Sample records for antibody test product

  1. Antibody Blood Tests (United States)

    ... What do I do if I have a negative blood test (or panel) but I’m still having symptoms? While it is rare, it is possible for patients to have a negative antibody test results and still have celiac disease. ...

  2. Anti-insulin antibody test (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

  3. Antiparietal cell antibody test (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  4. ANA (Antinuclear Antibody Test) (United States)

    Advertisement Proceeds from website advertising help sustain Lab Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising & Sponsorship: Policy | Opportunities ...

  5. HIV Antibody Test (United States)

    ... 2 has a higher prevalence in parts of Africa.) The level of p24 antigen and the amount ... getting pregnant may opt to get tested (see Pregnancy: HIV .) The ... Teens , Young Adults , Adults , and Adults 50 and Up . ...

  6. Terasaki-ELISA for murine IgE-antibodies.I.Quality of the detecting antibody: production and specificity testing of antisera specific for IgE

    NARCIS (Netherlands)

    Savelkoul, H.F.J.; Soeting, P.W.C.; Radl, J.; Linde-Preesman, van der A.A.


    In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified emp

  7. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.


    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  8. Production and Screening of Monoclonal Peptide Antibodies. (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina


    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  9. Sensitive neutralization test for rubella antibody. (United States)

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A


    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  10. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar


    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors...... such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  11. 21 CFR 866.3290 - Gonococcal antibody test (GAT). (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  12. 21 CFR 864.9175 - Automated blood grouping and antibody test system. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated blood grouping and antibody test system... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes...

  13. Heparin-Induced Thrombocytopenia Antibody Test (United States)

    ... Global Sites Search Help? Heparin-induced Thrombocytopenia PF4 Antibody Share this page: Was this page helpful? Also known as: Heparin-PF4 Antibody; HIT Antibody; HIT PF4 Antibody; Heparin Induced Antibody; ...

  14. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody (United States)



  15. Competency development in antibody production in cancer cell biology

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.S.


    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The main objective of this project was to develop a rapid recombinant antibody production technology. To achieve the objective, the authors employed (1) production of recombinant antigens that are important for cell cycle regulation and DNA repair, (2) immunization and specific selection of antibody-producing lymphocytes using the flow cytometry and magnetic bead capturing procedure, (3) construction of single chain antibody library, (4) development of recombinant vectors that target, express, and regulate the expression of intracellular antibodies, and (5) specific inhibition of tumor cell growth in tissue culture. The authors have accomplished (1) optimization of a selection procedure to isolate antigen-specific lymphocytes, (2) optimization of the construction of a single-chain antibody library, and (3) development of a new antibody expression vector for intracellular immunization. The future direction of this research is to continue to test the potential use of the intracellular immunization procedure as a tool to study functions of biological molecules and as an immuno-cancer therapy procedure to inhibit the growth of cancer cells.

  16. Production and assay of forskolin antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ho, L.T.; Ho, R.J.


    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  17. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc. (United States)


    ECBC-TR-1339 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY...CHARACTERIZATION: CHARACTERIZATION OF AN MS2 HUMAN IGG ANTIBODY PRODUCED BY ANAPTYSBIO, INC. DARPA ATP Standardized Test Bed for Antibody...Characterization: Characterization of an MS2 human IgG antibody produced by AnaptysBio DARPA ATP Standardized Test Bed for Antibody

  18. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.


    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  19. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing. (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús


    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  20. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. (United States)

    Koren, Eugen; Smith, Holly W; Shores, Elizabeth; Shankar, Gopi; Finco-Kent, Deborah; Rup, Bonita; Barrett, Yu-Chen; Devanarayan, Viswanath; Gorovits, Boris; Gupta, Shalini; Parish, Thomas; Quarmby, Valerie; Moxness, Michael; Swanson, Steven J; Taniguchi, Gary; Zuckerman, Linda A; Stebbins, Christopher C; Mire-Sluis, Anthony


    The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

  1. Production and Purification of Polyclonal Antibodies. (United States)

    Nakazawa, Masami; Mukumoto, Mari; Miyatake, Kazutaka


    Polyclonal antibodies consist of a mixture of antibodies produced by multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against an antigen recognize multiple epitopes on a target molecule, which results in a signal amplification in indirect immunoassays including immune-electron microscopy. In this chapter, we present a basic procedure to generate polyclonal antibodies in rabbits.

  2. Generation and applications of monoclonal antibodies for livestock production. (United States)

    Van Der Lende, T


    Monoclonal antibodies (MCAs) have found widespread applications in livestock production. Although the generation of murine MCAs is at present a routine, the production of homologous MCAs, especially important for in vivo applications, is still hampered by the lack of efficient homologous fusion partners for immortalization of antibody producing lymphocytes of livestock species. At present, MCAs are used in immunodiagnostic tests e.g. to monitor livestock reproduction and quality of livestock products. In the future MCAs will also be used in immunosensors for real-time and on-site applications in the same areas. The commercial application of MCAs for the immunomodulation of (pharmacologically induced) physiological processes underlying important (re)production traits is at present limited to the use of anti-PMSG MCAs in PMSG-induced superovulation. However, many potentially interesting applications are under investigation (e.g. immunopotentiation of growth hormone to enhance growth; immunocytolysis of adipocytes to increase lean meat production; immunoneutralization of GnRH for immunocastration; immunoimitation of hormone activity with anti-idiotype antibodies). Attempts to use specific MCAs for the sexing of embryos have been disappointing, mainly because of the relatively low accuracy. In the future, MCAs against membrane proteins which are specific for X- or Y-chromosome bearing spermatozoa might be used for bulk separation of livestock sperm. In general, it is expected that engineered (homologous) recombinant MCAs will largely contribute to the development of a new generation of rapid immunodiagnostic tests and effective immunomodulation applications. They will further increase the use of MCAs in livestock production.

  3. 21 CFR 866.5100 - Antinuclear antibody immunological test system. (United States)


    ... autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antinuclear antibody immunological test system. 866.5100 Section 866.5100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  4. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization. (United States)

    Hansen, Debra T; Robida, Mark D; Craciunescu, Felicia M; Loskutov, Andrey V; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F


    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.

  5. Novel indirect fluorescent antibody test for Lyme disease. (United States)

    Chambers, M A; Swango, L J; Wright, J C


    An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.

  6. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech


    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  7. Serum Treponema IgM Antibody Test for Syphilis Diagnosis

    Institute of Scientific and Technical Information of China (English)

    郑占才; 张荣富; 溪茜


    Objective: To evaluate the clinical utility of testing serum anti-treponema pallidum IgM antibody in the diagnosis of syphilis patients. Methods: Seventy-two cases of syphilis were tested for specific IgM antibody with ELISA, and the results were compared with RPR and TPPA.Results: The sensitivity of IgM antibody was 73.3 %(11/15) in primary syphilis, 88.9% (16/18) in sec-ondary syphilis, and there was no significant differ-ence between these values (x2=1.6363, P>0.10). The sensitivity of IgM antibody in diagnosing latent syphi-lis was only 26.1% (6/23), much lower than the detec-tion rate in symptomatic earlv svDhilis (x2=17.6189. P<0.005). RPR and TPPA were both 100% sensitive in latent and early symptomatic syphilis. Two were posi,five for IgM in the 16 cases who had received regular treatments 2 to 24 months before enrolled.Conclusions: Specific IgM antibody detection doees not appear superior to RPR and TPPA in diagnosing primary syphilis. The diagnosis of latent syphilis should mainly rely on RPR and TPPA, since there are low titers of IgM antibody at that stage. IgM antibody testing alone should not be recommended for monitor-ing syphilis development or treatment efficacy. Fur-ther studies should be concerned.

  8. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;


    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  9. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina (United States)


    ECBC-TR-1395 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR...Thompson James Carney RESEARCH AND TECHNOLOGY DIRECTORATE Candice Warner Melody Zacharko EXCET, INC. Springfield, VA 22151-2110...4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv

  10. 21 CFR 866.5110 - Antiparietal antibody immunological test system. (United States)


    ...), atrophic gastritis (inflammation of the stomach), and autoimmune connective tissue diseases (diseases... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antiparietal antibody immunological test system. 866.5110 Section 866.5110 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  11. Overcoming the susceptibility gap between maternal antibody disappearance and auto-antibody production. (United States)

    Yosipovich, Roni; Aizenshtein, Elina; Shadmon, Roy; Krispel, Simcha; Shuster, Efrat; Pitcovski, Jacob


    In the first 10-14 days of a chick's life, protection is conferred by maternal antibodies. Further broiler protection is achieved by active vaccination. However, the high level of maternal antibodies interferes with the induction of an effective immune response by vaccination at a young age. As a result, there is a gap between the reduction in protective maternal antibodies and elevation of self-produced antibodies following active vaccination. The major aim of this study was to test an approach consisting of passive and active vaccination to overcome this gap and to provide continuous resistance to infectious viral diseases during the broiler's growth period. Newcastle disease virus (NDV), which is one of the world's most prevalent infectious diseases of poultry, was tested as a model. Following subcutaneous injection of 18 hemagglutination-inhibiting (HI) units of anti-NDV immunoglobulin Y per 1-day-old chick, protective log2 antibody titers above 4 could be detected to at least 17 days of age. The combination of passive immunization on day 1 of age with attenuated live vaccination on day 10 led to high protective titers throughout the entire growth period, up to 41 days of age. Moreover, the HI titers in the group of birds immunized with the combined vaccination were significantly more homogeneous than those in the group vaccinated only with live virus. Thus, full protection against NDV of all broilers in flock during their entire growth period was achieved by a vaccination regime that combines passive immunization and live vaccination.

  12. Enhancement of antibody production to hepatitis B surface antigen by anti-idiotypic antibody.


    Kakumu, S; Murase, K.; A Tsubouchi; Yoshioka, K.; Sakamoto, N.


    Studies were undertaken to determine whether anti-idiotypic antibody (anti-Id) against antibody to hepatitis B surface antigen (anti-HBs) could modulate in vitro anti-HBs production by human peripheral blood mononuclear cells stimulated with pokeweed mitogen. Peripheral blood mononuclear cells from patients positive for serum anti-HBs produced significantly increased amounts of anti-HBs by the addition of IgG fraction of anti-anti-HBs as well as purified HBsAg in a soluble form when compared ...

  13. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes. (United States)

    Na, Hong; Laver, John D; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J Timothy; Kim, Philip M; Smibert, Craig A; Lipshitz, Howard D; Sidhu, Sachdev S


    Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

  14. Antibody production in early life supported by maternal lymphocyte factors. (United States)

    Shimamura, Michio; Huang, Yi-Ying; Goji, Hiroshi


    To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2(+/-) mouse neonates born to Rag-2(+/+), Rag-2(+/-)or Rag-2(-/-)mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG(1) production was evaluated. The levels of IgE and IgG(1) were higher in the pups born to Rag-2(+/+)and Rag-2(+/-) dams than to lymphocyte-deficient Rag-2(-/-) dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2(-/-) mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk.

  15. Cell culture processes for monoclonal antibody production


    LI Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert; Amanullah, Ashraf


    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizin...

  16. Production of monoclonal antibody for the detection of meat and bone meal in animal feed. (United States)

    Kim, Shin-Hee; Huang, Tung-Shi; Seymour, Thomas A; Wei, Cheng-i; Kempf, Stephen C; Bridgman, C Roger; Clemens, Roger A; An, Haejung


    For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.

  17. Production and characterization of antibodies to advanced glycation products on proteins. (United States)

    Nakayama, H; Taneda, S; Kuwajima, S; Aoki, S; Kuroda, Y; Misawa, K; Nakagawa, S


    Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

  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.


    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. [Detection of influenza B virus antibodies in different age groups using hemagglutination inhibition tests]. (United States)

    Sonuvar, S; Kocabeyoğlu, O; Emekdaş


    Antibody levels against influenza B virus were investigated by using hemagglutination-inhibition (HA-I) tests in 402 sera obtained from different age groups. Hemagglutination antigens were obtained by production of influenza B virus (B/Singapur/LLC 6201) in trypsinized Madin Darby Bovine Kidney (MDBK) cell cultured and they were used in tests. In 355 out of 402 sera (88.3%) antibodies against influenza B virus were detected at titers varying between 1/20 and 1/1280. However in 47 sera (11.7%) no antibodies were detected at 1/20 titer. High titers of antibody (1/640-1/1280) were not detected in none of the sera obtained from an age group between 1 and 14. However high titer antibodies were detected in 15.6% of the sera from an age group between 26 and 35, in the 17.3% of the sera from a group above 50 years of age. Our findings suggest that the increase in the rates of seropositivity against influenza B virus depends on getting older and, that the infections by this virus may be widely seen in our country.

  20. [Evaluation of latex agglutination test for anti-treponemal antibody in comparison with chemical luminescence tests]. (United States)

    Watanabe, Naomi; Nagatomo, Ritsuko; Okubo, Shigeo; Yokota, Hiromitsu; Ikeda, Hitoshi; Yatomi, Yutaka


    The performance of a latex agglutination test (Mediace TPLA) in the detection of anti-treponemal antibody was evaluated in comparison with chemical luminescence tests (LumipulsII-N and Architect TPAb) in 346 cases. Anti-treponemal antibody was further determined by immunochromatography and immunoblotting tests and additionally evaluated by a serological test for syphilis with lipoidal antigens. The total concordance rate between the latex agglutination test and chemical luminescence tests ranged from 96% to 97%: the positive concordance rate ranged from 96% to 97%, and the negative concordance rate, from 97% to 98%. The latex agglutination test showed two false positive cases, and each chemical luminescence test showed two false positive cases, respectively. In eight cases, only the latex agglutination test showed negative results; all specimens contained anti-treponemal antibodies. However, none of these was considered to be a false positive and each was treated as syphilis based on the results of confirmatory analysis with immunochromatography and immunoblotting tests and a serological test for syphilis. The discordant results in the latex agglutination test and chemical luminescence tests may be caused by the different antigenisity of each test. With detailed analysis of those sera treated as syphilis, each specimen was found to contain various antibodies against syphilitic antigens, suggesting that there was a different specificity of native and recombinant antigens. Based on the present results for the comparison between the latex agglutination test and chemical luminescence tests, it was considered that further investigation is necessary to clarify the anti-treponemal antibody profile of syphilis at the disease stage.

  1. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

    Directory of Open Access Journals (Sweden)

    Stefanie Claudia Pohl


    Full Text Available Bivalent single chain (scFv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4 revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.

  2. [Production and characteristics of monoclonal antibodies to the diphtheria toxin]. (United States)

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V


    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  3. Production and purification of polyclonal antibody against melatonin hormone

    Directory of Open Access Journals (Sweden)

    Fooladsaz K


    Full Text Available Nowadays immunochemical techniques have played a very important and valuable role in quantitative and qualitative assays of liquid compounds of the body. Producing antibody against immunogenes is the first step to make immunochemical kits. In this study production and purification of polyclonal antibody against melatonin has been considered. This hormone which has several important functions in physiological conditions such as migraine, cirrhosis, mammary gland cancer and other diseases, is the most important pineal gland secretion. This gland is a circumventricular organ of brain and according to histological and anatomical studies, it is a high secretory organ, that secretes active biological substances like melatonin, oxytocin, serotonin and ect. In this study, melatonin has been considered as hapten and has become an immunogen by being linked to the bovine serum Albumin. Then, by the immunization of three white New Zeland rabbits that had the booster injections in regular intervals, the antibody titer was detected to be 1/2000, by using checkboard curves, and with the use of melatonin linked to penicillinase as a labeled antigen, the titer was detected 1/200. Finally an antibody with high purification rate has been obtained, which can be used in immunochemical assays like RIA, ELISA, and EIA.

  4. [Comparison of eight screening tests for ant-HCV antibody]. (United States)

    Deguchi, Matsuo; Kagita, Masanori; Yamashita, Naoko; Nakano, Takasi; Tahara, Kazuko; Asari, Seishi; Iwatani, Yoshinori


    We compared eight HCV screening tests for detection of anti-HCV antibody; Ortho Quick Chaser HCV Ab (QC), Ortho HCV Ab ELISA III (ELISA), Ortho HVC Ab PA test III (PA), Lumipulse II Ortho HCV (LUMI), IMx HCV.DAINAPACKII (IMx), ARCHITECT HCV (ARCH), Immucheck.F-HCV C50 Ab (Immu), RANREAM HCV Ab Ex II (RAN). Sera from six hundred patients were examined by these eight screening tests. The positive rates of the eight screening tests were from 9.0% to 13.2%. Forty-five sera showed discrepant results between the eight screening tests, and about half of them showed weak positive reaction and/or false positive. Twenty-five of the forty-five sera were negative for ant-HCV antibody in the CHIRON RIBA III confirmatory test, and forty-four of them were negative for HCV-RNA in the PCR method. The agreement rates between the two reagents were from 95.5% to 99.2%, but were not always high between the two reagents that used similar antigen. The specificities and sensitivities evaluated by using the RIBA III confirmatory test were excellent in ELISA, LUMI, IMx, ARCH and Immu. Three BBI seroconversion panels were used to compare the positive readings in the initial stage of HCV infection by eight screening tests. ELISA and ARCH showed the earliest positive readings, and then IMx, LUMI = RAN, PA, QC and Immu in this order. These findings indicate that ELISA and ARCH were the most excellent in the sensitivity, specificity and early diagnosis of HCV infection. However, we must pay attention to the weak positive reaction in the screening tests, because there is a possibility of "false positive".

  5. Antibody (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  6. Fission product separations testing

    Energy Technology Data Exchange (ETDEWEB)

    Bostick, D.A.; DePaoli, S.M. [Oak Ridge National Lab., TN (United States)


    The initial goal of this task is to adequately understand the treatment needs of the end user in treating contaminated wastewater. These needs are then incorporated into the evaluation of new treatment technologies for wastewater treatment. Pertinent information is than supplied to the end user so that they can select a preferred process to meet their waste treatment needs. New sorbent materials, ion-exchange materials, or other processes of interest to DOE`s Office of Environmental Restoration (EM-40) will be evaluated initially for the removal of {sup 90}Sr and {sup 137}Cs from groundwater and process wastewater. Laboratory studies will strive to obtain a quantitative understanding of the behavior of these new materials and to evaluate their sorption efficiency in reference to a standard benchmark treatment technique. Testing of the new materials will begin by conducting scoping tests where new treatment materials are compared with standard, commercially available materials in batch shaker tests. Experimental data for the most promising sorbents will then be fit to an equilibrium model so that nuclide sorption can be predicted for variable wastewater composition. Additional testing with actual wastewater will be conducted with two or three of the most effective treatment methods. Once batch testing of a treatment method is completed, dynamic column tests will be performed to validate the equilibrium sorption model and to obtain the defining column operating parameters for scaling up the technology.

  7. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

    Directory of Open Access Journals (Sweden)

    Paula A. Moreno-González


    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  8. Interim Guidance for Interpretation of Zika Virus Antibody Test Results. (United States)

    Rabe, Ingrid B; Staples, J Erin; Villanueva, Julie; Hummel, Kimberly B; Johnson, Jeffrey A; Rose, Laura; Hills, Susan; Wasley, Annemarie; Fischer, Marc; Powers, Ann M


    Zika virus is a single-stranded RNA virus in the genus Flavivirus and is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses (1,2). Among flaviviruses, Zika and dengue virus share similar symptoms of infection, transmission cycles, and geographic distribution. Diagnostic testing for Zika virus infection can be accomplished using both molecular and serologic methods. For persons with suspected Zika virus disease, a positive real-time reverse transcription-polymerase chain reaction (rRT-PCR) result confirms Zika virus infection, but a negative rRT-PCR result does not exclude infection (3-7). In these cases, immunoglobulin (Ig) M and neutralizing antibody testing can identify additional recent Zika virus infections (6,7). However, Zika virus antibody test results can be difficult to interpret because of cross-reactivity with other flaviviruses, which can preclude identification of the specific infecting virus, especially when the person previously was infected with or vaccinated against a related flavivirus (8). This is important because the results of Zika and dengue virus testing will guide clinical management. Pregnant women with laboratory evidence of Zika virus infection should be evaluated and managed for possible adverse pregnancy outcomes and be reported to the U.S. Zika Pregnancy Registry or the Puerto Rico Zika Active Pregnancy Surveillance System for clinical follow-up (9,10). All patients with clinically suspected dengue should have proper management to reduce the risk for hemorrhage and shock (11). If serologic testing indicates recent flavivirus infection that could be caused by either Zika or dengue virus, patients should be clinically managed for both infections because they might have been infected with either virus.

  9. An ELISA test for the detection of antibodies to Legionella pneumophila.


    Wreghitt, T. G.; Nagington, J.; Gray, J


    An enzyme-linked immunosorbent assay (ELISA) test has been developed to detect antibodies to Legionella pneumophila serogroup 1. There is good correlation between indirect fluorescent antibody (IFA) and ELISA titres but ELISA is more sensitive.

  10. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research (United States)

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  11. EBF recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies. (United States)

    Pihl, Susanne; Michaut, Lydia; Hendriks, Jenny; Loebbert, Ralf; Ryding, Janka; Nemansky, Martin; Vermet, Laurent; Companjen, Arjen


    Long- and short-term stability testing of the analyte is one of the key parameters in bioanalytical method validation in support of pharmacokinetics. However, for immunogenicity testing, the scientific rationale for long- and short-term stability testing on quality control samples most often spiked with polyclonal antibody raised in a different species should be questioned. Therefore, the European Bioanalysis Forum (EBF) formed a Topic Team to discuss the scientific rationale for stability testing of anti-drug antibodies (ADAs). A review of EBF member companies' experience on ADA stability and on anti-vaccine antibodies from vaccine projects was the basis of this discussion. EBF recommends to perform short-term stability testing of the positive control, but not to perform long-term stability testing of ADAs in nonclinical and clinical studies.

  12. Clinical utility of antinuclear antibody tests in children

    Directory of Open Access Journals (Sweden)

    Kickingbird Lauren M


    Full Text Available Abstract Background Antinuclear antibody (ANA tests are frequently used to screen children for chronic inflammatory diseases such as systemic lupus erythematosus (SLE. However, the diagnostic utility of this test is limited because of the large number of healthy children who have low-titer positive tests. We sought to determine the clinical utility of ANA tests in screening children for rheumatic disease and to determine whether there are specific signs or symptoms that enhance the clinical utility of ANA tests in children. Methods We undertook a retrospective analysis of 509 new patient referrals. Charts of patients referred because of results of ANA testing were selected for further analysis. Children with JRA, SLE, and other conditions were compared using demographic data, chief complaints at the time of presentation, and ANA titers. Results One hundred ten patients were referred because of an ANA test interpreted as positive. Ten patients were subsequently diagnosed with SLE. In addition, we identified one patient with mixed connective tissue disease, and an additional child with idiopathic Raynaud's phenomenon. Eighteen children of the children referred for a positive ANA test had juvenile rheumatoid arthritis (JRA. Another 80 children with positive ANA tests were identified, the majority of whom (n = 39, 49% had musculoskeletal pain syndromes. Neither the presence nor the titer of ANA served to distinguish children with JRA from children with other musculoskeletal conditions. Children with JRA were readily identified on the basis of the history and physical examination. Children with SLE were therefore compared with children with positive ANA tests who did not have JRA, designated the "comparison group." Non-urticarial rash was more common in children with SLE than in children without chronic inflammatory disease (p = 0.007. Children with SLE were also older (mean ± sd = 14.2 ± 2.5 years than the comparison group (11.0 ± 3.6 years; p

  13. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  14. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.;


    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

  15. Consumer oriented product noise testing (United States)

    Blomberg, Les


    This paper explores the need for product noise measurements and how best to meet that need in the near future. Currently there is only a small market place for quieter consumer products. This is not because of lack of interest. No one really wants to announce to everyone in their house that they just flushed the toilet, few really want the entire neighborhood to know they are mowing their yard, etc. The small market place is primarily due to a lack of regulations on product noise, a lack of information easily available to consumers about which products are quieter, and market consolidation resulting in fewer manufacturers, most of whom are unwilling to emphasize their quieter products at the risk of eroding sales of their noisier ones (that currently have greater market share). In the absence of the EPA fulfilling its statutory requirement to regulate and label product noise under the Noise Control Act of 1972, and with the unwillingness of most industries to voluntarily publish accurate product noise data, there is a significant role for ``Consumer Oriented Product Noise Testing.'' This paper explores the Noise Pollution Clearinghouse's ongoing and planned product noise testing, evaluating its advantages, disadvantages, and limitations.

  16. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16. (United States)

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkafi-Mesrati, Lobna; Jaoua, Samir; Stephan, Dietrich


    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

  17. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. (United States)

    Shankar, Gopi; Devanarayan, Viswanath; Amaravadi, Lakshmi; Barrett, Yu Chen; Bowsher, Ronald; Finco-Kent, Deborah; Fiscella, Michele; Gorovits, Boris; Kirschner, Susan; Moxness, Michael; Parish, Thomas; Quarmby, Valerie; Smith, Holly; Smith, Wendell; Zuckerman, Linda A; Koren, Eugen


    Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

  18. Heterophilic antibodies interfering with radioimmunoassay. A false-positive pregnancy test

    Energy Technology Data Exchange (ETDEWEB)

    Vladutiu, A.O.; Sulewski, J.M.; Pudlak, K.A.; Stull, C.G.


    A young woman with amenorrhea had a consistently positive pregnancy test result (serum radioimmunoassay measurement of ..beta..-human chorionic gonadotropin hormone). No fetal or placental tissue was found after uterine curettage and exploratory laparotomy. The false-positive pregnancy test result was due to heterophilic antibovine and antigoat antibodies in the patient's serum. These antibodies interfered with radioimmunoassays using goat antibodies. This case shows that serum heterophilic antibodies can interfere with immunoassays and result in unnecessary diagnostic procedures and/or unnecessary treatment.


    Directory of Open Access Journals (Sweden)

    Vetvicka Vaclav


    Full Text Available The role of glucan in stimulation of immune reactions has been well-established. In this report, we focused on the antibody production in glucan-supplemented children with chronic respiratory problems. We measured the levels of salivary IgA, IgM and IgG in 40 children aged 8-12 years and evaluated the effects of 100 mg d-1 oral dose of glucan. We found a significant increase in production of all tested antibodies in the glucan-stimulated group, but a decrease of antibody production in the control group. A thirty-day oral application of yeast-based natural immunomodulator β-glucan strongly stimulated the mucosal immunity of children with chronic respiratory problems.

  20. Evaluation of screening tests for the detection of antistreptolysin O antibodies.


    Lue, Y. A.; Nicolas, C.; Kemawikasit, P.; Pierre, A.; McLean, T I; Simms, D H


    The accuracy of two screening tests, one utilizing serum and the other utilizing whole blood, was compared with the accuracy of the conventional macrotitration method for the detection of antistreptolysin O antibodies. Of the 569 specimens tested with the serum screening procedure and the macrotitration method, 235 and 282, respectively, were positive for antistreptolysin O antibodies. Comparative testing of 200 specimens with the peripheral blood screening test and the macrotitration method ...

  1. 21 CFR 866.5120 - Antismooth muscle antibody immunological test system. (United States)


    ... autoimmune connective tissue diseases (diseases resulting from antibodies produced against the body's own... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antismooth muscle antibody immunological test system. 866.5120 Section 866.5120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH...

  2. The generation of monoclonal antibodies and their use in rapid diagnostic tests (United States)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

  3. Effect of skin test on serum antibody responses to Mycobacterium bovis infection in cattle (United States)

    Recently, several serologic tests designed to detect immunodominant antibodies to M. bovis antigens (e.g., MPB83, MPB70, ESAT-6, and CFP10) have emerged for potential use with samples from cattle. Of these, a commercial ELISA to MPB83/MPB70 (M. bovis antibody ELISA) has gained approval for use in ca...

  4. Recombinant mouse interferon-gamma regulation of antibody production.



    Interferon-gamma produced in monkey cells by transfection with mouse interferon-gamma cDNA suppressed the mouse in vitro antibody response in a manner similar to that of natural mouse interferon-gamma. Significant suppression was obtained with as little as 1 U of interferon. Recombinant human interferon-gamma produced by cloning in a similar fashion was not suppressive. Both the suppressive and the antiviral activities of recombinant interferon-gamma were neutralized by antibodies to mouse na...

  5. Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression


    Haredy, AM; Nishizawa, A.; Honda, K.; T. Ohya; Ohtake, H; Omasa, T


    To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host c...

  6. Antibodies against Hepatitis A and Hepatitis B Virus in Intravenous Immunoglobulin Products. (United States)

    Lee, Soyoung; Kim, Han Wool; Kim, Kyung Hyo


    The worldwide seroprevalence of hepatitis A virus (HAV) and hepatitis B virus (HBV) has changed over the last two decades, indicating a declining incidence of HAV and HBV infections. Therefore, vaccinations against HAV and HBV are recommended for unimmunized people before traveling to an endemic area. Unfortunately, primary antibody deficiency (PAD) patients can only obtain humoral immunity through intravenous immunoglobulin G (IVIG) replacement and not from vaccination because of a defect in antibody production. However, few studies have analyzed the titers of antibodies against HAV or HBV in IVIG products. In this study, the titers of anti-HAV and anti-HBs antibodies were measured in nineteen lots of IVIG products from five manufacturers from three countries (A, B from Korea; C, D from Japan; and E from the USA), and trough titers in plasma were estimated. Concentrations of anti-HAV antibody ranged from 1,888-8,927 mIU/mL and estimated trough titers exceeded the minimal protective value in all evaluated IVIG products. Concentrations of anti-HBs antibody ranged from 438-965 mIU/mL in products A and B and were 157, 123, and 1,945 mIU/mL in products C, D, and E, respectively. Estimated trough titers in products A, B, and E exceeded the minimal protective value but those in products C and D did not reach this threshold. These data demonstrated that available IVIG products generally provide sufficient antibodies against HAV and HBV to protect patients with PAD, although the trough concentrations of anti-HBs antibody in two IVIG products did not reach the minimum protective value.

  7. Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders


    successfully been used to eradicate BVD in Sweden. Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples....... The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools. Results: During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV...... infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level...

  8. {sup 90}Nb - a potential PET nuclide. Production and labeling of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, V.; Roesch, F. [Mainz Univ. (Germany). Inst. of Nuclear Chemistry; Hauser, H.; Eisenhut, M. [German Cancer Research Center, Heidelberg (Germany). Radiopharmaceutical Chemistry; Vugts, D.J.; Dongen, G.A.M.S. van [VU University Medical Center, Amsterdam (Netherlands). Dept. of Nuclear Medicine and PET Research; VU University Medical Center, Amsterdam (Netherlands). Dept. of Otolaryngology/Head and Neck Surgery


    Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is {sup 90}Nb. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. {sup 90}Nb exhibits a high positron branching of 53% and an optimal energy of {beta}{sup +} emission of E{sub mean} = 0.35 MeV only. Consequently, efficient radionuclide production routes and Nb{sup V} labeling techniques are required. {sup 90}Nb was produced by the {sup 90}Zr(p,n){sup 90}Nb nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) {sup 90}Nb was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with {sup 90}Nb was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 C. The selected production route led to a high yield of 145 {+-} 10 MBq/{mu}A h of {sup 90}Nb with high radioisotopic purity of > 97%. This yield may allow for large scale production of about 10 GBq {sup 90}Nb. The separation procedure resulted in 76-81% yield. The Zr/{sup 90}Nb decontamination factor reaches 10{sup 7}. Subsequent radiolabeling of the two different conjugates with {sup 90}Nb gave high yields; after one hour incubation at room temperature, more than 90% of {sup 90}Nb-Df-mAb was

  9. Optimizing selection of large animals for antibody production by screening immune response to standard vaccines. (United States)

    Thompson, Mary K; Fridy, Peter C; Keegan, Sarah; Chait, Brian T; Fenyö, David; Rout, Michael P


    Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.

  10. Development and Testing of Recombinant Single Domain Antibodies (United States)


    single domain antibodies from a naïve shark library. Molecular Immunology. 44:1786-1794. Sherwood, L.J., Osborn, L., Carrion , R., Patterson, J., Hayhurst...for viral diagnostics in hot climates 9. Sherwood, L.J., Osborn, L, Carrion , R., Patterson, J.P and Hayhurst, A. April 22, 2007 Rapid assembly of...and Austin Life Sciences Association, New Bruanfels, TX. 11. Sherwood, L.J., Osborn, L., Carrion , R., Patterson, J.P. and Hayhurst, A. May 14, 2007

  11. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 sdAb Produced by U.S. Naval Research Laboratory (United States)


    Biosurveillance (JPMO BSV), Critical Reagents Program instituted a quality program for the standardization of test methods to support more...operational and performance metrics for future detection and diagnostic platforms. 15. SUBJECT TERMS Antibody Biosurveillance Antibody Technology... Biosurveillance (JPMO BSV), Critical Reagents Program (CRP) instituted a quality program for the standardization of test methods to more fully characterize

  12. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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    M Jedi-Tehrani


    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  13. Production and potential use of monoclonal antibodies against polio viruses.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; G. van Steenis (Bert); A.G. Hazendonk


    textabstractLymphocyte hybridomas secreting monoclonal antibodies against different strains of polio virus type 1, 2, or 3 have been produced. For this purpose Balb/C mice were immunized with purified and inactivated virus suspensions and their splenocytes were fused with P3X63Ag8 mouse myeloma cell


    Institute of Scientific and Technical Information of China (English)


    Objective To assess whether there was strong association between antiphospholipid antibodies(APA) and coronary heart disease(CHD), to study the environmental factors of APA production and APA pathogenic mechanism in patients with CHD.Methods Blood samples from 76 patients with CHD and 30 controls were tested for anticardiolipin antibodies IgG(ACA-IgG),human cytomegalovirus IgG,IgM(HCMV-IgG,IgM) by enzyme-link immunosorbant assay(ELISA) and 6-keto-PGF1a,endothelin(ET) by radioimmunoassay(RIA).Results A total of 27 patients(35.53%) were ACA positive in 76, as compared to 2 of 30(6.67%) healthy individuals, P<0.05. There was no difference in ACA among acute myocardial infarction(AMI,39.13%), old myocardial infarction(OMI,26.53%), unstable angina pectoris(UA,38.40%), P>0.05. The number of ACA positive subjects was higher in HCMV infection patients with CHD than no HCMV infectious patients with CHD. There was no PGI2 and ET level difference between ACA-IgG positive and negative CHD.Conclusion There are strong association between APA and CHD. The HCMV infection may be an environmental factor of APA production in CHD patients with raised ACA. The alteration of PGI2 and ET are not the pathogenic mechanism of ACA in patients with CHD.

  15. Standardization and demonstration of antibody-coated Candida in urine by direct immunofluorescence test. (United States)

    Talwar, P; Pal, S R; Kaur, P; Kaiwar, R; Jayashree, T; Rao, M S; Vaidyanathan, S; Taiwar, P


    Acetone, carbontetrachloride, ethyl alcohol, mixture of ethyl alcohol and acetone, and heat were assessed for fixative property for direct immunofluorescent (IF) staining of antibody-coated Candida cells. The results indicated that ethyl alcohol was the most suitable fixative for the test. Antisera containing 16 units of Candida albicans type A agglutinin were found essential to get optimal detectable fluorescence of antibody-coated yeast cells. IF test showed cross reactivity between the yeasts of C. albicans and C. tropicalis. However, there was no cross reactivity with the conidia of A. flavus. The direct IF test could demonstrate antibody-coated yeast cells and pseudomycelia in deposits of urine in the direct smear. It correlated well with microscopy and culture studies. At times, it could demonstrate the antibody-coated yeasts earlier than routine significant culture. It could also differentiate the significant from non-significant fungal isolates from urine.

  16. Belgian recommendations on ANA, anti-dsDNA and anti-ENA antibody testing. (United States)

    Van Blerk, M; Bossuyt, X; Humbel, R; Mewis, A; Servais, G; Tomasi, J P; Van Campenhout, C; Van Hoovels, L; Vercammen, M; Damoiseaux, J; Coucke, W; Van de Walle, P


    Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

  17. In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

    Directory of Open Access Journals (Sweden)

    Andreas Weinhäusel


    Full Text Available The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses. In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively. The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics.

  18. Fed-batch CHO cell culture for lab-scale antibody production

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Ley, Daniel; Andersen, Mikael Rørdam


    Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enable rapid and predictable tech......-transfer to manufacturing scale. In this chapter, we will describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production....

  19. Process performance and product quality in an integrated continuous antibody production process. (United States)

    Karst, Daniel J; Steinebach, Fabian; Soos, Miroslav; Morbidelli, Massimo


    Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 10(6) cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307. © 2016 Wiley Periodicals, Inc.

  20. Evaluation of toxin neutralisation in test systems for diphtheria antibody assessment. (United States)

    Vandenberg, J; van der Gun, J W; Hendriksen, C F


    Over the past years, various authors have reported that the amount of toxin used in toxin neutralisation (TN) assays for diphtheria appears to influence the resulting relative antibody titre. Antibody affinity is thought to be an influencing factor. To confirm this observation and study the underlying mechanism of toxin neutralisation, a panel of sera was generated, differing in species of origin (mouse, guinea pig, and rabbit) and in affinity by using different immunisation schedules. The panel was then tested in relevant TN test systems for diphtheria antibody titration, namely the VERO cell test, the Toxin Binding Inhibition (ToBI) assay and the in vivo skin test in guinea pigs. A hyperimmune equine reference serum was used as the standard. Antibody affinity was measured in two different affinity ELISAs, the ammonium thiocyanate elution ELISA and the diethylamine inhibition ELISA. The VERO cell test clearly demonstrates the phenomenon; the higher the toxin dose used in the assay, the higher the resulting relative potency. The difference in relative antibody titre decreases as antibody affinity increases. This is especially evident when an equine hyperimmune reference serum is used as the standard. When a species homologous reference is used, the phenomenon is less distinct. The ToBI test, however, does not show the phenomenon. This discrepancy between these two test systems is being further investigated, and comparison will be made with the in vivo TN test. The findings confirm and support earlier observations. It is still unclear exactly which mechanisms are involved in the toxin neutralisation process. Antibody subclasses and class switching could play a role and will be further studied.

  1. Selecting an acceptable and safe antibody detection test can present a dilemma. (United States)

    Combs, M R; Bredehoeft, S J


    The Transfusion Service at Duke University Hospital has changed antibody detection methods from the use of albumin in indirect antiglobulin tests to low-ionic-strength solution (LISS), and from LISS to polyethylene glycol (PEG) in an effort to enhance the rapid detection of clinically significant antibodies. In 1996, staffing issues required the consideration of automation. Although previous studies indicated that the gel test was not as sensitive as PEG for detection of clinically significant antibodies, we chose to implement the gel test to be used with the Tecan MegaFlex-ID. We performed a retrospective analysis of identified antibodies and transfusion reactions to compare the outcomes of one year's experience with gel and PEG. We found comparable detection of potentially clinically significant antibodies by both methods and significantly fewer unwanted or clinically insignificant antibodies detected with the use of gel. Fewer delayed serologic transfusion reactions and no transfusion-associated hemolytic events occurred in the year that gel was used. Although we initially found the selection of the gel test to be a dilemma, our ultimate decision appears to have successfully protected patient safety and balanced sensitivity with specificity.

  2. Production of monoclonal antibodies to human glomerular basement membrane.

    Directory of Open Access Journals (Sweden)



    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  3. Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence

    DEFF Research Database (Denmark)

    Nielsen, Søren Saxmose; Toft, Nils


    Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were...... to determine: (a) the correlation between repeated BTM reactions; and (b) the association between the BTM antibody ELISA-level and the within-herd prevalence of antibody-positive cows. Eight BTM samples per herd and approximately four milk samples per lactating cow per herd were collected from each of 108...... Danish Holstein herds over a period of one year. All samples were tested using a commercial indirect ELISA for detection of MAP specific antibodies. The individual cow's results were dichotomised and used to estimate the within-herd antibody prevalence at each test-date. These prevalences were...

  4. Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats. (United States)

    Amada, Takako; Yoshimatsu, Kumiko; Yasuda, Shumpei P; Shimizu, Kenta; Koma, Takaaki; Hayashimoto, Nobuhito; Gamage, Chandika D; Nishio, Sanae; Takakura, Akira; Arikawa, Jiro


    Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.

  5. Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation. (United States)

    Hammerschmidt, Nikolaus; Tscheliessnig, Anne; Sommer, Ralf; Helk, Bernhard; Jungbauer, Alois


    Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine.

  6. Time course of antibodies against IgG and type II collagen in adjuvant arthritis. Role of mycobacteria administration in antibody production. (United States)

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


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


    Directory of Open Access Journals (Sweden)



    Full Text Available A total of 1021 randomly selected serum samples of adult male goats that had been screened for trypanosome infection were assayed for sperm antibodies using the immunoperoxidase staining technique. The result of the trypanosome screening revealed that 586(57.39% goats were positive for trypanosome infection, while 435(42.61% were negative. The assay for sperm antibodies showed that 482(47.21% animals were positive, while 539(52.79% were negative. In the group that was positive for trypanosome infection, 364(62.12% animals were positive, whereas 222(37.88% were negative for sperm antibodies (P<0.001. The group that was negative for trypanosome infection, had a significantly lower number and proportion 118(27.13% of positive compared to 317(72.87% negative for sperm antibodies. Out of a total 482 goats that were positive for sperm antibodies, a significantly higher number, 364(75.52%, were positive than 118(24.48% that were negative for trypanosome infection (P<0.001. In the group that was found negative for sperm antibodies, a significantly lower proportion, 222(41.19%, was positive compared to 317(58.81% that were negative for trypanosome infection (P<0.001. Seropositivity to sperm antibodies was positively correlated to trypanosome infection (P<0.001. Further work on the pathogenesis of sperm antibody production in trypanosome infection is advocated.

  8. Large-scale production of monoclonal antibodies in suspension culture. (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B


    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.


    Institute of Scientific and Technical Information of China (English)

    王慧琴; 林嘉友; 刘景生


    This study focused on the influences of opioids on the generation of antibody againse sheep erythrocyte in vitro.It was found that morphine,a-CAO,DADLE,MENK were able to inhibit the capacity of murine spleen cells to generate antibody and leukotriene C4 and conversely,dynorphin was able to stimulate the capacity of murine spleen cells to generate antibody and leukotriene C4. Morphine,a-CAO,MENK,DA-DLE,dynorphin decreased intracellular cAMP level,increased [Ca2+]i and calmodulin activity.The effects were completely blocked by naloxone,the specific opioid antagonist.Our results showed that opioids regulate the production of antibody in murine spleen cells,and alter intracellular cAMP,[Ca2+]i calmodulin activity,and leukotriene C4 production by way of binding to different receptor types.

  10. Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression. (United States)

    Haredy, Ahmad M; Nishizawa, Akitoshi; Honda, Kohsuke; Ohya, Tomoshi; Ohtake, Hisao; Omasa, Takeshi


    To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host cell line. Clone CHO-ATF4-16 did not show any change in growth rate compared with the parental cells or mock-transfected CHO-DP12-SF cells. The expression levels of mRNAs encoding both the antibody heavy and light chains in the CHO-ATF4-16 clone were analyzed. This analysis showed that ATF4 overexpression improved the total production and specific production rate of antibody without affecting the mRNA transcription level. These results indicate that ATF4 overexpression is a promising method for improving recombinant IgG production in CHO cells.


    Directory of Open Access Journals (Sweden)

    Goknur TERZI


    Full Text Available In this study Brucella antibodies were investigated with agglutination test (Whey-AT and Milk Ring Test (MRT in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 % of cow milk and 6 samples (12 % of goat milk. In cow milk, 4 (8 % positive, 3 (6 % suspicious and 43 (86 % negative samples; in goat milk 3 (6 % positive, 2 (4 % suspicious and 45 (90 % negative samples were determined according to antibodies titre of serum agglutination test (Whey-AT. [TAF Prev Med Bull 2006; 5(3.000: 196-203

  12. Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses. (United States)

    Jarvis, Jodie A; Franke, Mary A; Davis, April D


    An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure.

  13. The clinical significance comparison of a latex agglutination based syphilis screening test at low antibody titer. (United States)

    Wang, Hua-Cheng; Chen, Cha; Wang, Li-Na; Long, Yi-Fei; Zhang, Wei-Zheng; Li, You-Qiang; Xiao, Qian; Yuan, Hui


    The rapid increase of syphilis underscores a tremendous need to carefully evaluate many new serological tests for syphilis and choose efficient and economical strategies for syphilis screening, especially in the case of primary infection with low antibody titer. Between 2011 and 2012, 73 patients' sera samples were included in this retrospective study. They were either TRUST or TPPA reactive, either LA (latex agglutination) based auto3 TP or CLIA (chemiluminescence assay) based Architect Syphilis TP assay reactive. The contradictory weak response samples were further examined by FTA-Abs method. TPPA could not give reactive results in samples with antibody concentration less than 10 mIU. Auto3 TP reagent shows good linearity at low antibody titers and was more sensitive than TPPA, while the former does not show significant superiority compared to the Architect Syphilis TP assay at low antibody titer, except that it is suitable for adaptation on diverse automated chemistry analyzers.

  14. Accuracy of diagnostic antibody tests for coeliac disease in children

    DEFF Research Database (Denmark)

    Giersiepen, Klaus; Lelgemann, Monika; Stuhldreher, Nina;


    The aim of this study was to summarise the evidence from 2004 to September 2009 on the performance of laboratory-based serological and point of care (POC) tests for diagnosing coeliac disease (CD) in children using histology as reference standard.......The aim of this study was to summarise the evidence from 2004 to September 2009 on the performance of laboratory-based serological and point of care (POC) tests for diagnosing coeliac disease (CD) in children using histology as reference standard....

  15. Evaluation Of Algorithms Of Anti- HIV Antibody Tests

    Directory of Open Access Journals (Sweden)

    Paranjape R.S


    Full Text Available Research question: Can alternate algorithms be used in place of conventional algorithm for epidemiological studies of HIV infection with less expenses? Objective: To compare the results of HIV sero- prevalence as determined by test algorithms combining three kits with conventional test algorithm. Study design: Cross â€" sectional. Participants: 282 truck drivers. Statistical analysis: Sensitivity and specificity analysis and predictive values. Results: Three different algorithms that do not include Western Blot (WB were compared with the conventional algorithm, in a truck driver population with 5.6% prevalence of HIV â€"I infection. Algorithms with one EIA (Genetic Systems or Biotest and a rapid test (immunocomb or with two EIAs showed 100% positive predictive value in relation to the conventional algorithm. Using an algorithm with EIA as screening test and a rapid test as a confirmatory test was 50 to 70% less expensive than the conventional algorithm per positive scrum sample. These algorithms obviate the interpretation of indeterminate results and also give differential diagnosis of HIV-2 infection. Alternate algorithms are ideally suited for community based control programme in developing countries. Application of these algorithms in population with low prevalence should also be studied in order to evaluate universal applicability.

  16. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus. (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi


    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.



    Goknur TERZI


    In this study Brucella antibodies were investigated with agglutination test (Whey-AT) and Milk Ring Test (MRT) in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 %) of cow milk and 6 samples (12 %) of goat milk. In cow milk, 4 (8 %) positive, 3 (6 %) suspicious and 43 (86 %) negative samples; in goat milk 3 (6 %) positive, 2 (4 %) suspicious and 45 (90 %) negativ...

  18. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays. (United States)

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H


    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

  19. Monoclonal antibody disulfide reduction during manufacturing: Untangling process effects from product effects. (United States)

    Hutterer, Katariina M; Hong, Robert W; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C


    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

  20. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu


    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  1. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris. (United States)

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R


    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).


    Directory of Open Access Journals (Sweden)

    Mahdi Fasihi-Ramandi


    Full Text Available There are many studies related to immunological and molecular methods for diagnosis of Vibrio cholera (V. cholerae. However, most assays dependent on enrichment of culture of bacteria, which need more time and involves the use of costly equipment and reagents. In this study Balb/c mice were immunized with recombinant Outer Membrane Protein (rOMPw of vibrio cholerae and splenocytes of hyper immunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using rOMPw as coating antigen. The monoclonal antibodies from ascitic fluids were purified and its reaction with rOMPw was assessed by ELISA. Polyclonal antibodies were also produced by immunization of rabbits with the above mentioned antigen. The rabbit sera was affinity purified using Hi-Trap protein G column. The result showed that monoclonal antibody specific to rOMPw has been successfully generated. The monoclonal antibody reacted with recombinant OMPw in ELISA and immunonoblat method. Rabbit polyclonal antibody was also bound to rOMPw by ELISA. The results of agglutination test with whole bacteria also showed that both mouse monoclonal and rabbit polyclonal antibodies reacted with whole vibrio cholera but not other related bacteria. The purpose of this study was to check out if anti OMPw antibodies could use as diagnostic assay for detection of V. cholerae. Our results demonstrated that anti recombinant OMPw monoclonal and polyclonal antibodies are able to diagnose whole bacteria in pure culture using agglutination test but not by home made immunochromatic strip test.

  3. The Statistical Precision of Medical Screening Procedures: Application to Polygraph and AIDS Antibodies Test Data


    Gastwirth, Joseph L.


    The increased use of screening tests for drug use or antibodies to the HTLV-III (AIDS) virus, as well as pre-employment polygraph testing, has raised concerns about the reliability of the results of these procedures. This paper reviews the mathematical model underlying the analysis of data from screening tests. In addition to the known formulas for the proportion of positive (negative) classifications that are correct, we provide a large sample approximation to their standard errors. The resu...

  4. Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation. (United States)

    Conway de Macario, E; Macario, A J; Tosi, R M; Celada, F; Landucci-Tosi, S


    A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were

  5. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)



    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  6. Production and Characterization of Anti-Her2 Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    A.S. Tabatabaei-Panah


    Full Text Available Objective: Breast cancer is the most common cancer among women in the world.Early diagnosis of this cancer is a key element for its treatment. One of the approachesfor diagnosis of breast cancer is detection of its tumour-associated markers. Hence,Her2 has been the main focus of the researches in the field.Materials and Methods: For diagnosis of Her2 overexpression, monoclonalantibodies (mAb reacting against Her2 were produced in this study. For thispurpose, two peptides from extracellular domain of Her2 were selected and themAbs reacting against them were produced by hybrodoma technology. Reactivityof these antibodies were then evaluated in different immunological assays includingELISA, Immunoflurescence (IF, western blot (WB and immunoprecipitation (IP.Results: Total of 5 clones were produced from two separate fusions, and antibodyisotyping revealed that all clones were IgM. These mAbs showed appropriatereactivities in the following assays: ELISA, immunofluresence by staining of breastcancer cell line (SKBR3, WB and IP by detecting the 185 KD band of Her2.Conclusion: In conclusion, it seems that the mAbs are useful diagnostic tools fordetection of Her2 expression in patients with breast cancer.

  7. Test Marketing in New Product Development (United States)

    Klompmaker, Jay E.; And Others


    Discusses the role of test marketing in new product development, based on interviews with marketing executives. Attempts to clarify when a test market should be done, what its aims should be, and how it should be used. (JG)

  8. Antidotes, antibody-mediated immunity and the future of pharmaceutical product development. (United States)

    Caoili, Salvador Eugenio C


    If new scientific knowledge is to be more efficiently generated and applied toward the advancement of health, human safety must be more effectively addressed in the conduct of research. Given the present difficulties of accurately predicting biological outcomes of novel interventions in vivo, the imperative of human safety suggests the development of novel pharmaceutical products in tandem with their prospective antidotes in anticipation of possible adverse events, to render the risks of initial clinical trials more acceptable from a regulatory standpoint. Antibody-mediated immunity provides a generally applicable mechanistic basis for developing antidotes to both biologicals and small-molecule drugs (such that antibodies may serve as antidotes to pharmaceutical agents as a class including other antibodies) and also for the control and prevention of both infectious and noninfectious diseases via passive or active immunization. Accordingly, the development of prophylactic or therapeutic passive-immunization strategies using antipeptide antibodies is a plausible prelude to the development of corresponding active-immunization strategies using peptide-based vaccines. In line with this scheme, global proliferation of antibody- and vaccine-production technologies, especially those that obviate dependence on the cold chain for storage and transport of finished products, could provide geographically distributed breakout capability against emerging and future health challenges.

  9. Production of antibodies with peptide-CpG-DNA-liposome complex without carriers

    Directory of Open Access Journals (Sweden)

    Kim Doo-Sik


    Full Text Available Abstract Background The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. Results We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. Conclusions Our overall results show that Lipoplex(O is a potent adjuvant and that complexes of peptide and Lipoplex(O are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

  10. Evaluation of Calypte AWARE HIV-1/2 OMT antibody test as a screening test in an Indian setting

    Directory of Open Access Journals (Sweden)

    Ingole N


    Full Text Available Purpose: Integrated counselling and testing centres (ICTC provide counselling and blood testing facilities for HIV diagnosis. Oral fluid tests provide an alternative for people whodo not want blood to be drawn. Also, it avoids the risk of occupational exposure. The goal of this study was to evaluate the utility of Calypte AWARE HIV-1/2 OMT antibody test as a screening test in an Indian setting. Materials and Methods: A cross-sectional study was carried out after ethics committee approval in 250 adult ICTC clients. Blood was collected and tested from these clients for HIV diagnosis as per routine policy and the results were considered as the gold standard. Also, after another written informed consent, oral fluid was collected from the clients and tested for the presence of HIV antibodies. Twenty five clients who had and 25 clients who had not completed their secondary school education (Group A and Group B, respectively were also asked to perform and interpret the test on their own and their findings and experiences were noted. Result: The sensitivity, specificity, PPV and NPV of the oral fluid antibody test were 100%, 98.51%, 94.11% and 100%, respectively. Seventy six percent of clients preferred oral fluid testing. Group B found it difficult to perform the test as compared to Group A and this difference was statistically significant (P ≤ 0.05. Conclusion: Oral fluid testing can be used as a screening test for HIV diagnosis; however, confirmation of reactive results by blood-based tests is a must.

  11. Real world experience with antiphospholipid antibody tests : how stable are results over time?

    NARCIS (Netherlands)

    Erkan, D; Derksen, WJM; Kaplan, [No Value; Sammaritano, L; Pierangeli, SS; Roubey, R; Lockshin, MD


    Objective: To determine the stability and the degree of variation of antiphospholipid antibody (aPL) results over time in a large cohort of well evaluated aPL positive patients; and to analyse factors contributing to aPL variation and the validity of aPL in a real world setting in which aPL tests ar

  12. Laboratory Evaluation of Three Rapid Diagnostic Tests for Dual Detection of HIV and Treponema pallidum Antibodies


    Humphries, Romney M.; Woo, Jennifer S.; Chung, Jun Ho; Sokovic, Anita; Bristow, Claire C; Jeffrey D Klausner


    The performance of three research-use-only, dual HIV and syphilis rapid diagnostic tests (RDTs) was evaluated for 150 patient serum samples and compared to reference HIV and Treponema pallidum antibody detection methods. The RDTs performed comparably, with sensitivities of 93 to 99% and specificities of 97 to 100%. The kappa statistic between the RDTs was 0.95.

  13. A Study of Oral Production Tests

    Institute of Scientific and Technical Information of China (English)



    Testing the ability to speak is a most important aspect of language testing. Testing of students’spoken English can bring in a positive washback for English teaching. However,it’s extremely difficult to test. This paper conducts a general analysis of oral production tests from overview,types and items of oral tests and scoring methods,which’s supposed to serve as an introduction to the issue under discussion.

  14. False positive hepatitis C antibody test results in left ventricular assist device recipients: increased risk with age and transfusions (United States)

    Lee, Doreen; Colovai, Adriana; Levy, Dana; Vasovic, Ljiljana; Roach, Keith W.; Shuter, Jonathan; Goldstein, Daniel; D’Alessandro, David; Jorde, Ulrich P.; Muggia, Victoria A.


    Left ventricular assist devices (LVADs) have been successfully used in patients with heart failure. However, LVADs may trigger immune activation, leading to higher frequencies of autoantibodies. We describe the clinical, epidemiological, and laboratory characteristics of LVAD recipients with false positive hepatitis C (FPHC) serology among 39 consecutive adult LVAD recipients who bridged to heart transplantation from January 2007 to January 2013 at Montefiore Medical Center. FPHC patients were identified as those with post-LVAD positive hepatitis C ELISA antibody tests and negative confirmatory testing with hepatitis C RNA PCR and/or radioimmunoblot assay. Ten (26%) patients previously seronegative for hepatitis C were found to have FPHC after device placement. Of the 39 patients, 32 had HeartMate II devices. The mean age at LVAD placement was 55 years. FPHC correlated with older age at the time of LVAD implantation and with receipt of packed red blood cell transfusions, but not with gender, fresh frozen plasma transfusions, panel reactive antibodies, globulin fraction, rheumatoid factor, or anticardiolipin antibodies. Clinicians should be aware of this increased risk of FPHC in older LVAD patients and those more heavily transfused in order to avoid unnecessary apprehension and possible delay in transplantation. Further studies should be done to evaluate the possible relationship between transfused blood products and immunomodulation. PMID:28203425

  15. 9 CFR 113.450 - General requirements for antibody products. (United States)


    ... transfer as well as for the prevention and/or alleviation of a specific viral or bacterial disease shall be... serial of product prepared for marketing. Recording charts shall bear full information concerning the... and identity as prescribed for live bacterial vaccines in § 113.64. (ii) Viral Master Seeds shall...

  16. Immunisation - Choice of host, adjuvants and boosting schedules with emphasis on polyclonal antibody production. (United States)

    Delahaut, Philippe


    Polyclonal antibodies are frequently used as immunodiagnostic tools in fundamental research. They are also used for routine diagnostic purposes in human and veterinary medicine and for quality control procedures in the food-processing industry. The antibody is a major component of the detection system. It binds with the molecule to be identified. This conjugate is subsequently revealed by means of binding the antibody with a radio-isotope, a fluorescent substance, an enzyme inducing a color change, or a biosensor based analytical system. Polyclonal antibodies are also used for treatment purposes in various pathologies. They might have immunomodulating or anti-inflammatory properties. Snake venom, rabies and tetanus antisera are examples of a therapeutic application; immunosuppressive antithymocyte serum used in order to avoid rejection in organ transplantation is another example from human medicine. These therapeutic aids need hyperimmunisation of animals. Since these are subject to a certain number of interventions such as injections and blood samplings, animal welfare prescriptions have to be taken into account. The optimisation of the immunisation protocol allows for reducing the numbers of animals used as well as reducing stress and pain while obtaining high quality antibodies. This article describes the critical steps in polyclonal antibody production with a particular focus on the choice of the animal species, the age of the subjects, the injection protocol and the sampling times.

  17. Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A.

    Directory of Open Access Journals (Sweden)

    Julie Prigent

    Full Text Available Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains. Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14 and insect cells (Sf9. After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.

  18. Characterization of a monoclonal antibody cell culture production process using a quality by design approach. (United States)

    Horvath, Brian; Mun, Melissa; Laird, Michael W


    The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.

  19. Diagnosis of canine echinococcosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay. (United States)

    Craig, P S; Gasser, R B; Parada, L; Cabrera, P; Parietti, S; Borgues, C; Acuttis, A; Agulla, J; Snowden, K; Paolillo, E


    Echinococcus granulosus is one of the most important and widespread of the helminth zoonoses. Diagnosis of E. granulosus infection in dogs currently relies on arecoline dosing and detailed examination of the purge for adult worms. Two immunodiagnostic tests (ELISA) based on genus specific coproantigen detection or serum antibody (IgG, IgA and IgE) detection were compared against arecoline purgation for the detection of Echinococcus in naturally infected dogs in Uruguay. The coproantigen ELISA had a sensitivity of 76.9% compared with 34.6% for the serum IgG ELISA when assessed against 26 purge positive dogs (purge worm count range 1-4331). Coproantigen reactivity was positively correlated (r = 0.65) to purge worm count, with a threshold at over 20 worms. There was no positive correlation of antibody levels with worm counts. In 26 matched Echinococcus positive dog samples, the overall sensitivity of serological detection increased to 69.2% when seroreactivity for IgA and IgE antibodies were included and to 96.2% for both coproantigen and antibody assays combined. The detection of current infection of individual dogs with E. granulosus by coproantigen ELISA has the potential to replace arecoline purgation, while specific serum antibody detection should be useful in assessing Echinococcus exposure in dog populations.

  20. Effects of glutamine and asparagine on recombinant antibody production using CHO-GS cell lines. (United States)

    Xu, Ping; Dai, Xiao-Ping; Graf, Erica; Martel, Richard; Russell, Reb


    A unique and nontraditional approach using glutamine and asparagine supplements for CHO-glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench-top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines.

  1. Improved production and function of llama heavy chain antibody fragments by molecular evolution

    NARCIS (Netherlands)

    Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.


    The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

  2. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.


    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA level

  3. Seroprevalence of Mycoplasma gallisepticum antibody by ELISA and serum plate agglutination test of laying chicken

    Directory of Open Access Journals (Sweden)

    Md. Zulfekar Ali


    Full Text Available Aim: Mycoplasma gallisepticum (MG is important avian pathogen responsible for chronic respiratory disease of chicken and turkeys, which result in large economic loss for the poultry industry. The objectives of this study were determination of seroprevalence of MG antibody of commercial layer chicken at laying period in selected areas of Bangladesh. Materials and Methods: A total of 563 blood samples were collected randomly from selected commercial layer chickens at laying period during the period from July to December, 2013. Indirect enzyme linked immunosorbent assay (iELISA and serum plate agglutination (SPA test were performed to detect the presence of antibodies against MG. Results: Of 563 samples, 64.47% and 56.13% showed an overall prevalence of MG antibodies in iELISA and SPA test respectively. Prevalence of MG was recorded the highest (69.63% at 50-55 weeks of age compared with lowest (53.26% at 56-61 weeks of age (p<0.05. Significant (p<0.05 effect of breed were observed in the seroprevalence of MG infection in layer birds in the present study. The overall, 68.77%, 63.74% and 59.37% prevalence were found respectively in sonali, ISA Brown and White leg horn. The prevalence of MG antibodies was the highest (70.13% in December followed by November (68%, October (65.67%, August (63.46%, September (58.54% and July (51.78% month. The seroprevalence of MG antibodies was higher (69.63% in most of the large flocks and lower (56.82% in small flocks. Conclusion: Therefore, might be suggested that the commercial layer farms should be routinely checked to monitor MG infection and the reactor birds should be culled since MG organism has the potential to transmit vertically. The correlation between MG antibody in month and flock size was not significant (p=0.359 and p=0.868, respectively.

  4. Development of mass spectrometry based techniques for the identification and determination of compositional variability in recombinant polyclonal antibody products. (United States)

    Persson, Pia; Engström, Anders; Rasmussen, Lone K; Holmberg, Erland; Frandsen, Torben P


    Recombinant polyclonal antibodies are a new class of protein biologics, combining a defined number of target-specific antibodies, developed for therapeutic use across various indications. Development, manufacture, and release of recombinant polyclonal antibodies as well characterized biological products have required development of new chemistry, manufacturing, and control (CMC) technologies. Sym001 is a recombinant polyclonal antibody product containing 25 unique antibodies specific for the Rhesus D antigen. Sym001 drug substance is manufactured using a single batch technology, Sympress. Here, we describe the development of two novel mass spectrometry based methods that allows identification of individual antibodies in the Sym001 drug substance, through the determination of unique marker peptides or antibody light chains. The two methods provide an unambiguous identification of the 25 unique antibodies comprised in the Sym001 drug substance. Furthermore, the light chain liquid chromatography-mass spectrometry (LC-MS) method has been developed to allow the determination of the relative distribution of the 25 antibodies. The light chain LC-MS method has demonstrated linearity, specificity, precision, and accuracy, thus qualifying it for use in the quality control of recombinant polyclonal antibodies for human use. The development of such quantitative methods is central for the development and quality control of additional therapeutic recombinant polyclonal antibody products.

  5. Comparison of indirect fluorescent antibody test and the modified agglutination test for the detection of Toxoplasma gondii antibodies in stray dogs from Southern Brazil. (United States)

    de Almeida, Jonatas Campos; Frehse, Michelle Salmon; Navarro, Italmar Teodorico; Garcia, João Luis; Biondo, Alexander Welker; Freire, Roberta Lemos


    The aim of the present study was to determine the prevalence of antibodies to Toxoplasma gondii by two serological techniques in sera of 364 stray dogs from Brazil by immunofluorescence antibody test (IFAT, cut off point 1:16) and to the modified agglutination test (MAT, cut-off points 1:25 and 1:50). A total of 175/364 (48.07%) sera were positive by IFAT, and 108/364 (29.67%) and 85/364 (23.35%) were positive by MAT with cutoff points 1:25 and 1:50, respectively were positive by MAT. Cohen's Kappa Coefficient between IFAT and MAT was 0.81 (excellent) and 0.66 (substantial) with cutoff points 1:25 and 1:50, respectively. Using IFAT as gold standard, MAT sensitivity and specificity were 78% and 99% for 1:25 and 61% and 99% for 1:50, respectively. The results document of the usefulness of MAT for serological diagnosis because it does not require species-specific conjugate.

  6. Spleen vagal denervation inhibits the production of antibodies to circulating antigens.

    Directory of Open Access Journals (Sweden)

    Ruud M Buijs

    Full Text Available BACKGROUND: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output. METHODOLOGY/PRINCIPAL FINDINGS: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS, activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA. CONCLUSIONS/SIGNIFICANCE: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

  7. Comparison of the latex agglutination test with the hemagglutination inhibition test, enzyme-linked immunosorbent assay, and neutralization test for detection of antibodies to rubella virus.


    Meegan, J M; Evans, B. K.; Horstmann, D. M.


    The ability of a rapid, latex agglutination test to diagnose rubella infection and to measure immune status was evaluated by comparison with the hemagglutination-inhibition (HAI) test, enzyme-linked immunosorbent assay (ELISA), and the neutralization (NT) test. The latex agglutination test accurately detected serological conversions in 74 pairs of sera representing 21 natural infections and 53 immunizations. The antibody levels of 276 sera from the general population were determined by latex ...

  8. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. (United States)

    Stöger, E; Vaquero, C; Torres, E; Sack, M; Nicholson, L; Drossard, J; Williams, S; Keen, D; Perrin, Y; Christou, P; Fischer, R


    This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

  9. Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

    Directory of Open Access Journals (Sweden)

    Røed Knut H


    Full Text Available Abstract Background The incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens. Methods We tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT. Fractions of seropositive cows were compared by calculating 95% CI. Results The results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples. Conclusions Neither the farmers' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area.

  10. In vitro-induced antibody production in chronic hepatitis C virus infection

    Directory of Open Access Journals (Sweden)

    Oliveira Jr. E.B.


    Full Text Available The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV antibody production (IVIAP in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males with HCV infection (anti-HCV and HCV-RNA positive. Alanine aminotransferase (ALT was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52% and cirrhosis in 11/52 (21% patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540, with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4. These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

  11. Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein. (United States)

    Wangman, Pradit; Senapin, Saengchan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Rukpratanporn, Sombat; Sithigorngul, Paisarn


    The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.

  12. Evaluation of a bovine antibody test for diagnosing Mycobacterium avium complex in patients with cystic fibrosis

    DEFF Research Database (Denmark)

    Qvist, Tavs; Pressler, Tacjana; Katzenstein, Terese L;


    INTRODUCTION: The aim of this study was to test a commercial bovine enzyme-linked immunosorbent assay for investigating antibody activity against Mycobacterium avium complex. METHODS: All patients at the Copenhagen Cystic Fibrosis (CF) Center who had culture for nontuberculous mycobacteria...... before and after culture conversion was performed in case patients. RESULTS: Out of 286 included subjects, six had clinical M. avium complex pulmonary disease at the time of sera sampling. These patients presented with higher antibody test values (P-value ... at ruling out pulmonary disease. Screening sera from patients with CF could guide clinicians to focus attention on patients at higher risk of M. avium complex pulmonary disease. Pediatr Pulmonol. 2016; 9999:XX-XX. © 2016 Wiley Periodicals, Inc....

  13. ConocoPhillips Gas Hydrate Production Test

    Energy Technology Data Exchange (ETDEWEB)

    Schoderbek, David; Farrell, Helen; Howard, James; Raterman, Kevin; Silpngarmlert, Suntichai; Martin, Kenneth; Smith, Bruce; Klein, Perry


    Work began on the ConocoPhillips Gas Hydrates Production Test (DOE award number DE-NT0006553) on October 1, 2008. This final report summarizes the entire project from January 1, 2011 to June 30, 2013.

  14. Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

    Full Text Available Large-scale production of fully human IgG (hIgG or human polyclonal antibodies (hpAbs by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

  15. Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2

    DEFF Research Database (Denmark)

    McNair, I.; Marshall, M.; McNeilly, F.


    A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assa...... than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus....

  16. Red cell antibodies and low ionic strength: a study with enzyme-linked antiglobulin test. (United States)

    Leikola, J; Perkins, H A


    Alloantibody uptake on red blood cells was quantified with an accurate and reproducible enzyme-linked antiglobulin test. The uptake of anti-D, anti-Fy2 and anti-JK3 was markedly accelerated by low ionic strength salt solution (LISS) with a final ionic strength of 0.05 M. Near maximum uptake occurred within ten minutes at room temperature which corresponded to 60 minutes in saline at 37 C. Papain treatment of red blood cells increased the amount of anti-D bound, and there was no difference whether or not the papain-treated cells were suspended in LISS. In contrast, the uptake of IgG anti-A and anti-Leb was not accelerated by LISS, nor did LISS increase the rate of binding of antiblogulin to IgG antibody-coated red blood cells. We suggest this may be explained by the fact that the ABH and Lewis antigens (as well as bound IgG antibodies) extend beyond the "ionic cloud" surrounding the red blood cell. Antibody binding in the presence of albumin was approximately the same as in saline; but if the albumin was first dialyzed against LISS, the reaction was markedly accelerated and the final antibody uptake somewhat higher than in LISS alone.

  17. Serological survey on canine coronavirus antibodies in giant pandas by virus neutralization test

    Institute of Scientific and Technical Information of China (English)

    QIAOJun; XIAXian-zhu; YANGSong-tao; LIDe-sheng; HUGui-xue; GAOYu-wei; SUNHe-ting; ZHAOZhong-pen; XlEZhi-jing; YANFang; HEWen-qi; HUANGGen


    In order to survey the infectious situation of canine coronavirus (CCV) in giant panda population, a virus neutralization test detecting specific antibodies against CCV in giant panda's sera was established by using two-fold dilutions of serum and 100 TCID50 of the virus. The 62 sera samples of giant pandas, which were gathered from zoos and reserve region of Sichuan Province, China were detected. The neutralization antibody titer of 1:4 was recognized as the positive criterion, 8 sera samples were detected to be positive, and the positive rate was 12.9%. The titers of neutralizing antibody ranged from 1:8 to 1:32. It was the first comprehensive investigation on neutralization antibodies against CCV in giant panda population in China. The results of study showed that the infection of CCV in giant panda population was universal, which has posed a threat to the health of giant panda. Therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against CCV infection.

  18. EOR production technique tested on Codell

    Energy Technology Data Exchange (ETDEWEB)

    Stremel, K.


    A production method of natural gas that eliminated marketing problems and allowed maximum recovery was dicussed. The method involved the dehydration of the gas, stripping of the propane and butane products, and the compression and reinjection of the dry gas to the well. This maintenance of pressure will allow storage of the gas until marketing conditions improve. The production method is being tested at pilot wells in Colorado by Petromax Energy Corporation.

  19. Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71. (United States)

    Qin, Bo; Zhang, Jianhua; Xie, Wenhao; Liu, Xuehong; He, Tingting; Chen, Jinkun; Dong, Xuejun


    A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest including prokaryotically expressed VP1 and two strains of anti-VP1 monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using ethyl-dimethyl-amino-propyl carbodiimide (EDC) to prepare sensitized latex beads. LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, whereas these values were 86.8, 96.7, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71.

  20. Validation of serological tests for the detection of antibodies against Treponema pallidum in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Sascha Knauf


    Full Text Available There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs and two non-treponemal tests (NTTs were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG, however, could be considered as a confirmatory test.

  1. Validation of serological tests for the detection of antibodies against Treponema pallidum in nonhuman primates. (United States)

    Knauf, Sascha; Dahlmann, Franziska; Batamuzi, Emmanuel K; Frischmann, Sieghard; Liu, Hsi


    There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue) may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO) recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs) and two non-treponemal tests (NTTs) were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis) from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA) was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG), however, could be considered as a confirmatory test.

  2. Evaluation of the Canadian Rheumatology Association Choosing Wisely recommendation concerning anti-nuclear antibody (ANA) testing. (United States)

    Ferrari, Robert


    The objective of this study was to evaluate the Canadian Rheumatology Association Choosing Wisely recommendation concerning anti-nuclear antibody (ANA) testing. Patients with joint pain/stiffness/swelling were assessed to determine if ANA testing was indicated. An a priori threshold was set before ANA testing would be considered. Those who did not have ANA testing ordered were followed for 1 year to determine if any of them went on to have a diagnosis of systemic lupus erythematosus (SLE) or other connective tissue disease. A parallel study was conducted with a similar a priori threshold for the use of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody testing in the diagnosis of rheumatoid arthritis (RA), and again, patients were followed for 1 year. A total of 866 subjects were examined, 509 females (58.8 %) and 357 males (41.2 %). The mean age of the group was 47.5 ± 16.8 years. The mean duration of symptoms was 12.0 ± 5.6 weeks. Of the 866 subjects, 68 met an a priori threshold for ordering ANA, RF, and anti-CCP testing. Of these 68, there was a newly diagnosed case of SLE, 4 newly diagnosed cases of RA, and 3 cases of polymyalgia rheumatica. The remaining 798 subjects were followed for approximately 1 year and none developed evidence of SLE, RA, or other connective tissue disease. In the evaluation of non-specific musculoskeletal symptoms, setting an a priori threshold for ordering serology in keeping with the spirit of the Canadian Rheumatology Association Choosing Wisely recommendation for antibody testing results in a very low risk of missing a case of systemic lupus erythematosus or rheumatoid arthritis.

  3. Comparative Studies on Detection of Antibodies against Infectious Bursal Disease Virus with Test Strips and Agar Gel Immunodiffusion Method

    Institute of Scientific and Technical Information of China (English)

    Jinliang ZHANG; Wentong ZHANG; Sishun HU; Dingren BI; Xiliang WANG; Yuncai XIAO


    [Objective] This study aimed to compare the detection results of antibodies against infectious bursal disease virus with test strips and agar gel immunodiffusion method. [Method] Antibodies against infectious bursal disease virus in chicken serum were detected using test strips developed in our laboratory, and the results were comparad~with that using traditional agar diffusion method. [Result] The comparative study of the two methods showed that the sensitivity of test strips was eight times over agar gel immunodiffusion; test strips showed higher detection rate in the deter- mination test of 216 clinical samples, with high specificity, easy preservation, and simple and rapid operation, thereby being more suitable for the monitoring of clinical antibodies. [Conclusion] Test strips could replace the existing serological methods, having great promotion and application value in antibody monitoring.

  4. Antithyroglobulin antibody (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  5. Production LHC HTS power lead test results

    CERN Document Server

    Tartaglia, M; Fehér, S; Huang, Y; Orris, D F; Pischalnikov, Y; Rabehl, Roger Jon; Sylvester, C D; Zbasnik, J


    The Fermilab Magnet test facility has built and operated a test stand to characterize the performance of HTS power leads. We report here the results of production tests of 20 pairs of 7.5 kA HTS power leads manufactured by industry for installation in feed boxes for the LHC Interaction Region quadrupole strings. Included are discussions of the thermal, electrical, and quench characteristics under "standard" and "extreme" operating conditions, and the stability of performance across thermal cycles.

  6. Comparison of latex and haemolysin tests for determination of anti-streptolysin O (ASO) antibodies.


    Curtis, G D; Kraak, W A; Mitchell, R G


    A latex agglutination test was compared with the micro-titration haemolysin inhibition method for the detection of anti-streptolysin O (ASO) antibodies in 428 serum samples. After slight modification of the latex method to produce maximal agglutination good agreement was shown between the results obtained by the two methods. The latex test had a sensitivity of 83.6%, a specificity of 93.3%, a predictive positive value of 86.5% and a predictive negative value of 91.6%. It was convenient, requi...

  7. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design. (United States)

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J


    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  8. [Production and characteristics of monoclonal antibodies against individual prekeratins in simple types of rat epithelium]. (United States)

    Troianovskiĭ, S M; Krutovskikh, V A; Bannikov, G A


    BALB/c mice were immunized with intermediate filaments (IF) from the rat colon mucosa, and their splenocytes were fused with myeloma cells to obtain hybridomas. Specific antibody production was assessed by indirect immunofluorescence on cultured rat hepatoma 27 containing prekeratins. The clones that stained IF in hepatoma and not in fibroblasts were judged positive. Clones E3 and E6 were shown to produce monoclonal antibodies against prekeratin with molecular mass of 40 kD (PK40), while clones E2 and E7 produced antibodies against prekeratin with molecular mass of 55 kD (PK55). This was established by immunoblotting with 125I-protein A in cell lysates from the colon, bladder, and hepatoma 27. Only PK55 was revealed in liver and salivary gland lysates. The above proteins were not detected in esophagus, fibroblast and skeletal muscle cell lysates. The monoclonal antibodies make it possible to study individual prekeratin expression in embryogenesis, differentiation and neoplastic transformation of simple epithelium.

  9. [Salmonella typhi vaccination response study reveals defective antibody production selective IgA deficiency patient]. (United States)

    Pleguezuelo, Daniel E; Gianelli, Carla


    Selective IgA deficiency (SIgAD) is the most prevalent immunodeficiency worldwide, progressing to common variable immunodeficiency only in few reported cases. We report the case of a Spanish female aged 22 and diagnosed of selective IgA deficiency, a long history of bronchitis, several episodes of pneumonia, bilateral bronchiectasis, normal IgG, IgM, IgG subclasses, and detectable pre-vaccination IgG antibodies against tetanus toxoid and Streptococcus pneumoniae. She was evaluated in our clinic in order to rule out common variable immunodeficiency. We observed good antibody response to tetanus toxoid, absence of circulating switched memory B cells, decreased response to pneumococcal polysaccharide antigens and a lack of response to Salmonella typhi vaccine. Most SIgAD patients presents with upper respiratory tract infections or mild diarrhea. Those with lower tract infections, pneumonia or untreatable diarrhea should follow B-cell subpopulations' study and antibody response to vaccines. Absence of response to Salmonella typhi vaccine allowed us to expose the defective antibody production.

  10. Production of Antibody Raised Against Lipopolysaccharide (LPS of Vibrio Cholerae Non-O1

    Directory of Open Access Journals (Sweden)

    H Shirzad


    Full Text Available Background: Cholera, an infectious disease caused by Vibrio cholerae, is primarily transmitted by ingestion of contaminated food or water. In severe cases, cholera may lead to severe dehydration, metabolic acidosis, and ultimately, hypovolemic shock and death. Methods: In this study V.cholerae non-O1 was cultured in suitable media. LPS was extracted from the surface of  bacteria by hot phenol-water method and then purified by high-speed centrifugation. For production of specific antibody against LPS, white newzeland rabbits were first immunized by whole cell bacteria and then immunized with highly purified LPS. The titre of the antiserum was determined by ELISA for each serogroup. Results: Results presented in this study indicate that serum anti-LPS antibodies raised against purified LPS of V.cholerae non-O1 can detect V.cholerae non-O1 .Conclusion: This antibody had low cross reactivity with V.cholerae O1, serotype Inaba or Ogawa. So, this antibody can be used for for detection of V. cholerae non-O1.

  11. Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

    Directory of Open Access Journals (Sweden)

    Telma M. Alves


    Full Text Available Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs anti-C. fetus subsp. venerealis of the IgM (1 and IgG (14 classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs: two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

  12. An imaging diagnosis of cerebral paragonimiasis: CT and MR findings and correlation with ELISA antibody test

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kee Hyun; Cha, Sang Hoon; Han, Moon Hee; Kim, Hong Dae; Cho, Seung Yull; Kong, Yoon; Kang, Hyung Keun; Kim, Myung Soon [Seoul National University College of Medicine, Seoul (Korea, Republic of)


    To evaluate the CT and MR findings of cerebral paragonimiasis(PW) and to assess the diagnostic value of the specific antibody test by enzyme-linked immunosorbent assay(ELISA) for PW, 55 CT scans and 13 MR images of 57 patients with cerebral PW were reviewed retrospectively, and correlated with the serum/ CSF antibody levels. We divided the patients into three groups, early active (n=21), chronic(n=32), and combined stage(n=4), on the basis of CT/MR findings. In the groups of early active stage the most common and characteristic finding was multiple, conglomerated, ring-like enhancing lesion in the unilateral cerebral hemisphere, which was seen in 52% on CT and 44% on MR. Other non-specific findings included a solitary ring-like or irregular enhancing lesions, ill-defined low density lesions without enhancement, localized hemorrhage with or without enhancing lesions. In the group of chronic stage, there were multiple calcifications of various shapes, most commonly 1-2 cm sized round shape, and associated encephalomalacia. MR was superior to CT in detecting hemorrhage and in characterizing the central contents of ring-shaped calcifications, while it was inferior to CT in identifying small calcifications. Antibody levels of serum and CSF were positive in 86% and 82% in early active group, and in 48% and 31% in chronic stage,retrospectively.The positive rate was significantly different between the two groups (P=0.001). CT/MR findings were characteristic in only approximately half the cases in early active cerebral PW which can be cured by Praziquantel therapy. Therefore, antibody test by ELISA is recommended as a complementary tool, particularly in patients with non-specific imaging findings.

  13. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees


    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  14. Expression of recombinant antibodies. (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas


    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  15. Comparison of five commercial serological tests for the detection of anti-Chlamydia trachomatis antibodies. (United States)

    Baud, D; Regan, L; Greub, G


    Screening for Chlamydia trachomatis-specific antibodies is valuable in investigating recurrent miscarriage, tubal infertility and extrauterine pregnancy. We compared here the performance of immunofluorescence (IF) to four other commercial tests in detecting IgG antibodies directed against C. trachomatis: two enzyme-linked immunosorbent assays (ELISAs) using the major outer membrane protein (MOMP) as the antigen, commercialised respectively by Medac and R-Biopharm (RB), one ELISA using the chlamydial heat shock protein 60 (cHSP60) as the antigen (Medac), as well as a new automated epifluorescence immunoassay (InoDiag). A total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all five tests. The prevalence of C. trachomatis-specific IgG antibodies as determined by the IF, cHSP60-Medac, MOMP-Medac, MOMP-RB and InoDiag was 14.3, 23.2, 14.3, 11.9 and 26.2%, respectively. InoDiag exhibited the highest sensitivity, whereas MOMP-RB showed the best specificity. Cross-reactivity was observed with C. pneumoniae using IF, MOMP-RB and InoDiag, and Parachlamydia acanthamoebae using the cHSP60 ELISA test. No cross-reactivity was observed between C. trachomatis and the other Chlamydiales (Neochlamydia hartmannellae, Waddlia chondrophila and Simkania negevensis). Given its high sensitivity, the new automated epifluorescence immunoassay from InoDiag represents an interesting alternative. The MOMP-based ELISA of R-Biopharm should be preferred for large serological studies, given the high throughput of ELISA and its excellent specificity.

  16. Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests. (United States)

    Gallardo, C; Soler, A; Nieto, R; Carrascosa, A L; De Mia, G M; Bishop, R P; Martins, C; Fasina, F O; Couacy-Hymman, E; Heath, L; Pelayo, V; Martín, E; Simón, A; Martín, R; Okurut, A R; Lekolol, I; Okoth, E; Arias, M


    The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs.

  17. A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the conjugation with KLH and the antibody production. (United States)

    Katayama, Hidekazu; Mita, Masatoshi


    A small peptide-keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for producing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide-KLH conjugates is difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate, we could obtain specific antibodies with a high antibody titer. This method might also be useful for the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such as vertebrate relaxins.

  18. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging. (United States)

    Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H


    Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

  19. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging

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    Peter T. Sage


    Full Text Available Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR cells to stimulatory T follicular helper (TFH cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

  20. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA


    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  1. Correlates of participation in AIDS education and HIV antibody testing by methadone patients. (United States)

    Magura, S; Grossman, J I; Lipton, D S; Amann, K R; Koger, J; Gehan, K


    The authors examined the factors associated with methadone patients' decisions about participating in a clinic-based AIDS prevention protocol. Despite the offer of incentives, only 27 percent attended AIDS education and only 12 percent obtained voluntary HIV antibody (ab) testing. However, AIDS education was attended by proportionately more of those who were at highest risk for AIDS because of current intravenous drug use. The availability of HIV-ab testing neither encouraged nor discouraged participation in AIDS education. Patients who were relatively more likely to choose HIV-ab testing were older, had been or were married, had plans to have children, believed the test to be useful, and believed that their counselors support their decision to be tested. Those who declined to be tested were reluctant to confront the emotional aspects of their risk status, were concerned about possible breaches of confidentiality, and doubted the value of testing. The implications of the findings for implementing AIDS prevention measures in methadone programs are discussed. Programs need either to require attendance at AIDS education or give patients an incentive to attend. HIV-ab testing should be available but should remain voluntary. A stronger medical rationale for testing is developing and may increase future participation. Methadone programs must continue to engage patients actively in AIDS risk reduction efforts.

  2. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Newsom-Davis, J.; Willcox, N.; Calder, L.


    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

  3. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico


    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

  4. Purification of Herpes Simplex Virus Type 1 for Production of High Titer Polyclonal Antibody against the Virus

    Directory of Open Access Journals (Sweden)

    Z Meshkat


    Full Text Available ABSTRACT: Introduction & Objective: Herpes simplex virus type 1 infection is one of the most prevalent viral infections worldwide. Different methods are being investigated for the virus’ detection, prevention and therapy. The aim of the present study was to purify the virus and to produce a high titer polyclonal antibody against the virus. Materials & Methods: This experimental study was done in the Virology Department of Tarbiat Modares University from 2001 to 2002. Virus purification was done using serial dilution and plaque purification protocols. A single plaque was chosen and propagated, and the virus titer was determined. In inoculated animals, the titer of produced antibody against the virus was measured by virus neutralization test. Results: Using virus neutralization test, it was found that the high level of antibody has been raised in animals against the virus. Conclusion: Considering the preparation of high titer antibody against the virus, the produced antibody can be used for the development and optimization of different diagnostic methods.

  5. Choosing wisely: Review and commentary on anti-nuclear antibody (ANA) testing. (United States)

    Fritzler, Marvin J


    Choosing Wisely®: Next Steps in Improving Healthcare Value is an initiative of the American Board of Internal Medicine (ABIM) Foundation. The driving forces for the Choosing Wisely (CW) campaign include rising and unstainable health care expenditures and evidence that there is lack of fiscal stewardship of health care resources. The American College of Rheumatology and the Canadian Rheumatology Association published their top five Choosing Wisely recommendations, the first of which pertained to antinuclear antibodies (ANA) and ANA subserology testing. Concerns about the wasteful use of these tests prompted an analysis of the expenditures attributable to ANA testing as a proportion of total health care expenditures and based on a financial model was in the range of 0.00125%. It is suggested that if the sole use of ANA testing is to add evidence to support a diagnosis when the pre-test probability is high, then the ANA test has limited clinical value. Accordingly, the goal of ANA testing needs to be reconsidered and expanded beyond an approach to simply confirming a diagnosis with 'intention to treat' to a goal of case finding of 'pre- or early disease' with an 'intent to prevent' disease. This an area where more significant inroads can be made in preventing end organ disease and thereby reducing health care expenditures HCE. One CW recommendation that bears emphasizing is that, with a few possible exceptions, repeat ANA or ANA subserology testing has little clinical value in monitoring disease activity or predicting a flare.

  6. Production of high titre antibody response against Russell's viper venom in mice immunized with ethanolic extract of fruits of Piper longum L. (Piperaceae) and piperine. (United States)

    Shenoy, P A; Nipate, S S; Sonpetkar, J M; Salvi, N C; Waghmare, A B; Chaudhari, P D


    Piper longum L. fruits have been traditionally used against snakebites in north-eastern and southern region of India. The aim of the study was to assess the production of antibody response against Russell's viper venom in mice after prophylactic immunization with ethanolic extract of fruits of Piper longum L. and piperine. The mice sera were tested for the presence of antibodies against Russell's viper venom by in vitro lethality neutralization assay and in vivo lethality neutralization assay. Polyvalent anti-snake venom serum (antivenom) manufactured by Haffkine Bio-Pharmaceutical Corporation Ltd. was used as standard. Further confirmation of presence of antibodies against the venom in sera of mice immunized with PLE and piperine was done using indirect enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion test. Treatment with PLE-treated mice serum and piperine-treated mice serum was found to inhibit the lethal action of venom both in the in vitro lethality neutralization assay and in vivo lethality neutralization assay. ELISA testing indicated that there were significantly high (ppiperine treated mice serum and the venom antigens. In double immunodiffusion test, a white band was observed between the two wells of antigen and antibodies for both the PLE-treated and piperine-treated mice serum. Thus it can be concluded that immunization with ethanolic extract of fruits of Piper longum and piperine produced a high titre antibody response against Russell's viper venom in mice. The antibodies against PLE and piperine could be useful in antivenom therapy of Russell's viper bites. PLE and piperine may also have a potential interest in view of the development of antivenom formulations used as antidote against snake bites.

  7. Contribution of V(H replacement products in mouse antibody repertoire.

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    Lin Huang

    Full Text Available VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

  8. Clinical appraisal of patients and detection of serum antibodies by ELISA and CIA tests in late periods of Trichinella sp. invasion

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    Kociecka W.


    Full Text Available Results of our studies using ELISA and competitive inhibition assay (CIA tests fully confirmed the previously experienced trichinellosis and reflected persistent stimulation of antibody production due to the antigen release from Trichinella larvae, which had survived longer and undergone gradual destruction in the muscles. The studies proved that the tests complement each other, yielding concordant results in 86.7 % of cases. Due to its higher specificity, the CIA test can help in interpreting pathological signs/symptoms and in evaluating humoral response activity at late and distant in time periods following the invasion.

  9. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

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    Davoud Koolivand


    Full Text Available The genomic region of Grapevine fanleaf virus (GFLV encoding the movement protein (MP was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3 to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG concentrations (1, 1.5, and 2 mM each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

  10. Production and characterization of antibodies against irradiated human erythrocytes membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Amancio, Francisco F. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)]|[Pernambuco Univ., Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia; Andrade Junior, Heitor F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical


    Gamma irradiation affects people in several situations, with few if any sensitive biological assay of its action. Nucleic acids and proteins are affected by radiation, but only the former was used in most dosimetric techniques. The irradiation of proteins promotes structural modifications attributed to free radicals from water radiolysis. Theoretically, antibodies induced by irradiated proteins could recognize these radical-related new epitopes, allowing their use as a probe. Human erythrocyte membrane proteins (HEMP), few and well defined molecules, are certainly exposed to radiation, being the ideal target. With this rationale, we study the production of antibodies in mice immunized with {sup 60} Co irradiated HEMPs. Menbranes from hypotonic lysis with differential centrifugation of A+ erythrocytes, were irradiated in a Gammacell 220 with 400, 800 and 1600 Gy, and used as immunogen for Balb/c mice, after SDS-PAGE. Irradiated HEMP induced antibodies recognize only irradiated human erthrocytes in an intact cell indirect immunofluorescence assay (ICIIFA). When used in Wester-blot against non-irradiated HEMPs, those sera recognize most proteins, suggesting a pool of abs directed both to native, as detected by Western Blot, or irradiated, as detected by ICIFA, HEMPs. Those data confirmed our assumptions, allowing the use of those abs in the search for a method of biological dosimetry. (author). 18 refs., 3 figs.

  11. 75 FR 28335 - Testing and Labeling Pertaining to Product Certification (United States)


    ... Safety Commission 16 CFR Part 1107 Testing and Labeling Pertaining to Product Certification; Proposed... Product Certification AGENCY: Consumer Product Safety Commission. ACTION: Proposed rule. SUMMARY: The... product complies with certification requirements under a reasonable testing program for...

  12. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

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    Pleckaityte Milda


    Full Text Available Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY, the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the sc

  13. Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Yaling HAN; Haiwei LIU; Jian KANG; Xiaozeng WANG; Ye HU; Lianyou ZHAO; Shaohua LI


    Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.


    Directory of Open Access Journals (Sweden)

    Ľubomíra Juhaniaková


    Full Text Available The aim of this work was to determine microbiological quality and water activity of confectionery products as corpus of desserts, stuffing cakes and finished cakes. In confectionery products microbiological parameters: total count of bacteria, coliforms bacteria, mesophilic aerobic bacteria, yeasts, microscopic filamentous fungi, counts of Staphylococcus aureus and Salmonella spp. were observed. These confectionery products were evaluated: 5 corpus of Kremeš, 5 stuffing of Kremeš and 5 Venček cake. For microbiological tests 15 samples of confectionery products were used. The numbers of total count of bacteria ranged from 2.9 to 3.65 log CFU.g-1, the number of mesophilic aerobic bacteria ranged from 2.00 to 3.28 log CFU.g-1, coliforms bacteria in confectionery products ranged from 0.00 to 3.15 CFU.g-1, number of yeasts ranged from 0.00 to 3.30 log CFU.g-1and the number of microscopic fungi ranged from 0.00 to 2.90 CFU.g-1. None of the samples showed any growth of coliforms bacteria, Staphylococcus aureus and Salmonella spp. From microscopic fungi were isolated genera Alternaria, Aspergillus and Penicillium. Eight from fifteen investigated samples of confectionary products were in accordance with the Codex Alimentarius of the Slovak Republic. The lowest water activity was found in Kremeš corpus samples (0.953 and higher in Venček samples (0.973.

  15. Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Zornig, Hanne D; Buhmann, Caecilie;


    Gender-specific and parity-dependent acquired antibody recognition is characteristic of variant surface antigens (VSA) expressed by chondroitin sulfate A (CSA)-adherent Plasmodium falciparum involved in pregnancy-associated malaria (PAM). However, antibody recognition of recombinant products...

  16. Evaluation of five screening tests licensed in Argentina for detection of hepatitis C virus antibodies

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    Viviana Ré


    Full Text Available This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV. The tests analyzed were: Detect-HCV™ (3.0 Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK and Serodia-HCV particles agglutination test (Japan have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays.

  17. Hepatitis B core antibody testing in Indian blood donors: A double-edged sword!

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    R N Makroo


    Full Text Available Background: Until lately, anti-HBc antibodies were considered an effective marker for occult Hepatitis B virus (HBV infection and have served their role in improving blood safety. But, with the development of advanced tests for HBV DNA detection, the role of anti-HBc in this regard stands uncertain. Materials and Methods: Anti-HBc and HBsAg ELISA and ID-NAT tests were run in parallel on donor blood samples between April 1, 2006 and December 31, 2010 at the Department of Transfusion Medicine, Indraprastha Apollo Hospitals, New Delhi. A positive ID-NAT was followed by Discriminatory NAT assay. Results: A total of 94 247 samples were tested with a total core positivity rate of 10.22%. We identified nearly 9.17% of donors who were reactive for anti-HBc and negative for HBsAg and HBV DNA. These are the donors who are potentially non-infectious and may be returned to the donor pool. Conclusion: Although anti HBc testing has a definite role in improving blood safety, centers that have incorporated NAT testing may not derive any additional benefit by performing anti-HBc testing, especially in resource-limited countries like ours.

  18. Proposed production test for reducing minimum downtime

    Energy Technology Data Exchange (ETDEWEB)

    Jaklevick, J.F.


    The object of the production test described in this report is to evaluate the operational aspects of a proposed method for reducing minimum downtime. The excess xenon poisoning, which occurs during the first 32--38 hours after the shutdown of a reactor from present equilibrium levels, will be partially overridden by a central enriched zone whose added reactivity contribution would be compensated during normal operation by means of poison splines.

  19. Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody.

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    Fatemeh Torkashvand

    Full Text Available Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44 cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.

  20. SSB peptide and DNA co-immunization induces inhibition of anti-dsDNA antibody production in rabbits

    Institute of Scientific and Technical Information of China (English)


    Background Patients with systemic lupus erythematosus often have various autoantibodies.The relationship between these antibodies is still poorly understood.The aim of the present study was to observe the anti-SSB antibody and anti-dsDNA antibody production profiles following immunization with synthetic SSB peptide alone,DNA alone or co-immunization with these two antigens.Methods SSB 214-225 aa peptide was synthesized by organic chemistry solid-phase peptide synthesis.Rabbits were immunized with the foliowing antigens:synthetic SSB peptide linked with keyhole limpet hemocyanin (KLH),DNA,SSB plus dsDNA,KLH and PBS.Antibodies were measured by ELISA.Histopathology and direct immufluorescence assays were also applied.Results Ainit-SSB and anti-dsDNA antibodies were produced following immunization with SSB peptide and DNA respectively.The level of SSB antibody in the co-immunization group was higher than that of the SSB peptide immunization group.The level of anti-dsDNA antibody in the co-immunization group was,however,lower than that in the DNA immunization group.Meanwhile,the level of anti-SSB antibody was higher than that of anti-DNA antibody in the co-immunization group.No morphological or immunological abnormalities were found in the heart,liver,kidney,spleen or skin tissues.Conclusion Inhibition of anti-dsDNA-antibody was induced by co-immunization with synthesized SSB peptide and DNA,which might explain,at least partly,the mild disease in some LE subsets associated with SSB antibody.

  1. Slant Hole Completion Test, Cozzette and Paludal production testing

    Energy Technology Data Exchange (ETDEWEB)

    Mann, R.L.; Malinowsky, M.S.


    The Slant Hole Completion Test has been successful in providing good technology transfer to the oil and gas industry. The gas-producing rate from the Cozzette horizontal open-hole interval was significantly greater than from offset vertical wells. The source of water production from the Cozzette is not presently understood. Diagnosis of the water production mechanism is vital to economic exploitation of the Cozzette gas resource using horizontal well technology. Stimulation in a high-angle cased and cemented wellbore, such as the Paludal 2, 3 and 4 intervals in SHCT No. 1, is more difficult because of high near-wellbore stress that results in higher treating pressures. Low gas production potential following the Paludal 2, 3 and 4 stimulation may result from damage to the natural fracture system resulting from casing cementing operations. Comparison of gas production rates in SHCT No. 1 and MWX No. 1 following similar stimulations in the Paludal 3 and 4 intervals, indicate there is no advantage to a hydraulic fracture treatment in a slant wellbore when compared to a vertical wellbore.

  2. Detection of leptospiral antibodies by microscopic agglutination test in north-east of Iran

    Institute of Scientific and Technical Information of China (English)

    Ehsanollah Sakhaee; Gholam Reza Abdollah pour


    Objective: To detect leptospiral antibodies by microscopic agglutination test (MAT) in north-east of Iran. Methods: This study was conducted to evaluate prevalence of human leptospiral infections by MAT, using six current reference strains of Leptospira interrogans in north-east of Iran. A total of 285 serum samples were collected from three north-east provinces of Iran, from December, 2009 to June, 2010. Results: Antibodies were detected at least against one serovar of Leptospira interrogans in 45 sera (15.79 %) among 285 samples at a dilution 1:100 or greater. Positive titers against more than one serovar were detected in 24 sera of the positive samples. Therefore, there were 75 positive reactions against different serovar of Leptospira interrogans. Positive titers were recorded against serovar icterohaemorrhagiae (31 samples), hardjo (26 samples), grippotyphosa (7 samples), pomona (5 samples), canicola (4 samples) and ballum (2 sample).Conclusions:In present study the most prevalent (Leptospira icterohaemorrhagiae) and the least prevalent (Leptospira ballum) serovar are different from previous studies. Maybe, species and prevalence of serovars change during the time in one area and between regions.

  3. The Anti-Acetylcholine Receptor Antibody Test in Suspected Ocular Myasthenia Gravis

    Directory of Open Access Journals (Sweden)

    Jung Jin Lee


    Full Text Available Aim. To estimate the clinical significance of anti-acetylcholine receptor antibody (anti-AChR-Ab levels in suspected ocular myasthenia gravis. Methods. In total, 144 patients complaining of fluctuating diplopia and ptosis were evaluated for serum levels of anti-acetylcholine receptor antibody and their medical charts were retrospectively reviewed. Subjects were classified into three groups: variable diplopia only, ptosis only, and both variable diplopia and ptosis. We investigated serum anti-AChR-Ab titer levels and performed thyroid autoantibody tests. Results. Patients’ chief complaints were diplopia (N=103, ptosis (N=12, and their concurrence (N=29. Abnormal anti-AChR-Ab was observed in 21 of 144 patients (14.1%. Between the three groups, mean age, number of seropositive patients, and mean anti-AChR-Ab level were not significantly different (P=0.224, 0.073, and 0.062, resp.. Overall, 27.5% of patients had abnormal thyroid autoantibodies. Conclusion. The sensitivity of anti-AChR-Ab was 14.1% in suspected ocular myasthenia gravis and seropositivity in myasthenia gravis patients showed a high correlation with the presence of thyroid autoantibodies.

  4. Evaluation of the LIAISON ANA screen assay for antinuclear antibody testing in autoimmune diseases. (United States)

    Ghillani, P; Rouquette, A M; Desgruelles, C; Hauguel, N; Le Pendeven, C; Piette, J C; Musset, L


    Antinuclear antibodies (ANA) are widely detected by immunofluorescence on HEp-2 cells in patients with connective tissue diseases and other pathological conditions. We evaluated the first-automated chemiluminescence immunoassay for the detection of ANA (LIAISON ANA screen, DiaSorin). This study was carried out simultaneously in two laboratories by testing 327 patient samples with clinically defined connective diseases, 273 routine samples for ANA screening, and 300 blood donors. A total of 268 out of 337 IIF-positive sera were positive with LIAISON ANA screen (79.5% of agreement) and 240 out of 263 IIF-negative sera were negative with LIAISON ANA screen (91.2% of agreement). After resolution of discrepant results, the concordance reached, respectively, 94.9% and 98.8%. The specificity was 99.3% and the sensitivity was 94%. Unlike results obtained by other ANA screening assays, we observed acceptable sensitivity and specificity. Despite the presence of HEp-2 cell extract, we failed to detect some antibodies as antinucleolar, antinuclear envelope, and antiproliferating cell nuclear antigen. This automated assay allows quick process to results and exhibits satisfactory sensitivity for the detection of the main ANA specificities of connective tissue diseases.

  5. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products. (United States)

    Gahoual, Rabah; Beck, Alain; Leize-Wagner, Emmanuelle; François, Yannis-Nicolas


    Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products.

  6. Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids. (United States)

    Kumar, Jaideep; Chaudhury, Ashok; Bera, Bidhan C; Kumar, Ritesh; Kumar, Rajender; Tatu, Utpal; Yadav, Suresh Chandra


    The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

  7. Model based adaptive control of a continuous capture process for monoclonal antibodies production. (United States)

    Steinebach, Fabian; Angarita, Monica; Karst, Daniel J; Müller-Späth, Thomas; Morbidelli, Massimo


    A two-column capture process for continuous processing of cell-culture supernatant is presented. Similar to other multicolumn processes, this process uses sequential countercurrent loading of the target compound in order maximize resin utilization and productivity for a given product yield. The process was designed using a novel mechanistic model for affinity capture, which takes both specific adsorption as well as transport through the resin beads into account. Simulations as well as experimental results for the capture of an IgG antibody are discussed. The model was able to predict the process performance in terms of yield, productivity and capacity utilization. Compared to continuous capture with two columns operated batch wise in parallel, a 2.5-fold higher capacity utilization was obtained for the same productivity and yield. This results in an equal improvement in product concentration and reduction of buffer consumption. The developed model was used not only for the process design and optimization but also for its online control. In particular, the unit operating conditions are changed in order to maintain high product yield while optimizing the process performance in terms of capacity utilization and buffer consumption also in the presence of changing upstream conditions and resin aging.

  8. Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products, and animal feed (United States)

    A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 µg/mL in raw milk, since the detection test line ...

  9. Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus. (United States)

    Alkahalaf, A N; Halvorson, D A; Saif, Y M


    Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

  10. Recombinant renewable polyclonal antibodies. (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


    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.

  11. [Effects of Celosia argentea and Cucurbita moschata extracts on anti-DNP IgE antibody production in mice]. (United States)

    Imaoka, K; Ushijima, H; Inouye, S; Takahashi, T; Kojima, Y


    We have already reported that the Perilla frutescens extract (PFE) suppressed anti-DNA IgE antibody production in mice. In this study, we prepared extracts of Celosia argentea L. (CAE) and Cucurbita moschata Duch (CME), which are Chinese herbal medicines like Perilla frutescens, and examined the effects on anti-DNP antibody responses in mice. To examine the effects of CAE & CME on primary antibody responses, CAE & CME were intraperitoneally injected the day before primary immunization of DNP-ovalbumin. Anti-DNP antibody production was markedly suppressed. Then, we examined the effects on secondary antibody responses. CEA & CME were injected only the day before secondary immunization. Anti-DNP IgE production was markedly suppressed, but IgG responses were not affected. It was also found that mitogenic activity occurred in CAE & CME dose dependently in vitro. These effects of CAE & CME were superior to that of PFE. These results suggest that CAE & CME may be more useful than PFE for the suppression of IgE antibody in certain allergic disorders.

  12. Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

    Directory of Open Access Journals (Sweden)

    M. A. Lauricella


    Full Text Available This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT and hemagglutination test (IHAT were standardized, and complement fixation test (CFT and direct agglutination test (DAT were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1 parasitemic dogs of two localities of endemic area (EA of Santiago del Estero province in Argentina (n = 134; (2 non-parasitemic dogs of the same area (n = 285; (3 dogs experimentally infected with T. cruzi in the patent period (n = 6; (4 non-infected dogs (n = 56 which were born in the city of Buenos Aires (BA, one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.

  13. Seroprevalence of bovine leptospiral antibodies by microscopic agglutination test in Southeast of Iran

    Institute of Scientific and Technical Information of China (English)

    Mohammad Khalili; Ehsanollah Sakhaee; Mohammad Reza Aflatoonian; Gholamreza Abdollahpour; Saeed Sattari Tabrizi; Elham Mohammadi Damaneh; Sajad Hossini-nasab


    Objective:To evaluate serological findings of bovine leptospirosis which is a zoonotic disease with worldwide distribution caused by Leptospira interrogans. Methods: One hundred and sixty seven sera were collected from 9 commercial dairy herds in jiroft suburbs, from July to October 2011. Microscopic agglutination test (MAT) was used to evaluates serological findings of bovine leptospirosis in Jiroft suburb dairy farms, Kerman province, Iran. Results:Antibodies were found by MAT at least against one serovar of Leptospira interrogans in 29 samples (17.36%) among 167 sera at a dilution 1:100 or higher, and Leptospira pomona was the most prevalent serovar. Positive titers against more than one serovar were detected in 6 sera of the positive samples. Conclusion:This study is the first report of leptospirosis in Southeast Iran and showed that Leptospira pomona was the most and Leptospira icterohaemorrhagiae the least prevalent serovars in Southeast Iran.

  14. Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

    Directory of Open Access Journals (Sweden)

    Ajda Biček


    Full Text Available Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of turkey, pheasant and peafowl. Chicken IgY light chain specific mAb 3E10 revealed the presence of common epitopes on immunoglobulins of turkey, pheasant and sparrow. Monoclonal antibody clone 1F5/3G2 was used to prepare horseradish peroxidase (HRP conjugate and immunoadsorbent column. Conjugated mAbs were demonstrated to be excellent secondary antibodies for diagnostics of certain infections in different avian species. Since they do not react with mammalian immunoglobulins using our mAbs as secondary antibodies in human serodiagnostics would minimize background staining that appears when using mouse detection system. In dot immunobinding assay (DIBA and immunoblot assay they recognized specific IgY antibodies against Mycoplasma synoviae, Mycoplasma gallisepticum and Newcastle disease virus in sera of infected or vaccinated birds. Immunoadsorption as a method for removal of IgY from samples in which Mycoplasma synoviae specific IgY was predominant immunoglobulin class enabled more exact demonstration of specific IgA and IgM antibodies. Herein we are presenting effective mAbs useful in diagnostics of avian and mammalian infections as well as in final steps of detection and purification of chicken antibodies and their subunits produced in vivo or in vitro as polyclonal, monoclonal or recombinant antibodies.

  15. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens. (United States)

    Geier, Christoph B; Piller, Alexander; Linder, Angela; Sauerwein, Kai M T; Eibl, Martha M; Wolf, Hermann M


    Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  16. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Directory of Open Access Journals (Sweden)

    Christoph B Geier

    Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  17. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies. (United States)

    Hu, Zhonglan; Chen, Zhenyu; Huang, Nongyu; Teng, Xiu; Zhang, Jun; Wang, Zhen; Wei, Xiaoqiong; Qin, Ke; Liu, Xiao; Wu, Xueping; Tang, Huan; Zhu, Xiaofeng; Cui, Kaijun; Li, Jiong


    Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.

  18. Wheat germ cell-free system-based production of hemagglutinin-neuraminidase protein of human parainfluenza virus type 3: generation and characterization of monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Satoko eMatsunaga


    Full Text Available Human parainfluenza virus 3 (HPIV3 commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of monoclonal antibodies against hemagglutinin-neuraminidase (HN of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we newly identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. This study provides the availability of our newly-developed MAbs as a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

  19. Antibody-independent Targeted Quantification of TMPRSS2-ERG Fusion Protein Products in Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    He, Jintang; Sun, Xuefei; Shi, Tujin; Schepmoes, Athena A.; Fillmore, Thomas L.; Petyuk, Vladislav A.; Xie, Fang; Zhao, Rui; Gritsenko, Marina A.; Yang, Feng; Kitabayashi, Naoki; Chae, Sung Suk; Rubin, Mark; Siddiqui, Javed; Wei, John; Chinnaiyan, Arul M.; Qian, Weijun; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Liu, Tao; Camp, David G.


    Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. The studies on TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies or an antibody-independent method that is sufficiently sensitive for detecting the truncated ERG protein products resulting from TMPRSS2-ERG gene fusions and alternative splicing. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays led to confident detection of 6 unique ERG peptides in either the TMPRSS2-ERG positive cell lines or tissues but not in the negative controls, indicating that ERG protein expression is highly correlated with TMPRSS2-ERG gene rearrangements. Significantly, our results demonstrated for the first time that at least two groups of ERG protein isoforms were simultaneously expressed at variable levels in TMPRSS2-ERG positive samples as evidenced by concomitant detection of two mutually exclusive peptides. Three peptides shared across almost all fusion protein products were determined to be the most abundant peptides, and hence can be used as “signature” peptides for detecting ERG overexpression resulting from TMPRSS2-ERG gene fusion. These PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products, thus improving our understanding of the role of TMPRSS2-ERG gene fusion in the biology of prostate cancer.

  20. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

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    Lilian Terezinha de Queiroz Leite


    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.Anticorpos monoclonais (AcM foram produzidos contra o extrato EDTA obtido de Leptospira interrogans, sorovar icterohaemorrhagiae. Pelo teste de precipitação foram caracterizados como IgM e IgG (IgGl e IgG2. A eletroforese em gel de poliacrilamida do extrato EDTA revelou diversas bandas quando corada pela prata. No "Western blot", as bandas em torno de 20 kDa reagiram com o AcM 47B4D6, foram oxidadas pelo periodato e não digeridas pela pronase, sugerindo que o determinante é de natureza carboidrato. O determinante reconhecido pelo AcM 47B4D6 estã localizado sob o envelope externo como revelado pela imunocitoquímica usando marcação com ouro coloidal. O AcM contra extrato EDTA do sorovar icterohaemorrahagiae não protegeu hamsters quando inoculados com lepstopira homóloga virulenta.

  1. Prebiotic and antimicrobials on performance, carcass characteristics, and antibody production in broilers

    Directory of Open Access Journals (Sweden)

    Maíra Fomentini


    Full Text Available ABSTRACT: To evaluate the effect of supplementation with mannan oligosaccharides, avilamycin and halquinol, alone or in combination, on the performance, carcass characteristics and antibody production in broilers (1-49 days old, male broiler chicks (n=1440; Cobb 500; one day old were housed and distributed into a completely randomized design into six treatments (eight replicates; 30 animals per pen. To produce the experimental diets, three types of performance enhancer additives were used. Halquinol (HAL, avilamycin (AVI and mannan oligosaccharides (MOS were included (alone or in combination in the basal diet (instead of corn starch. Effects of diet were observed on results of animal performance in the period 1-21 and 1-42 days old. Broilers fed with a diet without growth promoter showed lower weight gain in relation to those fed with diets with antimicrobials, MOS or a combination of them. In the period 1-49 days old, feed conversion increased in broilers fed with rations without promoter. At the end of the experimental period no influence of diets was observed on the carcass yield and cuts, and titles of specific antibodies to avian infectious bronchitis. The use of MOS and/or antimicrobials (AVI or HAL, alone or in combination, improves feed conversion of broilers reared until 49 days of age.

  2. A fully defined, fed-batch, recombinant NS0 culture process for monoclonal antibody production. (United States)

    Hermes, Paul A; Castro, Chris D


    To manufacture a glycoprotein, mammalian cells expressing the desired protein are often grown in fed-batch mode. Feeding an undefined, nonanimal hydrolysate helps the cells receive sufficient nutrition, but makes systems difficult to optimize. Even different lots of the same hydrolysate may have significant variability; furthermore, individual components may actually be detrimental to the cells. Switching to fully defined feeds could eliminate these issues. For monoclonal antibody (mAb) production by fed-batch NS0 cells, this article describes the replacement of a hydrolysate-based feed with a fully defined, animal-component-free feed system. The defined feed initially had 67 components, but additional experiments allowed a reduction to 25 components. The mAb titer is approximately 20% higher than in the undefined system, and the feed volume is circa 20% lower. The two systems generated antibodies with similar glycosylation profiles. Other benefits of the defined feed system include lower raw material costs, the ability to optimize key nutrient concentrations, greater confidence in raw material quality, and the elimination of potential, hydrolysate-associated endotoxin issues.

  3. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection. (United States)

    Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar


    Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

  4. Antinuclear antibody testing in pleural fluid for the diagnosis of lupus pleuritis. (United States)

    Porcel, J M; Ordi-Ros, J; Esquerda, A; Vives, M; Madroñero, A B; Bielsa, S; Vilardell-Tarrés, M; Light, R W


    We sought to determine whether measuring antinuclear antibodies (ANA) and their specificities [dsDNA, extractable nuclear antigens (ENA)] on pleural fluid may contribute to the differential diagnosis of pleural effusions. ANA were tested by indirect immunofluorescence on Hep-2 cells in the pleural fluid of 266 patients with effusions of different etiologies, including 15 lupus pleuritis. The cutoff value for diagnostic use was set at 1:160. Pleural fluid analysis of specific autoantibodies, such as anti-dsDNA and anti-ENA, was also performed if a positive ANA test was obtained. All patients with lupus pleurisy and 16 of 251 (6.4%) patients with pleural effusions secondary to other causes were ANA positive. Fifty-six percent of the positive ANAs in non-lupus pleural fluids were due to neoplasms. The pleural fluid ANA titers were low (pleuritis from non-lupus etiologies, the absence of pleural fluid anti-dsDNA or anti-ENA favored the latter. ANAs in pleural fluid provided no additional diagnostic information beyond that obtained by the measurement in serum and, therefore, these tests need not be routinely performed on pleural fluid samples. However, in patients with SLE and a pleural effusion of uncertain etiology, lack of ANAs or specific autoantibodies in pleural fluid argues against the diagnosis of lupus pleuritis.

  5. Evaluation of the indirect fluorescent antibody test for diagnosis of Babesia gibsoni infections in dogs. (United States)

    Yamane, I; Thomford, J W; Gardner, I A; Dubey, J P; Levy, M; Conrad, P A


    We determined the extent of the serologic cross-reactivity in the indirect fluorescent antibody (IFA) test for Babesia gibsoni, and the optimal cut-off titer for seropositivity in the test. The lowest titer to B gibsoni detected in a dog with naturally acquired clinical babesiosis was 1,280, and 7 of 12 dogs had titer between 10,240 and 20,480. Two experimentally infected normosplenic dogs developed high titer (40,960 to 81,920) to B gibsoni, and the same sera reacted in IFA tests for B canis (titer Neospora caninum (titer canis and T gondii had titer < or = 160 to B gibsoni. Dogs that were experimentally infected with N caninum had titer (80 to 10,240) to N caninum, but failed to have serologic reactivity to B gibsoni. Serologic titer of healthy dogs from Australia, a country where B gibsoni is not known to exist, was < or = 160 to B gibsoni. On the basis of these findings, a cut-off titer of 320 was considered to be appropriate for serodiagnosis of B gibsoni in dogs with clinical signs of babesiosis. A more conservative titer of 1,280 was established as the cut-off titer for seroepidemiologic studies based on relative costs and benefits of false-positive results and failure to isolate B gibsoni parasites after splenectomy and immunosuppression from a clinically normal dog with B gibsoni titer of 640.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures. (United States)

    Serrato, J Antonio; Palomares, Laura A; Meneses-Acosta, Angélica; Ramírez, Octavio T


    It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.

  7. The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach.

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    Scott R Fry


    Full Text Available BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1 has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6% and 96% (95% CI: 92.2% to 99.8 respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1% and 96.7% specificity (95% CI: 82.8% to 99.9% compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

  8. Entrapment of animal cells for the production of biomolecules such as monoclonal antibodies. (United States)

    Scheirer, W; Nilsson, K; Merten, O W; Katinger, H W; Mosbach, K


    An important problem in the production of monoclonal antibodies is the large-scale cultivation of hybridoma cells in vitro. Fragility of cells and suboptimal in vitro cultivation methods have led to poor results in larger scale production up to now. To lower the mechanical stress on the cells we tried to entrap the cells into microspheres made of polymer material. In addition to other materials, agarose as embedding medium was investigated and results with hybridoma and other, non anchorage-dependent cell lines are given. The conclusion of the results is that encapsulation of living cells is possible and entrapped cells remain viable and continue to produce the desired substance for at least several weeks. The substances are secreted through the polymer matrix. Handling of microspheres is shown to be easy and simple fermentation apparatus may be used for the production on a reliable technical scale. Some problems remain unsolved, such as the determination of viable cell count within the microspheres and cultivation in columns which seems to be the simplest form of continuous production process.

  9. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis. (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


    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.

  10. Production of Mouse Monoclonal Antibody against Morphine without Cross Reactivity with Heroin

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    S Kashaninan


    Conclusion: The study findings revealed that the produced antibody against morphine was comparable with other antibodies for specificity and affinity; therefore it is usable in design of diagnostic immunoassay in biologic fluids.

  11. Transgenic Production of an Anti HIV Antibody in the Barley Endosperm.

    Directory of Open Access Journals (Sweden)

    Goetz Hensel

    Full Text Available Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4 signal peptide and the endoplasmic reticulum (ER retention signal (SEKDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP, deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues, consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.

  12. Interference of infectious bursal disease virus on antibody production against Newcastle disease and infectious bronchitis virus

    Directory of Open Access Journals (Sweden)

    WM Cardoso


    Full Text Available This work has the objective of verifying the interference of infectious bursal disease virus in the antibody production against Newcastle disease virus and infectious bronchitis virus. The experiment was carried out with 640 day-old-chicks from a 42 weeks old hen flock. The birds were separated into eight experimental groups (n=80/group and were submitted to different combinations of vaccinations, with live vaccines, to Newcastle disease, avian infectious bronchitis, and infectious bursal disease with diverse combinations of days of vaccination. We verified that the utilization of polyvalent vaccinal programs have a different efficacy comparing to monovalent vaccinations when Newcastle disease, infectious bronchitis, and infectious bursal disease vaccinations are applied. This way, the use of vaccinations to infectious bursal disease in polyvalent vaccinal programs is desirable due to improvement of NDV response with the presence of IBV by the probable reduction of interference of IBV under NDV.

  13. A Comparative Study of Detection of Bordetella avium Antibodies in Turkeys by ELISA, SPAT, and AGID Test


    TÜRKYILMAZ, Süheyla; TÜRKYILMAZ, Kenan; KAYA, Osman


    The aims of this study were to develop a serum plate agglutination test (SPAT) antigen and agar gel immunodiffusion (AGID) test antigen for the serological detection of turkeys that have been exposed to Bordetella avium; to compare the sensitivity of commercial enzyme-linked immunosorbent assay (ELISA) with SPAT, and AGID test, and to survey B. avium antibodies in turkey flocks in Aydın, Turkey. For these purposes, serum samples collected from 300 turkeys were examined by ELISA, SPAT, and AGI...

  14. Perceived Empathy of Service Providers Mediates the Association between Perceived Discrimination and Behavioral Intention to Take Up HIV Antibody Testing Again among Men Who Have Sex with Men



    HIV antibody testing is a key measure of HIV prevention for men who have sex with men (MSM). The World Health Organization recommends sexually active and at-risk MSM to take up HIV antibody testing regularly. This study aimed to investigate the prevalence of behavioral intention to take up HIV antibody testing in the next six months among Hong Kong MSM who were ever-testers. An anonymous cross-sectional survey recruited 326 MSM who had taken up HIV antibody testing from gay-friendly venues an...

  15. Should we offer routine hepatitis C antibody testing in men who have sex with men?

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    Christopher Ward


    Full Text Available Introduction: There has been a significant rise in the number of HIV positive men who have sex with men (MSM co-infected with hepatitis C (HCV. Most infections are thought to occur through high risk sexual practices, exacerbated by drug use. Previous data has suggested no need for routine screening in HIV negative MSM. We looked at HCV antibody testing and HCV risk assessment in all MSM clinic attenders as part of a Public Health England initiative. Materials and Methods: Routine HCV antibody testing was offered to all MSM attending a large inner city sexual health clinic from April to June 2014. Patients were asked to fill in a questionnaire assessing HCV risk. Demographic data, HIV status and STI results were collected and compared. Results: We collected 471 HCV risk assessment questionnaires during the eight-week period. The median age was 34 (range 18–71 and 403 (85.6% were White British. Ten (2.1% patients were known to be HCV positive, of which 3 were on treatment and 5 (1.1% had cleared HCV. One hundred and forty-nine (31.6% patients were HIV negative, 254 (53.9% were HIV positive and 68 (14.5% had unknown HIV status at time of clinic visit. In the last three months 151 (32.1% reported unprotected receptive anal intercourse, 58 (12.3% reported group sex, 11 (2.3% reported receptive fisting and 32 (6.8% reported more than 10 partners. Eighty-seven (18.5% patients had shared notes/straws to snort drugs and 29 (6.2% reported injecting drugs or slamming. One hundred and forty-two (30.0% patients reported recreational drug use in the last 12 months, with cocaine, methadrone and ketamine most popular. One hundred and fifteen (24.4% patients reported sex under the influence of recreational drugs. There were no statistical differences between HIV positive and HIV negative patients in their risk, sexual behaviour and drug use. STI screens were performed on 269 patients with nine (3.3% new HIV diagnoses, four (1.5% early syphilis, and 28 (10


    Institute of Scientific and Technical Information of China (English)


    This paper analyses the principle of microwave humidity testing. According to the problems in the production procedure of ceramic products, a microwave humidity testing system is designed and analyzed for its advantages. Furthermore, the system has been applied to the production line that produces ceramic products and the testing results are also satisfying.

  17. Chikungunya virus RNA and antibody testing at a National Reference Laboratory since the emergence of Chikungunya virus in the Americas. (United States)

    Prince, Harry E; Seaton, Brent L; Matud, Jose L; Batterman, Hollis J


    Since first reported in the Americas in December 2013, chikungunya virus (CHIKV) infections have been documented in travelers returning from the Caribbean, with many cases identified by CHIKV antibody and/or RNA testing at our laboratory. We used our large data set to characterize the relationship between antibody titers and RNA detection and to estimate IgM persistence. CHIKV RNA was measured by nucleic acid amplification and CHIKV IgG/IgM by indirect immunofluorescence. Of the 1,306 samples submitted for RNA testing in January through September 2014, 393 (30%) were positive; for 166 RNA-positive samples, CHIKV antibody testing was also ordered, and 84% were antibody negative. Of the 6,971 sera submitted for antibody testing in January through September 2014, 1,811 (26%) were IgM positive; 1,461 IgM positives (81%) were also IgG positive. The relationship between the CHIKV antibody titers and RNA detection was evaluated using 376 IgM-positive samples (138 with RNA testing ordered and 238 deidentified and tested for RNA). RNA detection showed no significant association with the IgM titer but was inversely related to the IgG titer; 63% of the IgG negative sera were RNA positive, compared to 36% of sera with low IgG titers (1:10 to 1:80) and 16% with IgG titers of ≥1:160. Using second-sample results from 62 seroconverters, we estimated that CHIKV IgM persists for 110 days (95% confidence interval, 78 to 150 days) after the initial antibody-negative sample. These findings indicate that (i) RNA detection is more sensitive than antibody detection early in CHIKV infection, (ii) in the absence of RNA results, the IgG titer of the IgM-positive samples may be a useful surrogate for viremia, and (iii) CHIKV IgM persists for approximately 4 months after symptom onset.

  18. Rapid identification of vibrio-cholerae O1 by coaglutination test using mono-specifis antibody

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    Bazargan SA


    Full Text Available In our investigation, rabbit hyper-immune serum to V.cholerae ogawa was absorbed with V.cholerae inaba whole-cells and vice versa. Applying ammonium sulphate precipitation method, mono-specific g globulins were purified and concentrated from the absorbed whole serum. These antibodies were fixed on staphylococcus cowan 1 NCTC-8325 whole-cells, using different chemical fixatives. It was observed that maximum fixation of g globulin to protein-A was achieved by 1-propanol 50% at 3 hours, which revealed through single radial immuno-diffusion techniqe. The rectal swab samples were cultured in an enrichment bile-peptons broth. After 5 hours 37°C while agitations, one drop of each sample was mixed with one drop of vibrio-cholerae bivalent mono-specific coagglutination reagent (VBCR. The results were read after 2 to 3 minutes. Finally though statistical analysis sensitivity and specificity of coagglutination test were calculated to be 95.1% and 99.2% respectively, when compared to positive & negative controls and conventional culture methods. Using VBCR, coagglutination test can be therefore considered as a simple, reliable and rapid method to detect V.cholerae O1 in the stool of patients in endemic area and less equipped laboratories

  19. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    Energy Technology Data Exchange (ETDEWEB)

    Hovi, T.; Roivainen, M.


    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  20. Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum. (United States)

    Talbot, F; Robreau, G; Gueguen, F; Malcoste, R


    Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.

  1. Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes. (United States)

    Pinkert, C A; Manz, J; Linton, P J; Klinman, N R; Storb, U


    The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

  2. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production. (United States)

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng


    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

  3. Evaluation of bovine coronavirus antibody levels, virus shedding, and respiratory disease incidence throughout the beef cattle production cycle (United States)

    Objective- Determine how levels of serum antibody to bovine coronavirus (BCV) are related to virus shedding patterns and respiratory disease incidence in beef calves at various production stages. Animals- 890 crossbred beef calves from four separately managed herds at the U.S. Meat Animal Research C...


    Institute of Scientific and Technical Information of China (English)


    Objective To investigate the relationship bet wee n cytomegalovirus (CMV) infection and the production of anticardiolipin antibody (ACA) in renal transplant recipients.Methods Polymerase c hain reaction (PCR) was used qualitat ively for detection of CMV-DNA in 146 renal transplant recipients.Meanwhile,enz yme-linked immunosorbent assay (ELISA) was used for detection of ACA-IgG in bl ood serum samples from these recipients and 32 healthy individuals. Results The ACA positive rate was 17.1% among the 146 ren al transplant recipients,and that of the control group was 6.3%.There was no sig nificant difference.However,the ACA positive rate of the renal transplant recipi ents infected with CMV was 31.2%.It was clearly higher than that of those with n o infection of CMV and that of the control group (P<0.005). Con clusion The production of ACA was closely related to CMV infection.It m ight be one of the factors of chronic angiopathy of the transpl anted kidney due to CMV infection.

  5. Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages. (United States)

    Franco, D de Lima; Nascimento, R C; Ferreira, K S; Almeida, S R


    Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1β. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

  6. Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer. (United States)

    He, Jintang; Sun, Xuefei; Shi, Tujin; Schepmoes, Athena A; Fillmore, Thomas L; Petyuk, Vladislav A; Xie, Fang; Zhao, Rui; Gritsenko, Marina A; Yang, Feng; Kitabayashi, Naoki; Chae, Sung-Suk; Rubin, Mark A; Siddiqui, Javed; Wei, John T; Chinnaiyan, Arul M; Qian, Wei-Jun; Smith, Richard D; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D; Liu, Tao; Camp, David G


    Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing "signature" peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.

  7. Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis. (United States)

    Wang, Xin; Lu, Rui; Liu, Qiao-Feng; Chen, Jian-Ping; Deng, Qiang; Zhang, Ya-Lou; Zhang, Bing-Hua; Xu, Jia-Nan; Sun, Lei; Niu, Qin-Wang; Liang, Quan-Zeng


    Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

  8. Evaluation of Anti-Nuclear antibody test results in clinical practice

    Directory of Open Access Journals (Sweden)

    Nevreste Çelikbilek


    Full Text Available Objective: Aim of this study is to evaluate anti-nuclear antibody (ANA test results obtained between 2009 and 2011. Methods: Of a totally 5068 cases tested for ANA by indirect immunofluorescence method (IIFA, randomly chosen 982 ANA-positive cases were reviewed in terms of gender, level and pattern of fluorescence, anti-dsDNA (anti-double stranded DNA and anti-extractable nuclear antigen (ENA profile. Anti-dsDNA levels and anti-ENA profiles were determined by enzyme linked immune assay (ELISA and immune-blotting (IB, respectively. Results: Sex distribution of ANA positive patients was determined as 756 (77% females and 226 (23% males. Fifty per cent of the cases were from rheumatology department, 20% from gastroenterology and 30% from other units. Fluorescence levels were considered borderline or weak positive in 62.6% of the samples. The most frequent patterns were homogeneous (23%, speckled (22%, homogeneous-speckled (15.5% and nucleolar (13.5%. Anti-dsDNA were studied in 759 ANA positive patients and 66 (8.7% samples were found positive, being 44 of them (68.8% with homogeneous pattern and the rest with speckled, nucleolar, nuclear dots, centromeric or midbody patterns. Totally 131 (31.6% of 414 samples studied for anti-ENA profile were found positive. The first four frequent profiles were SSA (34.4%, SSA-SSB (16.8%, Scl70 (16% and Sm/RNP (9.2%. Conclusion: Our results are similar with the current related literature. It is known that autoantibodies can be detectable before clinical symptoms being apparent, especially in SLE. Therefore, borderline or weak fluorescence levels should also be reported and the patients having them should be followed-up carefully. J Microbiol Infect Dis 2015;5(2: 63-68

  9. 46 CFR 57.01-1 - Qualifications and production tests. (United States)


    ... production tests for all types of manual and machine arc and gas welding and brazing processes. (b) (Modifies... 46 Shipping 2 2010-10-01 2010-10-01 false Qualifications and production tests. 57.01-1 Section 57... BRAZING Scope § 57.01-1 Qualifications and production tests. (a) (Replaces QW 101 and QB 101.)...

  10. Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

    Institute of Scientific and Technical Information of China (English)

    Wan-gang GU; Jun-fa YUAN; Ge-lin XU; Li-juan LI; Ni LIU; Cong ZHANG; Jian-hong ZHANG; Zheng-li SHI


    BALB/c mice were immunized with purified White spot syndrome virus (WSSV).Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E.coll in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish.Westernblot suggested that all these monoclonal antibodies were against the conformational structure of VP28.The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling.These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.

  11. Product Sterility Testing . . . To Test or Not to Test? That Is the Question. (United States)

    Daniell, Elaine; Bryans, Trabue; Darnell, Kimbrell; Hansen, Joyce; Hitchins, Victoria M; Saavedra, Manuel


    The applications for sterility testing in the validation and routine control of sterilization of medical devices have changed dramatically over the years. As the definition of sterility assurance has evolved, so has the state of the science associated with product sterility testing. Historically, product sterility testing has been applied to such things as sterilization validation, sterilization lot release, packaging qualification, aseptic processing qualification, and determination of shelf life for the packaged medical device. In most of these cases, however, the results obtained from performing sterility testing on products do not provide the desired confirmation and assurance. Utilizing sterility testing on fully processed finished product is not appropriate for determination of sterilization process effectiveness, sterility assurance level, package integrity, or shelf life. The industry has developed more robust methods for validation of these applications to assure sterility and package performance. This article outlines the appropriate applications for sterility testing and highlight the applications currently in use that have significant limitations within the results and introduce undesirable risk to the validity of the data.

  12. Post-transplant donor-specific antibody production and graft outcome in kidney transplantation: results of sixteen-year monitoring by flow cytometry. (United States)

    Piazza, Antonina; Poggi, Elvira; Ozzella, Giuseppina; Borrelli, Laura; Scornajenghi, Alessandra; Iaria, Giuseppe; Tisone, Giuseppe; Adorno, Domenico


    Our data show that monitoring by sensitive flow cytometric techniques of the de novo production of anti-HLA antibodies in patients receiving kidney transplantation is a useful and noninvasive tool to identify the onset of an immune response towards the graft before any clinical manifestation of antibody-mediated graft injury. Consequently prospective posttransplant monitoring of anti-HLA donor-directed antibodies may offer the chance to realize an effective clinical intervention in order to prevent graft dysfunction and to prolong graft survival. The long follow-up period of the study allowed us to demonstrate a very low graft survival rate in patients who developed donor-specific HLA antibodies in comparison with patients who did not have antibodies, thus confirming the "humoral theory of transplantation". The posttransplant production of anti-HLA antibodies can predict not only graft failure but also chronic dysfunction of the graft. Moreover, our findings suggest that graft survival is influenced by the epitope- and locus-specificity of anti-HLA donor-directed antibodies. The interval between antibody appearance and loss of graft function was short in some patients but reached several years in others. Moreover, some patients showed consistent production of antibodies for many years and an uneventful clinical status. These findings suggest a mechanism of graft "accommodation" or the production of "harmless" antibodies. Immunosuppressive drug combinations able to inhibit T and B cell activation are useful tools to prevent the humoral immune response against graft and consequently to prolong graft survival.

  13. Specific Antibody Production by Blood B Cells is Retained in Late Stage Drug-naïve HIV-infected Africans

    Directory of Open Access Journals (Sweden)

    Lydie Béniguel


    Full Text Available Unseparated peripheral blood mononuclear cells (PBMCs obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL, were cultured in vitro and tested for their ability to produce antibodies (Abs reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10 but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp 160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations.

  14. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production. (United States)

    Strauss, Daniel M; Cano, Tony; Cai, Nick; Delucchi, Heather; Plancarte, Magdalena; Coleman, Daniel; Blank, Gregory S; Chen, Qi; Yang, Bin


    The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained.

  15. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. (United States)

    Adler, B; Faine, S; Gordon, L M


    The enzyme-liked immunosorbent assay (ELISA) was compared with the standard microscopic agglutination test (MAT) as a method for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. Peak antibody levels detected by the 2 tests occurred at different times following experimental infection of sheep. In serums from flocks of sheep with naturally acquired infection there was a 95% correlation between MAT and ELISA with respect to the presence or absence of antibody to serovar hardjo, although the levels of correlation of the titres of the 2 tests was low. The 2 tests appeared to measure different antigen-antibody systems. The ELISA would be a useful test for screening large numbers of serums for antibodies to L. interrogans serovar hardjo.

  16. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico


    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.O método de imunofluorescência direta (DIFMA, com anticorpos monoclonais gênero- específicos para Leishmania, foi avaliado na rotina diagnostica da leishmaniose cutânea no Equador. O método foi comparado com técnicas diagnosticas de rotina: o esfregaço, a cultura e o exame histopatolôgico. As amostras para o diagnóstico foram obtidas de um total de 90 lesões cutâneas ativas, de doentes das ãreas do Equador, endêmicas para leishmaniose cutânea. O DIFMA foi positivo em todas as lesões, com resultados significativamente superior aos métodos diagnósticos de rotina, isolado ou em combinação. A sensibilidade do DFIMA não diminui em lesões crônicas. O método mostra-se muito útil no diagnóstico de leishmaniose cutânea, pela sua sensibilidade, rapidez e facilidade de execução.

  17. 49 CFR 178.33b-8 - Production tests. (United States)


    ... 49 Transportation 2 2010-10-01 2010-10-01 false Production tests. 178.33b-8 Section 178.33b-8... Specifications for Inside Containers, and Linings § 178.33b-8 Production tests. (a) Burst Testing. (1) One out of... if the test container shall fail, the lot shall be rejected or ten additional containers may...

  18. Significance of serum antibody test for toxocariasis in healthy healthcare examinees with eosinophilia in Seoul and Gyeongsangnam-do, Korea. (United States)

    Kim, Hong Seok; Jin, Yan; Choi, Min-Ho; Kim, Jae-Hwan; Lee, Young Ha; Yoon, Cheong Ha; Hwang, Eui-Hyuk; Kang, Hun; Ahn, Sang-Yong; Kim, Gi Jin; Hong, Sung-Tae


    There have been numerous reports on the relationship between eosinophilia and toxocariasis. The present study investigated seropositive rates of toxocariasis among healthy people with or without eosinophilia in urban and rural areas, and assessed risk factors for positive antibody test. A total of 610 healthy people, who visited health check-up (Medicheck®, Korea Association of Health Promotion), 310 from Seoul and 300 from Gyeongsangnam-do, were subjected for this study. Their serum samples were tested by ELISA with the crude antigen of Toxocara canis larvae. Cross-reactions with other tissue invading helminth antigens were also investigated. Total antibody positive rate of toxocariasis was 8.7% of the 610 subjects. When the subjects were grouped into 3 by their eosinophil counts, the antibody positive rates significantly differed by the groups; 5.9% (18/306) in the group500/µL (P=0.028). A total of 22 serum samples cross-reacted with other tissue-invading helminth antigens. A questionnaire analysis recognized drinking alcohol and smoking as significant risk factors of toxocariasis. In conclusion, toxocariasis antibody positive rate is correlated with eosinophil counts. It is recommended that healthy subjects with eosinophilia by routine health examination and risk factors undergo Toxocara serology by multiantigen ELISA to investigate etiology.

  19. Production of anti-fullerene C60 polyclonal antibodies and study of their interaction with a conjugated form of fullerene (United States)

    Hendrickson, O. D.; Fedyunina, N. S.; Martianov, A. A.; Zherdev, A. V.; Dzantiev, B. B.


    The aim of this study was to produce anti-fullerene C60 antibodies for the development of detection systems for fullerene C60 derivatives. To produce anti-fullerene C60 antibodies, conjugates of the fullerene C60 carboxylic derivative with thyroglobulin, soybean trypsin inhibitor, and bovine serum albumin were synthesized by carbodiimide activation and characterized. Immunization of rabbits by the conjugates led to the production of polyclonal anti-fullerene antibodies. The specificity of the immune response to fullerene was investigated. Indirect competitive immunoenzyme assay was developed for the determination of conjugated fullerene with detection limits of 0.04 ng/mL (calculated for coupled C60) and 0.4 ng/mL (accordingly to total fullerene-protein concentration).

  20. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.


    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms a

  1. The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)


    Those antibodies elicited by different tether site for attachment to carrier protein have different specificity. Herein we reported that a monoclonal antibody against morphine with high specificity and affinity was successfully produced by using different linkers to couple to different carrier proteins.

  2. Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

    DEFF Research Database (Denmark)

    Pedersen, Thomas Skamris; Tian, Xinsheng; Thorolfsson, Matthias;


    PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregat...

  3. Chemo-enzymatic production of O-glycopeptides for the detection of serum glycopeptide antibodies

    DEFF Research Database (Denmark)

    Nøstdal, Alexander; Wandall, Hans H


    Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protocol...

  4. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms. (United States)

    Maragos, C M


    Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2β or Ab2γ). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin.

  5. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini


    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  6. Monoclonal antibodies in animal production; their use in diagnostics and passive immunization.

    NARCIS (Netherlands)

    Booman, P.


    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal

  7. A Novel Scheme for Production of Polyclonal Antibody against Estrogenic Bisphenols

    Institute of Scientific and Technical Information of China (English)


    A polyclonal antibody against the currently concerned estrogenic bisphcnol compoundswas produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used tosynthesize the complete antigen in which the characteristic bisphenol structure was exposed to thelargest extent. The produced polyclonal antibody showed high specificity and affinity forbisphenol A.

  8. Antibody screening tests variably overestimate the prevalence of hepatitis C virus infection among HIV-infected adults in Ghana. (United States)

    King, S; Adjei-Asante, K; Appiah, L; Adinku, D; Beloukas, A; Atkins, M; Sarfo, S F; Chadwick, D; Phillips, R O; Geretti, A M


    HIV coinfection with HCV has been poorly studied in sub-Saharan Africa, and the reliability of available seroprevalence estimates remains uncertain. The study aim was to determine HCV RNA prevalence in HIV-infected subjects receiving care in Kumasi, Ghana, and relate the findings to HCV antibody detection. From a population of 1520 HIV-infected adults, all HBsAg-positive subjects (n = 236) and a random subset of HBsAg-negative subject (n = 172) were screened for HCV RNA using pooled plasma; positive samples were genotyped by core and NS5B sequencing. HCV antibodies were detected by three commercial screening assays and confirmed by the line immunoassay. HCV RNA was detected in 4/408 subjects (1.0%, 95% confidence interval 0.0-1.9%), comprising 3/236 (1.3%; 0.0-2.8%) HBsAg-positive and 1/172 (0.6%; 0.0-1.8%) HBsAg-negative subjects. HCV RNA-positive subjects showed reactivity in all three antibody screening assays. Among HCV RNA-negative subjects, 5/67 (7.5%), 5/67 (7.5%) and 19/67 (28.4%) showed antibody reactivity by each screening assay, respectively, including two (3.0%) with reactivity by all three assays. Only one sample (1.5%) had confirmed antibody reactivity by line immunoassay indicating past HCV infection. HCV-positive subjects (three males, two females) were aged 30-46 years, by questionnaire-based interview reported surgical procedures and blood transfusion as risk factors for infection. HCV genotypes were 2 (subtypes 2j, 2l, 2k/unassigned) and 1 (subtype unassigned). Without further testing, HCV antibody screening assays variably overestimated HCV prevalence among HIV-infected subjects in Ghana. These findings inform the interpretation of previous seroprevalence estimates based upon screening assays alone.

  9. Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus

    Directory of Open Access Journals (Sweden)

    Zhang Shunchuan


    Full Text Available Abstract Duck virus enteritis (DVE is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.

  10. Antibody-mediated activation of FGFR1 induces FGF23 production and hypophosphatemia.

    Directory of Open Access Journals (Sweden)

    Ai-Luen Wu

    Full Text Available The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23 controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.

  11. Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests

    Directory of Open Access Journals (Sweden)

    Sofie Wallerström


    Full Text Available Introduction: Avian influenza viruses circulate in bird populations, and it is important to maintain and uphold our knowledge of the viral strains that are currently of interest in this context. Here, we describe the use of hemagglutinin-pseudotype retroviruses based on highly pathogenic influenza viruses for the screening of avian sera for influenza A antibodies. Our aim was also to determine whether the pseudovirus neutralization tests that we assessed were sensitive and simple to use compared to the traditional methods, including hemagglutination inhibition assays and microneutralization tests. Material and methods: H5 and H7 pseudovirus neutralization tests were evaluated by using serum from infected rabbits. Subsequently, the assays were further investigated using a panel of serum samples from avian species. The panel contained samples that were seropositive for five different hemagglutinin subtypes as well as influenza A seronegative samples. Results and discussion: The results suggest that the pseudovirus neutralization test is an alternative to hemagglutination inhibition assays, as we observed comparable titers to those of both standard microneutralizations assays as well as hemagglutinin inhibition assays. When evaluated by a panel of avian sera, the method also showed its capability to recognize antibodies directed toward low-pathogenic H5 and H7. Hence, we conclude that it is possible to use pseudoviruses based on highly pathogenic avian influenza viruses to screen avian sera for antibodies directed against influenza A subtypes H5 and H7.

  12. [Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies]. (United States)

    Krivitskaia, V Z; Sirotkin, A K; Samoĭlovich, M P; Sominina, A A


    Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.

  13. 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


    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.

  14. Toxicity Tests Applied to the Biocidal Products


    Karabay Yavaşoğlu, N.Ülkü


    Biocides are defined as chemical substances used to suppress, destroy, deter, render harmless, or exert a controlling effect on any harmful organism to human or animal health, or that cause damage to natural or manufactured materials. Biocidal products (BPs) containing biocides are disinfectants, products related to human and veterinary hygiene, products used for pests such as insects, rodents etc., repellents and industrial chemicals like anti-fouling paints for ship and material preservativ...

  15. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Haustraete Jurgen


    Full Text Available Abstract Background Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. Results Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. Conclusion Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression

  16. Detection of Schistosoma mansoni Antibodies in a Low-Endemicity Area Using Indirect Immunofluorescence and Circumoval Precipitin Test (United States)

    Carvalho do Espírito-Santo, Maria Cristina; Pinto, Pedro Luiz; Gargioni, Cybele; Viviana Alvarado-Mora, Monica; Pagliusi Castilho, Vera Lúcia; Pinho, João Ranato Rebello; de Albuquerque Luna, Expedito José; Borges Gryschek, Ronaldo Cesar


    Parasitological diagnostic methods for schistosomiasis lack sensitivity, especially in regions of low endemicity. The objective of this study was to determine the prevalence of Schistosoma mansoni infections by antibody detection using the indirect immunofluorescence assay (IFA-IgM) and circumoval precipitin test (COPT). Serum samples of 572 individuals were randomly selected. The IFA-IgM and COPT were used to detect anti-S. mansoni antibodies. Of the patients studied, 15.9% (N = 91) were IFA-IgM positive and 5.1% (N = 29) had COPT reactions (P < 0.001 by McNemar's test). Immunodiagnostic techniques showed higher infection prevalence than had been previously estimated. This study suggests that combined use of these diagnostic tools could be useful for the diagnosis of schistosomiasis in epidemiological studies in areas of low endemicity. PMID:24639303

  17. Development and validation of a point-of-care test for detecting hantavirus antibodies in human and rodent samples. (United States)

    Koishi, Andrea Cristine; Aoki, Mateus Nóbrega; Jorge, Taissa Ricciardi; Suzukawa, Andréia Akemi; Zanluca, Camila; Levis, Silvana; Duarte Dos Santos, Claudia Nunes


    Hantaviruses are etiologic agents of a zoonotic disease transmitted mainly from wild rodents to humans, causing Hemorrhagic Fever with Renal Syndrome in Eurasia and the Hantavirus Cardiopulmonary Syndrome in the Americas (HCPS), reaching a lethality rate of 40% in Brazil. Hantavirus diagnostic and seroprevalence are often based on the presence of IgM and IgG antibodies against the virus. Here we propose a rapid test assay able to identify hantavirus antibodies with sensibility and specificity similar to ELISA assays. We analyzed five groups of samples, including healthy human population and small mammals of endemic areas, suspected cases of HCPS, patients with non-related infections and a serum panel from a different geographical region. The test presented good rates of sensibility (87-100%) and specificity (97-100%) for all groups, being a promising tool suitable for both rodent and human hantavirus epidemiological surveys.

  18. Protective levels of canine distemper virus antibody in an urban dog population using plaque reduction neutralization test

    Directory of Open Access Journals (Sweden)

    O.I. Oyedele


    Full Text Available Blood samples from 50 dogs were collected at three veterinary clinics in Ibadan and Abuja, Nigeria and the serum from each sample was evaluated serologically for neutralizing antibodies against canine distemper virus (CDV by the highly sensitive plaque reduction (PRN neutralization assay. Thirteen dogs had plaque reduction neutralization titres of 0-100, seven had titres of 100-1 000 while 30 had titres ranging from 1 000-6 000. The PRN titres of vaccinated dogs were found to be significantly higher than unvaccinated dogs. The widespread use of the highly reproducible PRN test for the evaluation of antibody response to CDV may be very important in the generation of international CDV positive serum standards that should help to improve pre-and post-vaccination testing of dogs worldwide.

  19. An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

    Directory of Open Access Journals (Sweden)

    Elizabeth Abrahams-Sandi


    Full Text Available Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre > 16 was identified as the diagnostic threshold with the best performance, providing 93.7% sensitivity and 84.6% specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.

  20. A rapid latex agglutination test for the detection of anti-cysticercus antibodies in cerebrospinal fluid (CSF

    Directory of Open Access Journals (Sweden)

    ROCHA Sérgio M.


    Full Text Available Simple and rapid latex-based diagnostic tests have been used for detecting specific antigens or antibodies in several diseases. In this article, we present the preliminary results obtained with a latex agglutination test (LAT for diagnosing neurocysticercosis by detection of antibodies in CSF. A total of 43 CSF samples were assayed by the LAT: 19 CSF samples from patients with neurocysticercosis and 24 CSF samples from patients with other neurologic disorders (neurosyphilis, n = 8; neurotoxoplasmosis, n = 3; viral meningitis, n = 4, chronic headache, n = 9. The LAT exhibited 89.5% sensitivity and 75% specificity. The use of LAT seems to be an additional approach for the screening of neurocysticercosis with advantage of simplicity and rapidity. Further studies could be performed using purified antigens and serum samples.

  1. Lyme disease antibody (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  2. Relevance of microbial finished product testing in food safety management

    NARCIS (Netherlands)

    Zwietering, M.H.; Jacxsens, L.; Membre, J.M.; Nauta, M.; Peterz, M.


    Management of microbiological food safety is largely based on good design of processes, products and procedures. Finished product testing may be considered as a control measure at the end of the production process. However, testing gives only very limited information on the safety status of a food.

  3. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva


    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  4. Mechanistic modeling of ion-exchange process chromatography of charge variants of monoclonal antibody products. (United States)

    Kumar, Vijesh; Leweke, Samuel; von Lieres, Eric; Rathore, Anurag S


    Ion-exchange chromatography (IEX) is universally accepted as the optimal method for achieving process scale separation of charge variants of a monoclonal antibody (mAb) therapeutic. These variants are closely related to the product and a baseline separation is rarely achieved. The general practice is to fractionate the eluate from the IEX column, analyze the fractions and then pool the desired fractions to obtain the targeted composition of variants. This is, however, a very cumbersome and time consuming exercise. A mechanistic model that is capable of simulating the peak profile will be a much more elegant and effective way to make a decision on the pooling strategy. This paper proposes a mechanistic model, based on the general rate model, to predict elution peak profile for separation of the main product from its variants. The proposed approach uses inverse fit of process scale chromatogram for estimation of model parameters using the initial values that are obtained from theoretical correlations. The packed bed column has been modeled along with the chromatographic system consisting of the mixer, tubing and detectors as a series of dispersed plug flow and continuous stirred tank reactors. The model uses loading ranges starting at 25% to a maximum of 70% of the loading capacity and hence is applicable to process scale separations. Langmuir model has been extended to include the effects of salt concentration and temperature on the model parameters. The extended Langmuir model that has been proposed uses one less parameter than the SMA model and this results in a significant ease of estimating the model parameters from inverse fitting. The proposed model has been validated with experimental data and has been shown to successfully predict peak profile for a range of load capacities (15-28mg/mL), gradient lengths (10-30CV), bed heights (6-20cm), and for three different resins with good accuracy (as measured by estimation of residuals). The model has been also

  5. Synthesis and Characterization of Hapten-Protein Conjugates for Antibody Production against Cyanogenic Glycosides. (United States)

    Bolarinwa, Islamiyat Folashade


    Consumption of cyanogenic plants can cause serious health problems for humans. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of cyanide poisoning from the consumption of improperly processed cyanogenic plants. Hapten-protein conjugates were synthesized with amygdalin and linamarin by using a novel approach. Polyclonal antibodies were generated by immunizing four New Zealand White rabbits with synthesized amygdalin-bovine serum albumin and linamarin-bovine serum albumin immunogen. This is the first time an antibody was produced against linamarin. Antibody titer curves were obtained from all the four rabbits by using a noncompetitive enzyme-linked immunosorbent assay. High antibody titer was obtained at dilutions greater than 1:50,000 from both immunogens. This new method is an important step forward in preventing ingestion of toxic cyanogenic glycosides.

  6. Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa). (United States)

    Boadella, Mariana; Lyashchenko, Konstantin; Greenwald, Reena; Esfandiari, Javan; Jaroso, Raquel; Carta, Tania; Garrido, Joseba M; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian


    New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or cross-reacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.

  7. Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy


    Maciej Żaczek; Marzanna Łusiak-Szelachowska; Ewa Jończyk-Matysiak; Beata Weber-Dąbrowska; Ryszard Międzybrodzki; Barbara Owczarek; Agnieszka Kopciuch; Wojciech Fortuna; Paweł Rogóż; Andrzej Górski


    In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties towards applied phages (K rate). Among 20 examined patients receiving...

  8. A new rapid diagnostic test for detection of anti-Schistosoma mansoni and anti-Schistosoma haematobium antibodies

    Directory of Open Access Journals (Sweden)

    Coulibaly Jean T


    Full Text Available Abstract Background Parasitological methods are widely used for the diagnosis of schistosomiasis. However, they are insensitive, particularly in areas of low endemicity, and labour-intensive. Immunoassays based on detection of anti-schistosome antibodies have the merit of high sensitivity and recently a rapid diagnostic test (RDT, incorporating Schistosoma mansoni cercarial transformation fluid (SmCTF for detection of anti-schistosome antibodies in blood has been developed. Here, we assessed the diagnostic performance of the SmCTF-RDT for S. mansoni and S. haematobium infections by comparing it with microscopy for egg detection. Methods A cross-sectional survey was carried out in Azaguié, south Côte d’Ivoire. 118 pre-school-aged children submitted two stool and two urine samples, which were subjected to the Kato-Katz and urine filtration methods for the detection of S. mansoni and S. haematobium eggs, respectively. Urine was also subjected to a commercially available cassette test for S. mansoni, which detects circulating cathodic antigen. A finger-prick blood sample was used for the SmCTF-RDT for detection of anti-S. mansoni and anti-S. haematobium antibodies. Results The prevalence of both anti-S. mansoni and anti-S. haematobium antibodies was more than three times higher than the prevalence of infection estimated by egg detection under a microscope. Using quadruplicate Kato-Katz as the reference standard for the diagnosis of S. mansoni infection, the sensitivity, negative predictive value (NPV, and positive predictive value (PPV of the SmCTF-RDT was 75.0%, 84.2% and 22.5%, respectively. When two urine filtrations were considered as the reference standard for the diagnosis of S. haematobium infection, the sensitivity, NPV and PPV of SmCTF-RDT was 66.7%, 94.9% and 5.1%, respectively. The specificity of SmCTF-RDT, when using egg-detection as the reference standard, was estimated to be 34.4%. This low specificity may be a reflection of the

  9. Production and Application of Monoclonal Antibodies Against Methamphetamine (MA) and Morphine (MP)

    Institute of Scientific and Technical Information of China (English)


    Three monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher thanThree monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher than 108

  10. 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: [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: [Institute of Agri-Food and Land Use, Queen' s University Belfast, Belfast BT9 5AG, Northern Ireland (United Kingdom)


    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.

  11. Comparison between the Counter Immunoelectrophoresis Test and Mouse Neutralization Test for the Detection of Antibodies against Rabies Virus in Dog Sera

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    Luzia Helena Queiroz da Silva


    Full Text Available The detection of rabies antibodies is extremely valuable for epidemiological studies, determination of immune status in man, animals, and for the diagnosis of the disease. Several serological procedures have been described for this purpose. The present study reports a comparison between counterimmunoelectrophoresis test (CIET and mouse neutralization test (MNT in the detection of antibodies against rabies virus from 212 serum samples of vaccinated dogs. The agreement between both techniques was 79.7% and a significative association was demonstrated. The correlation coefficients between MNT and the CIET titers was determined considering 88 samples showing positive results in both techniques [CIET = 2 and MNT = 5 (0.13 IU/ml] and resulted r² = 0.7926 (p < 0.001. The performance of CIET system was technically simple, cheap and rapid, and thereby it could be useful for serological monitoring of dog vaccination campaigns as well as for individual analysis.

  12. Development of a Time and Cost Benefit Antibody Binding Test-Based Method for Determination of Rabies Vaccine Potency. (United States)

    Asgary, Vahid; Mojtabavi, Nazanin; Janani, Alireza; Mousavi, Tahereh; Hadjati, Jamshid; Khosravy, Mohammad Sadeq; Ahangari Cohan, Reza


    This study is an improvement on the antibody binding test, known as ABT method, to develop a simple and fast method in comparison with NIH for determination of rabies vaccine potency. In the current study, several commercial human and veterinary vaccines were tested using both modified ABT and NIH methods. The ED50 was calculated using the probit method and the relative potency of each vaccine was measured based on the reference vaccine. The test was repeated four times to calculate the reproducibility of the method. Statistical analysis indicated that there was no significant difference between the result obtained from NIH and modified ABT method for either human or veterinary vaccines (p > 0.05). In addition, the linearity of the method (R(2)) was calculated as 0.94 by serial dilution of a test vaccine. Coefficient variances were determined as less than and more than 10% for the human and veterinary rabies vaccines, respectively. In conclusion, the findings suggest that the modified method could be considered as an alternative approach for rabies vaccine potency determination in in-process quality control tests at industrial scale. It is a time and cost benefit method and accuracy may further be increased by employing monoclonal antibodies against trimeric form of G glycoprotein. However, the use of serum samples may be useful compared with an artificial mix of antibodies because other components from the serum samples could have a positive impact on cell sensitivity and mimic more the complexity of the immune response. Although the modified test has solved a fundamental problem, it is still not sensitive enough for veterinary vaccine assessment and needs further modifications to obtain the acceptability criteria.

  13. High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler. (United States)

    Choo, Chiou-Yu; Tian, Yuan; Kim, Wan-Seop; Blatter, Erich; Conary, Jon; Brady, Ciaran P


    A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.

  14. The Utilization of a New Immunochromatographic Test in Detection of Helicobacter pylori Antibody from Maternal and Umbilical Cord Serum

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    Fu-Chen Kuo


    Full Text Available Background. Helicobacter pylori (H. pylori was linked with several extragastrointestinal diseases, including preeclampsia and intrauterine growth restriction of fetus. One of the signals which can be transferred from mother to fetus is the H. pylori IgG antibody. Aims. We utilized a commercial immunochromatographic kit to detect the antibody in maternal and cord serum. Methods. Three hundred and forty-six females were enrolled and the blood samples were collected on antenatal examination and on delivery. The maternal H. pylori infection was determined by stool H. pylori antigen test. Results. One hundred and five females (30.3% were H. pylori-infected, and the prevalence was higher in immigrants (43.5% than in Taiwanese (28.7%, P=0.058. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the kit were 77.1%, 88.0%, 73.6%, 89.8%, and 84.7%, respectively. This kit also had similar performance in cord serum. Comparing to the maternal result on delivery, this kit offered a consistent performance in antenatal maternal serum (kappa coefficient 0.92 and in cord serum (kappa coefficient 0.88. Conclusions. H. pylori IgG antibody can be transferred through the placenta into the fetal circulation. However, accuracy of the test kit needs to be evaluated before utilization in screening.

  15. Continuous production of monoclonal antibody in a packed-bed bioreactor. (United States)

    Golmakany, Naghmeh; Rasaee, Mohammad Javad; Furouzandeh, Mehdi; Shojaosadati, Seyed Abbas; Kashanian, Soheila; Omidfar, Kobra


    In the present study the growth and MAb (monoclonal antibody) production of a mouse x mouse hybridoma cell producing anti-digoxin MAb was evaluated. The hybridoma cells entrapped within the support matrix Fibra-Cel were cultured in batch and continuous mode following special protocols. Cell-culture studies were performed in a 1-litre spinner basket containing 3 g.litre-1 support matrix. Batch culture was operated with the cell density of 42x10(6) cells. During the 7 days of culture, the medium was sampled daily in order to assess glucose and MAb concentrations and the lactate dehydrogenase released into the culture medium. After a culture period of 72 h, the cell density and MAb concentration were found to be 10.4x10(7) cells/3 g of NWPF (non-woven polyester fibre) discs and 250 microg/ml respectively. This yield gradually decreased to 0.55x10(6) cells/3 g of packaging material and 60 microg/ml respectively at the end of the batch culture. In the continuous-culture studies, the batch culture was initially operated for 64.5 h and then continuous flow was started at the dilution rates of 0.15, 0.2, 0.25 and 0.3 day-1 and finally stabilized at 0.25 day-1 within 288 h (12 days). The MAb concentration at steady state was found to be 116-120 microg/day per ml, and the yield of operation was 62.5 mg/day per ml, which was 3.5 times higher than that of batch culture. In conclusion, a packed-bed bioreactor with the support matrix Fibra-Cel, operated in continuous-feeding mode, is more efficient for large-scale MAb production than a batch culture. On the other hand, by using a continuous-culture system, a better supply of nutrients and removal of inhibitory metabolites and proteolytic enzymes was obtained.

  16. Product acceptance environmental and destructive testing for reliability.

    Energy Technology Data Exchange (ETDEWEB)

    Dvorack, Michael A.; Kerschen, Thomas J.; Collins, Elmer W.


    To determine whether a component is meeting its reliability requirement during production, acceptance sampling is employed in which selected units coming off the production line are subjected to additional environmental and/or destructive tests that are within the normal environment space to which the component is expected to be exposed throughout its life in the Stockpile. This report describes what these tests are and how they are scored for reliability purposes. The roles of screens, Engineering Use Only tests, and next assembly product acceptance testing are also discussed, along with both the advantages and disadvantages of environmental and destructive testing.

  17. Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus. (United States)

    McLean, C S; Churcher, M J; Meinke, J; Smith, G L; Higgins, G; Stanley, M; Minson, A C


    A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

  18. Product Offerings Testing through Customer Satisfaction

    Directory of Open Access Journals (Sweden)

    Tina Vukasovic


    Full Text Available Consumer satisfaction is imperative to a successful business, the reason for the choice of topic for this paper being explained thereby. Market changes have resulted in consumer’s enormous growth of power, which was recognized by many companies who adapted their business to meeting those expectations. Adaptation, however, also resulted in the need for constant measuring and evaluation. According to the above-mentioned, this paper measures consumer satisfaction with the product offer of the drugstore chain X The survey results have shown that X’s offer has not completely come up to the expectations of a smaller number of interviewees. In relation to the measuring ranks of consumer satisfaction defined, the greatest number of consumers has turned out to be satisfied with the product offer, whereas the percentage of those who find it excellent is smaller than the percentage of those who assess it as average.

  19. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Directory of Open Access Journals (Sweden)

    Frank Sainsbury

    Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

  20. Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production (United States)

    Davoudi Vijeh Motlagh, Atefeh; Siadat, Seyed Davar; Abedian Kenari, Saeid; Mahdavi, Mehdi; Behrouzi, Ava; Asgarian-Omran, Hossein


    Background Outer membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains. Objectives The aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate. Methods Protein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines. Results Anti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice. Conclusions We conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity. PMID:27942362

  1. Immunomodulatory therapies for relapsing-remitting multiple sclerosis: monoclonal antibodies, currently approved and in testing. (United States)

    Craddock, Jessica; Markovic-Plese, Silva


    Relapsing-remitting multiple sclerosis (RRMS), a CNS inflammatory demyelinating disease, is one of the most prevalent causes of chronic disability in young adults. Studies of the disease pathogenesis have identified multiple therapeutic targets. The number of approved disease modifying therapies has almost doubled within the past 5 years, which creates a challenge for medical professionals to stay abreast of their use in everyday practice. This manuscript provides an overview of available injectable, oral, and intravenous therapies for RRMS, and offers guidance in selecting an appropriate therapy. Focus is on the recently approved and emerging monoclonal antibody therapies, because they offer more selective and superior therapeutic efficacy compared with injectable and oral disease modifying therapies. We discuss the outlook for monoclonal antibodies and their role in RRMS treatment in the future.

  2. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay


    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya


    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-ba...

  3. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings (United States)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.


    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

  4. Immune response in mice to ingested soya protein: antibody production, oral tolerance and maternal transfer

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Pedersen, Susanne Brix; Frøkiær, Hanne


    While allergic reactions to soya are increasingly investigated, the normal immune response to ingested soya is scarcely described. In the present study, we wanted to characterise the soya-specific immune response in healthy mice ingesting soya protein. Mice fed a soya-containing diet (F0) and mice...... of the first (F1) and second (F2) offspring generation bred on a soya protein-free diet were used either directly or were transferred between the soya-containing and soya protein-free diet during pregnancy or neonatal life. The mice were compared as to levels of naturally occurring specific antibodies analysed...... by ELISA, and to the presence of oral tolerance detected as a suppressed antibody and cell-proliferation response upon immunisation with soya protein. F0 mice generated soya-specific antibodies, while oral tolerance to the same soya proteins was also clearly induced. When F0 dams were transferred to soya...

  5. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

    Directory of Open Access Journals (Sweden)

    Lilian Terezinha de Queiroz Leite


    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

  6. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair. (United States)

    Esser-von Bieren, Julia; Volpe, Beatrice; Sutherland, Duncan B; Bürgi, Jérôme; Verbeek, J Sjef; Marsland, Benjamin J; Urban, Joseph F; Harris, Nicola L


    Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  7. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies. (United States)

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei


    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

  8. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

    Directory of Open Access Journals (Sweden)

    Julia Esser-von Bieren


    Full Text Available Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/- or activating Fc receptors (Fcrg-/- displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb, whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3 by macrophages (MΦ and myofibroblasts (MF within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  9. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor. (United States)

    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E


    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

  10. Relevance of microbial finished product testing in food safety management

    DEFF Research Database (Denmark)

    Zwietering, Marcel H.; Jacxsens, Liesbeth; Membré, Jeanne Marie


    -active way by implementing an effective food safety management system. For verification activities in a food safety management system, finished product testing may however be useful. For three cases studies; canned food, chocolate and cooked ham, the relevance of testing both of finished products...

  11. A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein. (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; McEachern, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa


    Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV-NiV-GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV-NiV-GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV-NiV-GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test.

  12. Passive haemagglutination test for human neurocysticercosis immunodiagnosis: I. Standardization and evaluation of the passive haemagglutination test for the detection of anti-Cysticercus cellulosae antibodies

    Directory of Open Access Journals (Sweden)

    Mirthes Ueda


    Full Text Available A passive haemagglutination test (PHA for human neurocysticercosis was standardized and evaluated for the detection of specific antibodies to Cysticercus cellulosae in cerebrospinal fluid (CSF. For the assay, formaldehyde-treated group O Rh-human red cells coated with the cysticerci crude total saline extract (TS antigen were employed. A total of 115 CSF samples from patients with neurocysticercosis was analysed, of these 94 presented reactivity, corresponding to 81.7% sensitivity, in which confidence limit of 95% probability (CL95% ranged from 74.5% to 88.9%. Eighty-nine CSF samples derived from individuals of control group presented as nonreactive in 94.4% (CL95% from 89.6% to 99.2%. The positive and negative predictive values were 1.4% and 99.9%, respectively, considering the mean rate of that this assay provide a rapid, highly reproducible, and moderately sensitive mean of detecting specific antibodies in CSF samples.

  13. Methodological issues in testing the marginal productivity theory

    NARCIS (Netherlands)

    P.T. Gottschalk (Peter); J. Tinbergen (Jan)


    textabstractPrevious tests of the marginal productivity theory have been criticized on several grounds reviewed by the authors. One important deficiency has been the small number of factor inputs entered in the production functions. In 1978 Gottschalk suggested a method to estimate production functi

  14. Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures. (United States)

    Lonoce, Chiara; Salem, Reda; Marusic, Carla; Jutras, Philippe V; Scaloni, Andrea; Salzano, Anna Maria; Lucretti, Sergio; Steinkellner, Herta; Benvenuto, Eugenio; Donini, Marcello


    Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

  15. Intravenous immunoglobulin prevents murine antibody-mediated acute lung injury at the level of neutrophil reactive oxygen species (ROS production.

    Directory of Open Access Journals (Sweden)

    John W Semple

    Full Text Available Transfusion-related acute lung injury (TRALI is a leading cause of transfusion-associated mortality that can occur with any type of transfusion and is thought to be primarily due to donor antibodies activating pulmonary neutrophils in recipients. Recently, a large prospective case controlled clinical study of cardiac surgery patients demonstrated that despite implementation of male donors, a high incidence of TRALI still occurred and suggested a need for additional interventions in susceptible patient populations. To examine if intravenous immunoglobulin (IVIg may be effective, a murine model of antibody-mediated acute lung injury that approximates human TRALI was examined. When BALB/c mice were injected with the anti-major histocompatibility complex class I antibody 34-1-2s, mild shock (reduced rectal temperature and respiratory distress (dyspnea were observed and pre-treatment of the mice with 2 g/kg IVIg completely prevented these symptoms. To determine IVIg's usefulness to affect severe lung damage, SCID mice, previously shown to be hypersensitive to 34-1-2s were used. SCID mice treated with 34-1-2s underwent severe shock, lung damage (increased wet/dry ratios and 40% mortality within 2 hours. Treatment with 2 g/kg IVIg 18 hours before 34-1-2s administration completely protected the mice from all adverse events. Treatment with IVIg after symptoms began also reduced lung damage and mortality. While the prophylactic IVIg administration did not affect 34-1-2s-induced pulmonary neutrophil accumulation, bone marrow-derived neutrophils from the IVIg-treated mice displayed no spontaneous ROS production nor could they be stimulated in vitro with fMLP or 34-1-2s. These results suggest that IVIg prevents murine antibody-mediated acute lung injury at the level of neutrophil ROS production and thus, alleviating tissue damage.

  16. Does the antibody production ability affect the serum anti-Helicobacter pylori Ig G titer?

    Institute of Scientific and Technical Information of China (English)

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


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

  17. Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T. (Kirin Brewery Co., Ltd., Gunma (Japan))


    Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation.

  18. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG


    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  19. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach. (United States)

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V


    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The


    Directory of Open Access Journals (Sweden)

    Nurhadi Nurhadi


    Full Text Available Citrus tristeza virus (CTV is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA. Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE. The specific coat protein (CP band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

  1. Perceived empathy of service providers mediates the association between perceived discrimination and behavioral intention to take up HIV antibody testing again among men who have sex with men.

    Directory of Open Access Journals (Sweden)

    Jing Gu

    Full Text Available HIV antibody testing is a key measure of HIV prevention for men who have sex with men (MSM. The World Health Organization recommends sexually active and at-risk MSM to take up HIV antibody testing regularly. This study aimed to investigate the prevalence of behavioral intention to take up HIV antibody testing in the next six months among Hong Kong MSM who were ever-testers. An anonymous cross-sectional survey recruited 326 MSM who had taken up HIV antibody testing from gay-friendly venues and internet in Hong Kong. Of the participants, 40.8% had had unprotected anal intercourse with regular or non-regular male sex partners in the last six months; they were at risk of HIV transmission despite experience in HIV antibody testing. Only 37.2% showed a strong intention to take up HIV antibody testing again in the next six months. Adjusted analysis showed that both perceived discrimination toward Hong Kong MSM (AOR = .60, 95% CI: .36-.98 and the CARE Measure assessing perceived empathy of service providers (AOR = 1.05, 95% CI: 1.02-1.08 were significantly associated with intention for retesting. Perceived discrimination, however, became statistically non-significant (AOR = .68, 95% CI: .41-1.14, when both CARE Measure and perceived discrimination entered into the adjusted model. It is warranted to increase HIV retesting rate by removing perceived discrimination and reducing the negative effect of perceived discrimination through enhancement of empathy of service providers.

  2. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains. (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail


    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  3. Antinuclear antibody panel (United States)

    ... page: // Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  4. Bayesian estimation of sensitivity and specificity of Coxiella burnetii antibody ELISA tests in bovine blood and milk

    DEFF Research Database (Denmark)

    Paul, Suman; Toft, Nils; Agerholm, Jørgen S.;


    Serological tests for Coxiella burnetii (the causative agent of Q fever) antibodies are usually based on enzyme linked immunosorbent assay (ELISA) although this method is not thoroughly evaluated. The objective of this study was to determine the sensitivity and specificity of an ELISA for detection...... of the ELISA methods on milk and blood were equal at 0.99. No conditional dependence was observed between the specificity estimates of the two test methods. However, the sensitivity estimates of both tests were significantly reduced when conditional covariances ≥40 were used. Collection of milk samples from...... to positive (S/P) cut-off of 40 for both blood and milk ELISAs. At this cut-off, sensitivity of milk ELISA was 0.86 (95% posterior credibility interval [PCI] [0.76; 0.96]). This was slightly but insignificantly higher than sensitivity of blood ELISA (0.84; 95% PCI [0.75; 0.93]). The specificity estimates...

  5. Production of anti-double-stranded DNA antibodies in activated lymphocyte derived DNA induced lupus model was dependent on CD4+ T cells. (United States)

    Wen, Z; Xu, L; Xu, W; Xiong, S


    Our previous study demonstrated that activated lymphocyte derived DNA (ALD-DNA) could function as an autoantigen to induce production of anti-double-stranded DNA (anti-dsDNA) antibodies in syngeneic BALB/c mice. Here we carefully evaluated the potential role of T cells in the induction of anti-dsDNA antibody. We demonstrated that ALD-DNA could effectively induce production of anti-dsDNA antibodies in vivo and in vitro. In contrast, ALD-DNA could not induce the generation of anti-dsDNA antibodies in nude mice. We further showed that in vivo depletion of CD3(+) T cells blocked the induction of anti-dsDNA antibodies in BALB/c mice. Notably, we demonstrated that CD4(+) but not CD8(+) T cells conferred ALD-DNA to induce anti-dsDNA antibodies. Finally, we demonstrated that adoptive transfer of CD4(+) T cells could rescue ALD-DNA induced anti-dsDNA antibodies in nude mice. Our results suggested that T helper cells were required for ALD-DNA to induce anti-dsDNA antibodies. These findings could further our understanding about the immunogenic properties of DNA and throw new light on SLE pathogenesis.

  6. 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 (United States)

    Munhoz, Danielle D.; Cardoso, Lucas T. A.; Luz, Daniela E.; Andrade, Fernanda B.; Horton, Denise S. P. Q.; Elias, Waldir P.; Piazza, Roxane M. F.


    Background 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. Methodology 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. Principal findings 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. Conclusion 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. PMID:25254981

  7. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp

    Institute of Scientific and Technical Information of China (English)



    A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cellfusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50%inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118%for an intra-assay and 96%-113%for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9%and 5.5%-14.9%, respectively.%本文题目:抗土霉素单克隆抗体的制备及其在虾中土霉素残留的检测应用Production of a monoclonal antibody against oxytetracycline and its application for oxy-tetracycline residue detection in shrimp研究目的:抗土霉素单克隆抗体的制备和特性分析,并用于间接竞争酶联免疫吸附测定法(icELISA)分析虾样品中土霉素的残留。创新要点:本研究建立分泌抗土霉素单克隆抗体的杂交瘤细胞株,筛选出对虾中土霉素残留检测具有较高灵敏度的单克隆抗体2-4F。研究方法:用细胞杂交技术使土霉素-牛血清白蛋白偶联物免疫的BALC/c雌性小鼠的脾细胞与骨髓瘤细胞融合,建立三株分泌抗土霉素单克隆抗体的杂交瘤细胞株(2-4F、7-3G和11-11A),并制备它们的单克隆抗体。通过 icELISA 法分析单克隆抗体对土霉素的半抑制质量浓度(IC50)和交叉反

  8. Probability of a false-negative HIV antibody test result during the window period: a tool for pre- and post-test counselling. (United States)

    Taylor, Darlene; Durigon, Monica; Davis, Heather; Archibald, Chris; Konrad, Bernhard; Coombs, Daniel; Gilbert, Mark; Cook, Darrel; Krajden, Mel; Wong, Tom; Ogilvie, Gina


    Failure to understand the risk of false-negative HIV test results during the window period results in anxiety. Patients typically want accurate test results as soon as possible while clinicians prefer to wait until the probability of a false-negative is virtually nil. This review summarizes the median window periods for third-generation antibody and fourth-generation HIV tests and provides the probability of a false-negative result for various days post-exposure. Data were extracted from published seroconversion panels. A 10-day eclipse period was used to estimate days from infection to first detection of HIV RNA. Median (interquartile range) days to seroconversion were calculated and probabilities of a false-negative result at various time periods post-exposure are reported. The median (interquartile range) window period for third-generation tests was 22 days (19-25) and 18 days (16-24) for fourth-generation tests. The probability of a false-negative result is 0.01 at 80 days' post-exposure for third-generation tests and at 42 days for fourth-generation tests. The table of probabilities of falsely-negative HIV test results may be useful during pre- and post-test HIV counselling to inform co-decision making regarding the ideal time to test for HIV.

  9. Anti-enrofloxacin Antibody Production by Using Enrofloxacin-screened HSA as an Immunogen

    Institute of Scientific and Technical Information of China (English)

    LIU Chune; LIN Hong; CAO Limin; JIANG Jie


    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  10. The use of monoclonal antibodies for the characterization and production of Mycobacterium leprae antigens

    Directory of Open Access Journals (Sweden)

    J. Ivanyi


    Full Text Available Similar immunizations of mice and hybridoma technology were used by several investigators to raise monoclonal antibodies which identified a limited range of epitopes and antigenic molecules. Further studies would have the scope for revealing yet more novel structures. The existing MABs are agreed standard reagents, avaiable to investigators and valuable for several applications. At least six epitopes specific for M. leprae were defined in molecular terms. Monoclonal antibody based immunoassays proved to be invaluable for the screening of recombinant DNA clones and for the topographic study of individual epitopes. Purification of antigens using affinity chromatography requires further development of techniques whilst serology of leprosy is open for clinical and epidemiological evaluation.

  11. Production and Characterisation of Anti-Cardiac Troponin-I Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Kh. H. Haider


    Full Text Available Cardiac troponin-I (cTn-I was isolated from bovine left ventricular tissue and used as immunogen. Sixteen murine hybridoma lines were produced with two of them. I D 12 and 5F4, showing a high specificity for cTn-I; both of these monoclonal antibodies (McAbs were isotyped as IgG I with kappa - light chains. The specificity of the McAbs for cTn-1 was confirmed by ELISA, western blotting and by the ability of the antibodies to block actomyosin ATPase inhibition by cTn-I. The McAbs may be useful for human ill vivo imaging of myocardial infarcts and other pathological conditions related to cardiac myocyte damage.

  12. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin


    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  13. Anti-DNA antibodies--quintessential biomarkers of SLE. (United States)

    Pisetsky, David S


    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  14. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. (United States)

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya


    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  15. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein. (United States)

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana


    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  16. Effect of operating conditions in production of diagnostic Salmonella Enteritidis O-antigen-specific monoclonal antibody in different bioreactor systems. (United States)

    Ayyildiz-Tamis, Duygu; Nalbantsoy, Ayse; Elibol, Murat; Deliloglu-Gurhan, Saime Ismet


    In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.

  17. High Production of IL-18 by Dendritic Cells Induced by Sera from Patients with Primary Antibody Deficiency

    Directory of Open Access Journals (Sweden)

    Maryam Nourizadeh


    Full Text Available Predominantly antibody deficiencies are a category of primary immunodeficiency diseases, whichconsist of several rare disorders such as common variable immunodeficiency (CVID and X-linked agammaglobulinemia (XLA. We evaluated the effects of CVID and XLA patients’ sera as a source of microenviromental factors on maturation and function of monocyte-derived DCs.Blood was collected from 10 CVID and 5 XLA patients before immunoglobulin replacementtherapy and also from 8 healthy volunteers in order to obtain necessary sera for this study. Monocyte derived DCs were generated from blood cells obtained from healthy volunteers in the presence of GM-CSF, IL-4 and 10% serum concentrations from cases and controls. Immature DCs were incubated with monocyte conditioned medium (MCM and TNF-α in order to generate mature DCs. Interleukin 18 (IL-18 production by CD40L-activated mature DCs was measured after 24 hours of culture in vitro.IL-18 production by DCs generated in the presence of CVID and XLA patients’ sera were6.75±2.59 and 7.08±1.75 ng/ml, respectively, which were significantly higher than normal serumconditioned DCs (3.55±0.68 ng/ml.These results suggest that the sera of patients with predominantly antibody deficiencies maycontain soluble factor(s that can induce a significant increase in IL-18 production by DCs.

  18. The role of evolutionarily conserved germ-line DH sequence in B-1 cell development and natural antibody production. (United States)

    Vale, Andre M; Nobrega, Alberto; Schroeder, Harry W


    Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire.

  19. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    Directory of Open Access Journals (Sweden)

    Kiefer Hans


    Full Text Available Abstract Background RAI3 is an orphan G-protein coupled receptor (GPCR that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA, western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147 and normal breast tissues (n = 44 using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50 as well as lymph node metastases (n = 3 for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic

  20. Effect of days in milk and milk yield on testing positive in milk antibody ELISA to Mycobacterium avium subsp. paratuberculosis in dairy cattle

    DEFF Research Database (Denmark)

    Nielsen, Søren Saxmose; Toft, Nils


    Milk samples are becoming more used as a diagnostic specimen for assessment of occurrence of antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). This study assessed the effect of days in milk (DIM) and milk yield on testing positive in a commercial MAP specific milk antibody ELISA...... from the first couple of DIM should be excluded from MAP testing until further information on their significance is established. Milk yield also had a significant effect on odds of testing positive due to its diluting effect. Inclusion of milk yield in the interpretation of test results could improve...

  1. [Comparison of the indirect immunofluorescent (IFAT), ELISA test and the comercial Chagatek test for anti-Trypanosoma cruzi antibodies detection]. (United States)

    Enciso, Clara; Montilla, Marleny; Santacruz, María M; Nicholls, Rubén Santiago; Rodríguez, Adriana; Mercado, Marcela; Puerta, Concepción


    Chagas disease is a public health problem in Colombia, particularly in the eastern region. Because of human migration from rural areas to urban centers, the possibility of transfusional transmission becomes increasingly important. However the risk can be minimized by a careful screening of blood donors by means of serological tests. Colombian blood banks use comercial, foreign serological tests for screening for T. cruzi infection. The purpose of the current study was to compare the IFAT and ELISA tests (both use antigen obtained from Colombian strains) with the comercially available Chagatek tests. Sera of blood donors were classified in two groups on the basis of the IFAT: group I, 15 positive patients and group II, 14 negative patients. Sera from each group were tested by the ELISA and Chagatek tests. The ELISA test detected 100% of the patients as positive in group I and 7% (1/14) of patients as positive in group II. The Chagatek test detected 93% (14/15) of the patients as positive in group I and 50% (7/14) in group II. The kappa index for concordance between the ELISA and IFAT tests was 0.93 (95% C.I.: 0.80-1.00); between IFAT and Chagatek 0.43 (95% C.I.: 0.26-0.62), and between ELISA and Chagatek 0.49 (95% C.I.: 0.31-0.67). These results highlighted the importance of using autochtonous Colombian strains as antigens in screening tests for blood donors.

  2. Evaluation of Multiplex-Based Antibody Testing for Use in Large-Scale Surveillance for Yaws: a Comparative Study. (United States)

    Cooley, Gretchen M; Mitja, Oriol; Goodhew, Brook; Pillay, Allan; Lammie, Patrick J; Castro, Arnold; Moses, Penias; Chen, Cheng; Ye, Tun; Ballard, Ronald; Martin, Diana L


    WHO has targeted yaws for global eradication by 2020. The program goals are to interrupt the transmission in countries where yaws is endemic and to certify countries as yaws free where yaws was endemic in the past. No new rapid plasmin reagin (RPR) seroreactivity in young children is required for certification of elimination at a country level. We sought to evaluate whether antibody responses to specific treponemal antigens measured in a high-throughput multiplex bead array (MBA) assay differentiate past versus current infection and whether a nontreponemal lipoidal antigen test can be incorporated into the MBA. Serum and dried blood spot specimens collected for yaws surveillance projects in Ghana, Vanuatu, and Papua New Guinea (PNG) were run on MBA to measure antibodies against recombinant p17 (rp17) and treponemal membrane protein A (TmpA) treponemal antigens. Results were compared to standard treponemal laboratory (TPPA or TPHA [TPP(H)A]) and quantitative RPR test data. Of 589 specimens, 241 were TPP(H)A(+)/RPR(+), 88 were TPP(H)A(+)/RPR(-), 6 were TPP(H)A(-)/RPR(+), and 254 were negative for both tests. Compared to TPP(H)A, reactive concordance of rp17 was 93.7%, while reactive concordance of TmpA was only 81.9%. TmpA-specific reactivity showed good correlation with RPR titers (R(2) = 0.41; P RPR testing (cardiolipin) were not detected in the MBA. Our results suggest that TmpA can be used as a treponemal antigen marker for recent or active infection and potentially replace RPR in a high-throughput multiplex tool for large-scale yaws surveillance.

  3. Cost-Effectiveness Analysis of Different Testing Strategies that Use Antibody Levels to Detect Chronic Hepatitis C in Blood Donors (United States)

    Granados-García, Víctor; Contreras, Ana M.; García-Peña, Carmen; Salinas-Escudero, Guillermo; Thein, Hla-Hla; Flores, Yvonne N.


    Aim. We conducted a cost-effectiveness analysis of seven hepatitis C virus (HCV) testing strategies in blood donors. Methods. Three of the seven strategies were based on HCV diagnosis and reporting guidelines in Mexico and four were from previous and current recommendations outlined by the CDC. The strategies that were evaluated determine antibody levels according to the signal-to-cut-off (S/CO) ratio and use reflex Immunoblot (IMB) or HCV RNA tests to confirm true positive (TP) cases of chronic HCV infection. Costs were calculated from the perspective of the Mexican Institute of Social Security (IMSS). A decision tree model was developed to estimate the expected number of true positive cases and costs for the base-case scenarios and for the sensitivity analyses. Results. Base-case findings indicate an extended dominance of the CDC-USA2 and CDC-USA4 options by the IMSS Mexico3 and IMSS-Mexico1 alternatives. The probabilistic sensitivity analyses results suggest that for a willingness-to-pay (WTP) range of $0–9,000 USD the IMSS-Mexico1 strategy is the most cost-effective of all strategies ($5,000 USD per TP). The IMSS-Mexico3, IMSS-Mexico2, and CDC-USA3 strategies are also cost-effective strategies that cost between $7,800 and $8,800 USD per TP case detected. The CDC-USA1 strategy was very expensive and not cost-effective. Conclusions. HCV antibody testing strategies based on the classification of two or three levels of the S/CO are cost-effective procedures to identify patients who require reflex IMB or HCV RNA testing to confirm chronic HCV infection. PMID:27159320

  4. 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.


    Full Text Available Chapter 1 reviews the literature about the immunological aspects of taeniid cestode infections and the existing vaccines against Taenia solium cysticercosis in pigs. One of the most promising vaccines is TSOL18, a protein that has been identified in the oncosphere of Taenia solium and expressed as a recombinant molecule in E. coli. Repeated experimental trials have shown that this vaccine is able to protect up to 100% of the immunised pigs against a challenge infection with T. solium. Antibodies raised by the vaccine are capable of killing the parasite in in vitro cultures and it is believed that antibody and complement mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. The identification of the villages with a high risk of T. solium infection, which could subsequently be used in the vaccine trial, is reported in chapter 2. A survey was conducted in 150 households owning 1756 pigs in the rural areas of Mayo-Danay division in the far north region of Cameroon. A questionnaire survey was carried out to collect information on the pig farming system and to identify potential risk factors for T. solium cysticercosis infection in pigs. Blood samples were collected from 398 pigs with the aim of estimating the sero-prevalence of Taenia solium cysticercosis. The results showed that 90.7% of the pigs were free roaming during the dry season and that 42.7% of households keeping pigs in the rural areas had no latrine facility. Seventy six percent of the interviewed pig owners affirmed that the members of the household used open field defecation. ELISA for antigen and antibody detection showed an apparent prevalence of porcine cysticercosis of 24.6% and 32.2%, respectively. A Bayesian approach using the conditional dependence between the two diagnostic tests indicated that the true sero-prevalence of cysticercosis in Mayo-Danay was 26.6%. Binary logistic regression analysis indicated that the

  5. Empirical testing of forecast update procedure forseasonal products

    DEFF Research Database (Denmark)

    Wong, Chee Yew; Johansen, John


    of a toy supply chain. The theoretical simulation involves historical weekly consumer demand data for 122 toy products. The empirical test is then carried out in real-time with 291 toy products. The results show that the proposed forecast updating procedure: 1) reduced forecast errors of the annual...

  6. Antibodies against glucan, chitin, and Saccharomyces cerevisiae mannan as new biomarkers of Candida albicans infection that complement tests based on C. albicans mannan. (United States)

    Sendid, B; Dotan, N; Nseir, S; Savaux, C; Vandewalle, P; Standaert, A; Zerimech, F; Guery, B P; Dukler, A; Colombel, J F; Poulain, D


    Antibodies against Saccharomyces cerevisiae mannan (ASCA) and antibodies against synthetic disaccharide fragments of glucans (ALCA) and chitin (ACCA) are biomarkers of Crohn's disease (CD). We previously showed that Candida albicans infection generates ASCA. Here, we explored ALCA and ACCA as possible biomarkers of invasive C. albicans infection (ICI). ASCA, ALCA, ACCA, and Candida mannan antigen and antibody detection tests were performed on 69 sera obtained sequentially from 18 patients with ICIs proven by blood culture, 59 sera from CD patients, 47 sera from hospitalized subjects colonized by Candida species (CZ), and 131 sera from healthy controls (HC). ASCA, ALCA, and ACCA levels in CD and ICI patients were significantly different from those in CZ and HC subjects (PICI patients, these levels increased as infection developed. Using ASCA, ALCA, ACCA, and Platelia Candida tests, 100% of ICIs were detected, with the kinetics of the antibody response depending on the patient during the time course of infection. A large number of sera presented with more than three positive tests. This is the first evidence that the detection of antibodies against chitin and glucans has diagnostic value in fungal infections and that these tests can complement more specific tests. Future trials are necessary to assess the value of these tests in multiparametric analysis, as well as their pathophysiological relevance.

  7. The World Health Organization Recommendations for Trachoma Surveillance, Experience in Nepal and Added Benefit of Testing for Antibodies to Chlamydia trachomatis pgp3 Protein: NESTS Study (United States)

    Zambrano, Andrea I.; Sharma, Shekhar; Crowley, Kathryn; Dize, Laura; Muñoz, Beatriz E.; Mishra, Sailesh K.; Rotondo, Lisa A.; Gaydos, Charlotte A.; West, Sheila K.


    Background The World Health Organization (WHO) now requires a second surveillance survey for trachoma after an impact assessment has found follicular trachoma (TF) <5% to determine if re-emergence has occurred. Using new WHO guidelines, we undertook surveillance surveys, and determined the prevalence of infection and antibody positivity, in two districts in Nepal. Methods 20 clusters were randomly selected within each district, 15 were randomly selected for antibody testing. In each cluster, we randomly selected 50 children ages 1–9 years and 100 adults ≥15 years. TF and trachomatous trichiasis (TT) were evaluated. Conjunctival swabs to test for chlamydial infection using GenXpert platform were obtained, and dried blood spots were collected to test for antibodies to Chlamydia Trachomatis pgp3 using the Luminex platform. Findings 3 cases of TF were found in the two districts, and one case of infection. Pgp3 antibody positivity was 2·4% (95% confidence interval: 1·4%, 3·7%), and did not increase with age (P = 0.24). No clustering of antibody positivity within communities was found. TT prevalence was <1/1,000 population. Interpretation The surveillance surveys, as proposed by WHO, showed no evidence for re-emergence of trachoma in two districts of Nepal. The low level and no significant increase by age in seroprevalence of antibodies to C trachomatis pgp3 antigen deserve further investigation as a marker of interruption of transmission. PMID:27654497


    Directory of Open Access Journals (Sweden)

    Edgar Antonio Reyes Montaño


    Full Text Available Producing polyclonal antibodies (IgY inchickens has advantages over those obtainedin other animal models, since theyhave been used as a tool for studyingdifferent proteins (NMDA glutamate receptorin our case, specifically the NR1subunit. We produced specific antibodiesagainst expression products by thealternative splicing of the gene encodingNMDA receptor NR1 subunit in adult ratbrain. Three peptides corresponding tothe splicing sites (N1, C1 and C2’ cassetteswere designed, synthesised and usedindividually as antigens in hens. Specificimmunoglobulins were purified fromyolks. The antibodies were then used forpurifying the NMDA receptor NR1 subunitusing affinity chromatography couplingthe three antibodies to the support.R

  9. Modeling of cell culture damage and recovery leads to increased antibody and biomass productivity in CHO cell cultures. (United States)

    Naderi, Saeideh; Nikdel, Ali; Meshram, Mukesh; McConkey, Brendan; Ingalls, Brian; Budman, Hector; Scharer, Jeno


    The development of an efficient and productive cell-culture process requires a deep understanding of intracellular mechanisms and extracellular conditions for optimal product synthesis. Mathematical modeling provides an effective strategy to predict, control, and optimize cell performance under a range of culture conditions. In this study, a mathematical model is proposed for the investigation of cell damage of a Chinese hamster ovary cell culture secreting recombinant anti-RhD monoclonal antibody (mAb). Irreversible cell damage was found to be correlated with a reduction in pH. This irreversible damage to cellular function is described mathematically by a Tessier-based model, in which the actively growing fraction of cells is dependent on an intracellular metabolic product acting as a growth inhibitor. To further verify the model, an offline model-based optimization of mAb production in the cell culture was carried out, with the goal of minimizing cell damage and thereby enhancing productivity through intermittent refreshment of the culture medium. An experimental implementation of this model-based strategy resulted in a doubling of the yield as compared to the batch operation and the resulting biomass and productivity profiles agreed with the model predictions.

  10. Summary receiver operating characteristics (SROC) and hierarchical SROC models for analysis of diagnostic test evaluations of antibody ELISAs for paratuberculosis. (United States)

    Toft, Nils; Nielsen, Søren S


    Critical, systematic reviews of available diagnostic test evaluations are a meticulous approach to synthesize evidence about a diagnostic test. However, often the review finds that data quality is poor due to deficiencies in design and reporting of the test evaluations and formal statistical comparisons are discouraged. Even when only simple summary measures are appropriate, the strong correlation between sensitivity and specificity and their dependence on differences in diagnostic threshold across studies, creates the need for tools to summarise properties of the diagnostic test under investigation. This study presents summary receiver operating characteristics (SROC) analysis as a means to synthesize information from diagnostic test evaluation studies. Using data from a review of diagnostic tests for ante mortem diagnosis of paratuberculosis as an illustration, SROC and hierarchical SROC (HSROC) analysis were used to estimate overall diagnostic accuracies of antibody ELISAs for bovine paratuberculosis while accounting for covariates: the target condition (infectious or infected) used in the test evaluation (one for the evaluation of Se and one for Sp); and the type of test (serum vs. milk). The methods gave comparable results (regarding the estimated diagnostic log odds ratio), considering the small sample size and the quality of data. The SROC analysis found a difference in the performance of tests when the target condition for evaluation of Se was infected rather than infectious, suggesting that ELISAs are not suitable for detecting infected cattle. However, the SROC model does not take differences in sample size between study units into account, whereas the HSROC allows for both between and within study variation. Considering the small sample size, more credibility should be given to the results of the HSROC. For both methods the area under the (H)SROC curve was calculated and results were comparable. The conclusion is that while the SROC is simpler and easier

  11. Poor sensitivity of rapid tests for the detection of antibodies to the hepatitis B virus: implications for field studies (United States)

    Cruz, Helena Medina; Scalioni, Leticia de Paula; de Paula, Vanessa Salete; Miguel, Juliana Custódio; do Ó, Kycia Maria Rodrigues; Milagres, Flavio Augusto Pádua; Cruz, Marcelo Santos; Bastos, Francisco Inácio; Flores, Priscila Pollo; Leal, Erotildes; Motta-Castro, Ana Rita Coimbra; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo


    Rapid tests (RTs) can be used as an alternative method for the conventional diagnosis of hepatitis B virus (HBV). This study aims to evaluate antibodies to HBsAg (anti-HBs) and antibodies to HBeAg (anti-HBe) RTs under different Brazilian settings. The following three groups were included: GI: viral hepatitis outpatient services; GII: low resource areas; and GIII: crack users and beauticians. Imuno-rápido anti-HBsAg™ and Imuno-rápido anti-HBeAg™ RTs were evaluated and showed specificities greater than 95% in all groups. The sensitivity values to anti-HBs were 50.38%, 51.05% and 46.73% and the sensitivity values to anti-HBe were 76.99%, 10.34% and 11.76% in the GI, GII and GIII groups, respectively. The assays had a low sensitivity and high specificity, which indicated their use for screening in regions endemic for HBV. PMID:28146158

  12. Immunofluorescent Antibody Test (IFAT for Trypanosoma cruzi in dogs from urban and rural areas of Pelotas, RS

    Directory of Open Access Journals (Sweden)

    Charlene Nascimento dos Santos


    Full Text Available Chagas disease (CD is a zoonosis with the protozoan Trypanosoma cruzi as the causative agent. Dogs are considered the main domestic reservoir for T. cruzi in most Latin American countries and in some areas of the United States. In southern Brazil, despite being an endemic area of the disease, the prevalence in dogs is still unknown. This study aimed to evaluate the frequency of T. cruzi antibodies in dogs from urban and rural areas of Pelotas, RS, Brazil. A total of 227 canine sera were used for serological tests, of which 99 were from urban areas and 128 were from rural areas of Pelotas. Information regarding the environment and the possible risk factors (origin, rural contact, age, breed, confinement and gender to which the dogs were exposed were recorded. Indirect immunofluorescence was used to assess the presence of specific immunoglobulins (IgG anti-T. cruzi in the serum of dogs. Of the 227 sera analyzed, 81 (35.7% exhibited anti-T. cruzi, which represented 34.3% of the dogs from the urban area and 36.7% of the dogs from the rural area. Among the variables analyzed, rural contact and male gender showed an association with seropositivity for T. cruzi. The detection of T. cruzi antibodies in the serum of these dogs emphasizes the need to study trypanosomiasis in this important domestic reservoir of Chagas disease.

  13. Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

    DEFF Research Database (Denmark)

    Fregeneda-Grandes, J.M.; Olesen, Niels Jørgen


    Three serological tests, enzyme linked immunosorbent assay (ELISA), 50 % plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaerma virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected ...... VHSV. When the sera were examined for the presence of VHSV antibodies by ELISA or WB, 61 % were found to be positive. When conducting WB analysis, the viral glycoprotein was the protein most frequently recognized, followed by the viral nucleoprotein.......Three serological tests, enzyme linked immunosorbent assay (ELISA), 50 % plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaerma virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected...

  14. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng


    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  15. Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis. (United States)

    Nakajima, Mie; Kodama, Michi; Yanase, Haruko; Iwanaga, Toshihiko; Mulenga, Albert; Ohashi, Kazuhiko; Onuma, Misao


    There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

  16. Monoclonal Antibodies Recognising Sialyl-Tn: Production and Application to Immunochemistry

    Directory of Open Access Journals (Sweden)

    Peter L. Devine


    Full Text Available In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAcα2,6GaINAcα 1-O-Ser/Thr on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 301, both IgM against ovine submaxillary mucin (OSM; >98% of glycans as sialyl-Tn. These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn but did not react with desialylated OSM or BSM. Sialic acid at I mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D I also showed similar reactivity to sialyl-Tn reactive mab Bn.3, and detected Bn.3 capturedOSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (>200 kDa species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.

  17. A Listening Test of Contro11ed-Productive Ability

    Institute of Scientific and Technical Information of China (English)



    The purpose of the study is to estimate a new testing method of evaluating the student's ability of Listening Comprehension. The problem to be addressed is whether there is evidence to show that the method can discriminate between the testees, separating the above average students from the average and the below-average studentsThus, the controlled-productive listening test will be" found to be reliable, valid (in that the test distinguished between different proficiency students )and practical.

  18. Development and Production of a Leishmania Skin Test (United States)


    manufacturing process of Leishmania tropica Skin Test Antigen (LtSTA) was made during this contract period to increase the yield and robustness of the...interest group. 15. SUBJECT TERMS LtSTA = Leishmania tropica Skin Test Antigen 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT... tropica Skin Test Antigen (LtSTA), is a sterile injectable microfluidized lysate of Leishmania tropica (WR#1063:C1A) promastigotes. The product is heat

  19. Central location test vs. home use test: Contrasting results depending on product type

    NARCIS (Netherlands)

    Boutrolle, I.; Delarue, J.; Arranz, D.; Rogeaux, M.; Köster, E.P.


    Marketing professionals and sensory scientists have several hedonic testing methods at their disposal to assess product acceptability. The central location test (CLT) which usually takes place in a standardised location under controlled conditions is more frequently used than the home use test (HUT)

  20. Antimycobacterial susceptibility testing methods for natural products research

    Directory of Open Access Journals (Sweden)

    Juan Gabriel Bueno Sánchez


    Full Text Available The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need of continuous developments on new and efficient methods to determine the susceptibility of isolates of M. tuberculosis in the search for novel antimicrobial agents. Natural products constitute an important source of new drugs, but design and implementation of antimycobacterial susceptibility testing methods are necessary for evaluate the different extracts and compounds. A number of biological assay methodologies are in current use, ranging from the classical disk diffusion and broth dilution assay format, to radiorespirometric (BACTEC, dye-based, and fluorescent/luminescence reporter assays. This review presents an analysis on the in vitro susceptibility testing methods developed for determinate antitubercular activity in natural products and related compounds (semi-synthetic natural products and natural products-derived compounds and the criteria to select the adequate method for determination of biological activity of new natural products.

  1. Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in white tailed deer (odocoileus virginianus) from Iowa and Minnesota using four serologic tests (United States)

    The white-tailed deer (Odocoileus virginianus) is considered one of the most important wildlife reservoir of Neospora caninum and Toxoplasma gondii in the US. Sera from white-tailed deer from Minnesota and Iowa were tested for antibodies to N. caninum by four serologic tests including the indi...

  2. Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

    Directory of Open Access Journals (Sweden)

    Marco Tamba


    Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

  3. Evaluation of a new latex agglutination test for detection of streptolysin O antibodies.


    Gerber, M. A.; Caparas, L S; Randolph, M F


    Acute- and convalescent-phase serum specimens were collected from 50 patients with group A streptococcal pharyngitis. The anti-streptolysin O (ASO) titer for each serum specimen was determined by using both the standard neutralization assay and the latex agglutination (LA) test (Rheumagen ASO; Biokit Inc., New Britain, Conn.). When the ASO titers derived by the two methods were compared, the correlation coefficient was 0.93. When the ability of the LA test to demonstrate a significant ASO tit...

  4. Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans. (United States)

    Capodicasa, Cristina; Chiani, Paola; Bromuro, Carla; De Bernardis, Flavia; Catellani, Marcello; Palma, Angelina S; Liu, Yan; Feizi, Ten; Cassone, Antonio; Benvenuto, Eugenio; Torosantucci, Antonella


    There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

  5. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    L.C. Kreutz


    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

  6. Production of a human single-chain variable fragment antibody against esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ming-Yan Xu; Xiao-Hu Xu; Geng-Zhen Chen; Xiao-Ling Deng; Jonathan Li; Xiao-Jun Yu; Mei-Zhen Chen


    AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×106 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup Ⅳ of immunoglobulin, and variable light (VL) gene from the κchain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

  7. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)


    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  8. Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum. (United States)

    Fraser, P D; Misawa, N; Sandmann, G; Johnson, J; Schuch, W; Bramley, P M


    Monoclonal antibodies have been prepared against the tomato (Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a beta-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against beta-galactosidase-phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature phytoene synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG/la 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.

  9. Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances

    Energy Technology Data Exchange (ETDEWEB)

    Witkowski, Peter T. [Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany); Charite Universitaetsmedizin, CCM, Institut fuer Virologie, Helmut Ruska Haus, Chariteplatz 1, 10117 Berlin (Germany); Schuenadel, Livia, E-mail: [FU-Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustrasse 3, 14195 Berlin (Germany); Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany); Wiethaus, Julia; Bourquain, Daniel R.; Kurth, Andreas; Nitsche, Andreas [Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany)


    Research highlights: {yields} Real-time data acquisition by RT-CES requires low operative effort. {yields} Time to result is reduced by using RT-CES instead of conventional methods. {yields} RT-CES enables quantification of virus titers in unknown samples. {yields} RT-CES is a useful tool for high-throughput characterization of antiviral agents. {yields} An RT-CES-based virus neutralization test was established. -- Abstract: Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence{sup TM}, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC{sub 50}) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.

  10. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer. (United States)

    Horenstein, A L; Poiesi, C; DeMonte, L; Camagna, M; Mariani, M; Albertini, A; Malavasi, F


    An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

  11. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells. (United States)

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C


    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  12. Fission product plateout/liftoff/washoff test plan. Revision 1

    Energy Technology Data Exchange (ETDEWEB)

    Acharya, R.; Hanson, D.


    A test program is planned in the COMEDIE loop of the Commissariat a l`Energy Atomique (CEA), Grenoble, France, to generate integral test data for the validation of computer codes used to predict fission product transport and core corrosion in the Modular High Temperature Gas-Cooled Reactor (MHTGR). The inpile testing will be performed by the CEA under contract from the US Department of Energy (DOE); the contract will be administered by Oak Ridge National Laboratory (ORNL). The primary purpose of this test plan is to provide an overview of the proposed program in terms of the overall scope and schedule. 8 refs, 3 figs.

  13. Clinical factors associated with a Candida albicans Germ Tube Antibody positive test in Intensive Care Unit patients

    Directory of Open Access Journals (Sweden)

    Martín-Mazuelos Estrella


    Full Text Available Abstract Background Poor outcomes of invasive candidiasis (IC are associated with the difficulty in establishing the microbiological diagnosis at an early stage. New scores and laboratory tests have been developed in order to make an early therapeutic intervention in an attempt to reduce the high mortality associated with invasive fungal infections. Candida albicans IFA IgG has been recently commercialized for germ tube antibody detection (CAGTA. This test provides a rapid and simple diagnosis of IC (84.4% sensitivity and 94.7% specificity. The aim of this study is to identify the patients who could be benefited by the use of CAGTA test in critical care setting. Methods A prospective, cohort, observational multicentre study was carried out in six medical/surgical Intensive care units (ICU of tertiary-care Spanish hospitals. Candida albicans Germ Tube Antibody test was performed twice a week if predetermined risk factors were present, and serologically demonstrated candidiasis was considered if the testing serum dilution was ≥ 1:160 in at least one sample and no other microbiological evidence of invasive candidiasis was found. Results Fifty-three critically ill non-neutropenic patients (37.7% post surgery were included. Twenty-two patients (41.5% had CAGTA-positive results, none of them with positive blood culture for Candida. Neither corrected colonization index nor antifungal treatment had influence on CAGTA results. This finding could corroborate that the CAGTA may be an important biomarker to distinguish between colonization and infection in these patients. The presence of acute renal failure at the beginning of the study was more frequent in CAGTA-negative patients. Previous surgery was statistically more frequent in CAGTA-positive patients. Conclusions This study identified previous surgery as the principal clinical factor associated with CAGTA-positive results and emphasises the utility of this promising technique, which was not

  14. 9 CFR 114.9 - Outline of Production guidelines. (United States)


    .... Production of polyclonal antibody components. 1. If purchased, list suppliers, criteria for acceptability...-antibody reactions, and other diagnostics whose production methods are amenable to description as described.... Principle of the test (ELISA, latex agglutination, etc.). 2. Antigen or antibody detection test. 3....

  15. Large Scale Production and Characterization of Anti-Human IgG Monoclonal Antibody in Peritoneum of Balb/c MICE

    Directory of Open Access Journals (Sweden)

    B. Baradaran


    Full Text Available Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers .For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human IgG were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After ten days, approximately 3 ml ascitic fluid was harvested from the peritoneum of each mouse. Ascitic fluid was assayed for the titer of monoclonal antibody in reaction with human IgG and its cross reactivity in reaction with IgM & IgA. The titer of mAb was 100,000 and didn't show cross reactivity with IgM & IgA. Immunobloting was done for confirming the ELISA method. In immunobloting, only one sharp band in the heavy chain position of IgG was developed. The subclass of antibody was IgG1 and its light chain was kappa. Ascitic fluid was purified by ion exchange chromatography and the purified monoclonal antibody was conjugated with HRP. The conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of all other infectious diseases.

  16. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

    Directory of Open Access Journals (Sweden)

    H. Sun


    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

  17. Evaluation of a safranin-O-stained antigen microagglutination test for francisella tularensis antibodies.



    A microagglutination test for Francisella tularensis, with 0.025-ml amounts of diluted sera and 0.025-ml amounts of safranin-O-stained antigen in U-bottom microtitration plates, was compared with a tube agglutination test by using 137 sera. There was 86.3% agreement (+/- 1 dilution variation) between the microagglutination results and the tube agglutination results for sera with tube agglutination titers of greater than or equal to 20. There was 100% agreement (+/- 1 dilution variation) for s...

  18. Peptide-based antibodies against glutathione-binding domains suppress superoxide production mediated by mitochondrial complex I. (United States)

    Chen, Jingfeng; Chen, Chwen-Lih; Rawale, Sharad; Chen, Chun-An; Zweier, Jay L; Kaumaya, Pravin T P; Chen, Yeong-Renn


    Complex I (NQR) is a critical site of superoxide (O2-*) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYICGEETALIESIEGK(219) of 51-kDa protein and (361)VDSDTLCTEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O2-* generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated O2-*generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.

  19. Intranasal administration of HIV-DNA vaccine formulated with a polymer, carboxymethylcellulose, augments mucosal antibody production and cell-mediated immune response. (United States)

    Hamajima, K; Sasaki, S; Fukushima, J; Kaneko, T; Xin, K Q; Kudoh, I; Okuda, K


    We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1)IