced against common acute lymphoblastic leukemia (C-ALL) cell lines. SJ-9A4 reacted with C-ALL, B-cell chronic lymphocytic leukemia (B-CLL), platelets and C-ALL neuroblastoma (NB) and the K562 cell lines. It had no significant reactivity with erythrocytes, granulocytes, circulating T or B lymphocytes, monocytes, granulocytic cell lines or a Ewing's sarcoma cell line. SJ-9A4 was shown to recognize the same region as two other MoAb to the p24 antigen, BA-2 and DU-ALL-1, as demonstrated by their ability to inhibit the binding of labeled SJ-9A4 to NALM-1 and NB cells. Other MoAb: J5, PI 153/3 and monoclonal anti-HLA-DR antibodies gave no inhibition. A solid phase indirect radioimmunometric assay (IRA) was developed which enabled the detection of P24 from C-ALL cells, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) or wheat germ agglutinin (WGA) and SJ-9A4 simultaneously. When BA-2 and DU-ALL-1 were used in place of SJ-9A4, similar IRA results were obtained. Using the RCA1/SJ-9A4-IRA, P24 from as few as 1.6 X 10(4) cells of a C-ALL cell line could be detected; however, similar extracts of NB cell lines were negative despite high levels of SJ-9A4 binding to intact cells. The presence of P24 in NB extracts was demonstrated by (1) preincubation of NB extracts with SJ-9A4 which blocked MoAb binding to P24 and (2) immunoadsorption of P24 from solubilized membranes of 35S-methionine (met) labeled NB cells. Treatment of NB cells with neuraminidase did not result in IRA binding when either RCA1 or WGA were used as the solid phase lectin indicating that the differences in lectin affinity are not due to over sialation of NB membrane glycoproteins. These findings demonstrate a difference in the glycosylation of P24 from C-ALL and NB cells.

  18. Discovery of Human IgGs against α-Cobratoxin for Development of Recombinant Antibody-based Antivenom

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Engmark, Mikael; Redsted Rasmussen, Arne

    , in which large mammals (typically horses) are immunized with snake venom and antiserum is derived from the animals blood. The incompatibility with the human immune system of these animal derived antivenoms leads to a range of side effects,such as serum sickness, anaphylaxis, and sometimes even death......More than 5.5 million people are bitten by venomous snakes per year on a global basis. This leads to approx. 125,000 deaths and 3 times as many amputations. Particularly Sub-Saharan Africa is affected by the problem. Current antivenoms are still being produced by a method developed in the 1890’s...

  19. Monoclonal antibodies against the major allergen of Plantago lanceolata pollen, Pla l 1: affinity chromatography purification of the allergen and development of an ELISA method for Pla l 1 measurement.

    Science.gov (United States)

    Calabozo, B; Duffort, O; Carpizo, J A; Barber, D; Polo, F

    2001-05-01

    Plantago lanceolata (English plantain) pollen is a relevant cause of pollinosis in temperate regions. The major allergen of this pollen, Pla l 1, is recognized by the specific IgE from more than 80% of plantain-sensitive patients. It displays significant sequence homology with the major olive-pollen allergen Ole e 1. The objective was to develop a monoclonal antibody-based ELISA to quantify Pla l 1, and to assess the correlation of Pla l 1 content with the biologic activity of plantain pollen extracts. We also aimed to establish the specificity of the monoclonal antibodies against the potentially cross-reactive allergen Ole e 1, and to investigate the presence of Pla l 1-like proteins in psyllium and melon that have been reported to cross-react with P. lanceolata pollen. After fusion of myeloma cells with spleen cells from a BALB/c mouse, two Pla l 1-specific monoclonal antibodies secreting hybridomas were selected, and the antibodies characterized. One of them (2A10) was used as the capture antibody in an ELISA for Pla l 1 quantitation. An anti-P. lanceolata rabbit serum was used as the second antibody. Pla l 1 was purified by immunoaffinity chromatography and used as the standard in the assay. The ELISA developed was highly reproducible and sensitive, with a detection limit of 0.1 ng/ml, and a practical working range of 0.4-12 ng/ml. The specificity was demonstrated against a large battery of allergens, including Ole e 1. The concentration of Pla l 1 was measured in 19 extracts of P. lanceolata pollen, and a good correlation was observed between the Pla l 1 content and the allergenic activity of the extracts. Pla l 1 was not detected in psyllium or melon extracts. The results prove the usefulness of the Pla l 1-ELISA for the standardization of extracts of P. lanceolata pollen intended for clinical use.

  20. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  1. Detection of pantothenic acid-immunoreactive neurons in the rat lateral septal nucleus by a newly developed antibody.

    Science.gov (United States)

    Mangas, Arturo; Yajeya, Javier; Gonzalez, Noelia; Husson, Marianne; Geffard, Michel; Coveñas, Rafael

    2016-01-01

    The available immunohistochemical techniques have documented restricted distribution of vitamins in the mammalian brain. The aim of the study was to develop a highly specific antiserum directed against pantothenic acid to explore the presence of this vitamin in the mammalian brain. According to ELISA tests, the anti-pantothenic acid antiserum used showed a good affinity (10-8 M) and specificity. The antiserum was raised in rabbits. Using an indirect immunoperoxidase technique, the mapping of pantothenic acid-immunoreactive structures was carried out in the rat brain. Pantothenic acid-immunoreactive perikarya were exclusively found in the intermediate part of the lateral septal nucleus. These cells were generally small, round, fusiform or pyramidal and showed 2-3 long (50-100 μm) immunoreactive dendrites. Any immunoreactive axons containing pantothenic acid were detected. The very restricted anatomical distribution of the pantothenic acid suggests that this vitamin could be involved in some specific neurophysiological mechanisms.

  2. Antibodies: an alternative for antibiotics?

    Science.gov (United States)

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.

  3. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    SANT'ANNA Anadayr L.M.

    2002-01-01

    Full Text Available 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.

  6. Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Minoru Tada

    Full Text Available Antibody-dependent cellular cytotoxicity (ADCC is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs. Among the human Fcγ receptors (FcγRs, FcγRIIIa is well known as the only receptor expressed in natural killer (NK cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1 and panitumumab (IgG2. We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.

  7. Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

    Science.gov (United States)

    Guo, Yancheng; Ngom, Babacar; Le, Tao; Jin, Xiue; Wang, Liping; Shi, Deshi; Wang, Xiliang; Bi, Dingren

    2010-09-15

    A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

  8. Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

    Directory of Open Access Journals (Sweden)

    Xiaoxiang Fu

    2015-08-01

    Full Text Available Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA was then developed. The median inhibitory concentration (IC50 of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

  9. Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercaria- I. analysis of antibody and T-lymphocyte responses in mouse strains developing differing levels of immunity

    International Nuclear Information System (INIS)

    James, S.L.; Labine, M.; Sher, A.

    1981-01-01

    The kinetics of cellular and humoral responses directed against schistosomula were examined in mice of three inbred strains which demonstrate differences in the degree of resistance induced by immunization with irradiated cercariae. T-Cell reactivity was observed during the first 4 weeks after vaccination but declined to control levels thereafter. Anti-schistosomulum antibody was first detected 2 weeks after vaccination, peaked by 6 weeks, and persisted as late as 15 weeks. In sera obtained at 6 weeks, antibody activity was detected in affinity chromatography-purified fractions containing IgM, IgA, IgG 1 , IgG 2 /sub a/, and IgG 3 immunoglobulins. In general, the cellular and humoral responses observed in C57Bl/6J mice, which consistently developed a high level of immunity after vaccination, were not significantly different from those observed in C3H/HeJ or CBA/J mice, which achieved only low to moderate levels of immunity. Thus, although antibody production appears to correlate more closely than T lymphocyte responsiveness with the typical long-term resistance pattern observed in this model, the absence of striking differences in parasite-specific antibody levels between mice of these different strains suggests that additional mechanisms may be involved in the development of immunity after vaccination

  10. Urinary and serum soluble CD25 complements urinary soluble CD163 to detect active renal anti-neutrophil cytoplasmic autoantibody-associated vasculitis: a cohort study.

    Science.gov (United States)

    Dekkema, Gerjan J; Abdulahad, Wayel H; Bijma, Theo; Moran, Sarah M; Ryan, Louise; Little, Mark A; Stegeman, Coen A; Heeringa, Peter; Sanders, Jan-Stephan F

    2018-03-01

    Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV. sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163. usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells. Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

  11. The effect of dexamethasone-induced immunosuppression on the development of faecal antibody and recovery from and resistance to rotavirus infection.

    Science.gov (United States)

    Oldham, G; Bridger, J C

    1992-04-01

    Rotavirus-naive and rotavirus-immune gnotobiotic calves were treated with high doses of dexamethasone (DX) to suppress the immune system. Calves were then infected with a virulent rotavirus inoculum, J-160, to investigate the role of immune responses both in recovery from primary rotavirus infection and in resistance to secondary rotavirus infection. Treatment of calves with DX markedly suppressed in vitro responsiveness of peripheral blood lymphocytes to mitogens within 48 h of the start of DX treatment. Suppression was similar in rotavirus-naive and rotavirus-immune calves. In contrast, the effect of DX treatment on specific antibody responses differed depending on when DX treatment started in relation to rotavirus infection. When DX treatment commenced prior to primary rotavirus infection both systemic and local specific antibody responses were inhibited. These calves, in which mitogen and antibody responses were suppressed, exhibited greater clinical signs than did control calves after infection with virulent rotavirus, but virus excretion was affected in only one of the two calves. When DX treatment was started after primary rotavirus infection but before secondary infection, systemic and local antibody responses to the primary infection and to the challenge infection were not affected. These calves resisted challenge with virulent virus as did DX-untreated rotavirus-immune calves, even though mitogen responses were suppressed. We conclude that in a primary rotavirus infection, virus excretion ceased when both antibody and mitogen responses were suppressed. Resistance to secondary rotavirus infection occurred when mitogen responsiveness was suppressed, but when antibody levels were normal. Thus, no evidence was obtained that fully functional cell-mediated immune mechanisms are essential for resistance to rotavirus infection. Evidence was provided for the ability of parenteral treatment with DX to suppress mucosal as well as systemic antibody responses.

  12. Radiolabeled antibodies for cancer imaging and therapy.

    Science.gov (United States)

    Barbet, Jacques; Bardiès, Manuel; Bourgeois, Mickael; Chatal, Jean-François; Chérel, Michel; Davodeau, François; Faivre-Chauvet, Alain; Gestin, Jean-François; Kraeber-Bodéré, Françoise

    2012-01-01

    Radiolabeled antibodies were studied first for tumor detection by single-photon imaging, but FDG PET stopped these developments. In the meantime, radiolabeled antibodies were shown to be effective in the treatment of lymphoma. Radiolabeling techniques are well established and radiolabeled antibodies are a clinical and commercial reality that deserves further studies to advance their application in earlier phase of the diseases and to test combination and adjuvant therapies including radiolabeled antibodies in hematological diseases. In solid tumors, more resistant to radiations and less accessible to large molecules such as antibodies, clinical efficacy remains limited. However, radiolabeled antibodies used in minimal or small-size metastatic disease have shown promising clinical efficacy. In the adjuvant setting, ongoing clinical trials show impressive increase in survival in otherwise unmanageable tumors. New technologies are being developed over the years: recombinant antibodies and pretargeting approaches have shown potential in increasing the therapeutic index of radiolabeled antibodies. In several cases, clinical trials have confirmed preclinical studies. Finally, new radionuclides, such as lutetium-177, with better physical properties will further improve the safety of radioimmunotherapy. Alpha particle and Auger electron emitters offer the theoretical possibility to kill isolated tumor cells and microscopic clusters of tumor cells, opening the perspective of killing the last tumor cell, which is the ultimate challenge in cancer therapy. Preliminary preclinical and preliminary clinical results confirm the feasibility of this approach.

  13. Fabrication of highly stable glyco-gold nanoparticles and development of a glyco-gold nanoparticle-based oriented immobilized antibody microarray for lectin (GOAL) assay.

    Science.gov (United States)

    Huang, Li-De; Adak, Avijit K; Yu, Ching-Ching; Hsiao, Wei-Chen; Lin, Hong-Jyune; Chen, Mu-Lin; Lin, Chun-Cheng

    2015-03-02

    The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Biophysical characterization of antibodies with isothermal titration calorimetry

    Directory of Open Access Journals (Sweden)

    Verna Frasca

    2016-07-01

    Full Text Available Antibodies play a key role in the immune response. Since antibodies bind antigens with high specificity and tight affinity, antibodies are an important reagent in experimental biology, assay development, biomedical research and diagnostics. Monoclonal antibodies are therapeutic drugs and used for vaccine development. Antibody engineering, biophysical characterization, and structural data have provided a deeper understanding of how antibodies function, and how to make better drugs. Isothermal titration calorimetry (ITC is a label-free binding assay, which measures affinity, stoichiometry, and binding thermodynamics for biomolecular interactions. When thermodynamic data are used together with structural and kinetic data from other assays, a complete structure-activity-thermodynamics profile can be constructed. This review article describes ITC, and discusses several applications on how data from ITC provides insights into how antibodies function, guide antibody engineering, and aid design of new therapeutic drugs.

  15. Antibody repertoire development in swine

    Czech Academy of Sciences Publication Activity Database

    Butler, J. E.; Sun, J.; Wertz, N.; Šinkora, Marek

    2006-01-01

    Roč. 30, - (2006), s. 199-221 ISSN 0145-305X Institutional research plan: CEZ:AV0Z50200510 Keywords : swine * b cells * immunoglobulins Subject RIV: EE - Microbiology, Virology Impact factor: 3.399, year: 2006

  16. Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

    Science.gov (United States)

    Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

    2010-08-01

    Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

  17. Plasma-enhanced antibody immobilization for the development of a capillary-based carcinoembryonic antigen immunosensor using laser-induced fluorescence spectroscopy.

    Science.gov (United States)

    Yu, Qiaoling; Zhan, Xuefang; Liu, Kunping; Lv, Hao; Duan, Yixiang

    2013-05-07

    In this study, antibody immobilization using a microwave-induced H2O/Ar plasma pretreatment was achieved for the first time. Plasma was used to activate the surface of a capillary-based immunosensor by increasing the density of silicon hydroxyls and dangling bonds to ensure better silanization. The capture antibodies were covalently immobilized after the silanized surface reacted with glutaraldehyde and antibodies. A Cy3-labeled detection antibody was used in combination with the antigen captured by the immunosensor to complete the sandwich-type immunoassay, and the signals were measured using a laser-induced fluorescence system. Microwave-induced H2O/Ar plasma pretreatment of the carcinoembryonic antigen (CEA) immunosensor improved the antibody immobilization, and there was an obvious improvement in the linear detection range, i.e., 1 order of magnitude compared with a commercial enzyme-linked immunosorbent assay (ELISA). This novel immobilization method dramatically improved the detection limit (0.5 pmol/L CEA) and sensitivity. Assay validation studies indicated that the correlation coefficient reached 0.9978, and the relative standard deviations were Ar plasma was demonstrated to be a sensitive tool for CEA diagnostics.

  18. Stratification of antibody-positive subjects by antibody level reveals an impact of immunogenicity on pharmacokinetics.

    Science.gov (United States)

    Zhou, Lei; Hoofring, Sarah A; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J; Chirmule, Narendra; Starcevic, Marta

    2013-01-01

    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

  19. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter

    Science.gov (United States)

    Repessé, Yohann; Peyron, Ivan; Dimitrov, Jordan D; Dasgupta, Suryasarathi; Moshai, Elika Farrokhi; Costa, Catherine; Borel-Derlon, Annie; Guillet, Benoit; D’Oiron, Roseline; Aouba, Achille; Rothschild, Chantal; Oldenburg, Johannes; Pavlova, Anna; Kaveri, Srinivas V; Lacroix-Desmazes, Sébastien

    2013-01-01

    Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46–3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30–3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development PMID:23716558

  20. Radiommunoassay for triiodothyronine in serum. Development of the solid phase technic and comparison with two liquid phase RIA systems: the polyethylene glycol (PEG) and double antibody methods

    International Nuclear Information System (INIS)

    Hamada, M.M.

    1985-01-01

    A solid phase radioimmunoassay (RIA) system for triiodothyronine (T 3 ) was established by immobilizing triiodothyronine antibodies on the inner wall of reaction tubes. The antibody-coated tubes were made via reaction of antibody with glutaraldeyde residue pre coated on the inner wall of the tubes by alkaline self-polimerization. The quality of the coated tubes was tested through its performance in RIA methodology, by analysing the following RIA parameters: minimum detectable dose (MMD), nonspecific binding (NSB), X 50%, slope of the standard curve, intra and inter assay precision, accuracy of the method and figure of merit. The quality and characteristics of the reagents used in the RIA were analysed. (M.A.C.) [pt

  1. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana

    1994-01-01

    A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody...... response was studied with serum samples collected in 1992 from 56 CF patients in a cross-sectional study and with serum samples from 18 CF patients in a longitudinal study. Anti-beta-lactamase immunoglobulin G antibodies were present in all of the serum samples from the patients with chronic...... bronchopulmonary P. aeruginosa infection (CF + P) but in none of the CF patients with no or intermittent P. aeruginosa infection. Anti-beta-lactamase antibodies were present in serum from CF + P patients after six antipseudomonal courses (median) and correlated with infection with a beta-lactam-resistant strain...

  2. Epitope analysis of anti-myeloperoxidase antibodies in patients with ANCA-associated vasculitis.

    Directory of Open Access Journals (Sweden)

    Shen-Ju Gou

    Full Text Available OBJECTIVE: Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA directing myeloperoxidase (MPO in ANCA-associated vasculitis (AAV. The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV. METHODS: Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands. RESULTS: Sera from 45 of the 77 (58.4% patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset. CONCLUSION: The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis.

  3. Production of antibodies which recognize opiate receptors on murine leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

    1988-01-01

    An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

  4. Antibodies Against Melanin

    African Journals Online (AJOL)

    1973-01-06

    Jan 6, 1973 ... Departments of Internal Medicine and Anatomical Pathology, University of Stellenbosch and MRC. Pigment Metabolism Research Unit, ... at the production of antibodies against natural melanoprotein. and a consideration of our negative .... the random polymerization of several monomers, antibody formed ...

  5. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    OpenAIRE

    Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-01-01

    International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...

  6. Clinical response, pharmacokinetics, development of human anti-chimaeric antibodies, and synovial tissue response to rituximab treatment in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Thurlings, R. M.; Teng, O.; Vos, K.; Gerlag, D. M.; Aarden, L.; Stapel, S. O.; van Laar, J. M.; Tak, P. P.; Wolbink, G. J.

    2010-01-01

    To analyse whether persistence of synovial B lineage cells and lack of clinical response to rituximab treatment in patients with rheumatoid arthritis (RA) are associated with low rituximab serum levels and anti-rituximab antibody (ARA) formation. Fifty-eight patients with RA were treated with

  7. Clinical response, pharmacokinetics, development of human anti-chimaeric antibodies, and synovial tissue response to rituximab treatment in patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Thurlings, R.M.; Teng, O.; Vos, K.; Gerlag, D.M.; Aarden, L.; Stapel, S.O.; van Laar, J.M.; Tak, P.P.; Wolbink, G.J.

    2010-01-01

    Objectives: To analyse whether persistence of synovial B lineage cells and lack of clinical response to rituximab treatment in patients with rheumatoid arthritis (RA) are associated with low rituximab serum levels and anti-rituximab antibody (ARA) formation. Methods: Fifty-eight patients with RA

  8. The development of AIDS or AIDS-related conditions in a cohort of HIV antibody-positive homosexual men during a 3-year follow-up period

    DEFF Research Database (Denmark)

    Pedersen, C; Kolby, P; Sindrup, J

    1989-01-01

    One hundred and thirty-three homosexual men seropositive for the antibody against human immunodeficiency virus (HIV) were enrolled in a prospective study in 1984-85. The 3-year cumulative incidences of the acquired immunodeficiency syndrome (AIDS) and AIDS-related conditions, by life-table analyses...

  9. In palindromic rheumatism, hand joint involvement and positive anti-CCP antibodies predict RA development after 1 year of follow-up

    NARCIS (Netherlands)

    Emad, Yasser; Anbar, Ashraf; Abo-Elyoun, Ihab; El Shaarawy, Nashwa; Al-Hanafi, Hadeel; Darwish, Hatem; Gamil, Mona; Rasker, Johannes J.

    2014-01-01

    This study aimed to determine the frequency of rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibodies in a cohort of patients with palindromic rheumatism (PR) and to find determinants for progression to rheumatoid arthritis (RA). All new cases of PR (n = 90) were included

  10. Patients with humoral primary immunodeficiency do not develop protective anti-influenza antibody titers after vaccination with trivalent subunit influenza vaccine

    NARCIS (Netherlands)

    van Assen, Sander; Holvast, Albert; Telgt, Denise S.C.; Benne, Cornelis A.; de Haan, Aalzen; Westra, Johanna; Kallenberg, Cornelis; Bijl, Marc

    Yearly influenza vaccination is recommended for patients with humoral primary immunodeficiency (hPID). However, humoral responses following vaccination can be expected to be reduced in these patients.The efficacy of influenza vaccination in patients with hPID, anti-influenza antibody responses was

  11. Cambridge Healthtech Institute's 4th Annual Recombinant Antibodies Conference.

    Science.gov (United States)

    Casey, Joanne L; Coley, Andrew M

    2003-08-01

    The 4th Annual Recombinant Antibodies Conference was immediately following the 5th Annual 'Molecular Display: The Chemistry Set for Proteins and Small Molecules' conference, both held in Cambridge, MA and organised by Cambridge Healthtech Institute. The former conference focused on development of new approaches for recombinant antibody development, with particular emphasis on improved methods for selection and optimisation allowing rapid validation and development of human antibodies for the clinic. There were many impressive presentations describing emerging technologies such as new antibody-like scaffolds, covalent P2 antibody display, de-immunisation of antibodies and measuring affinities of as many as 400 clones simultaneously using proteomic microarray platforms. The conference also highlighted the latest applications of library technologies for proteomics and target discovery, and the generation of therapeutic molecules as antibodies alone or as drug, toxin or radionuclide conjugates.

  12. Antibody engineering: methods and protocols

    National Research Council Canada - National Science Library

    Chames, Patrick

    2012-01-01

    "Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient...

  13. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  14. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene</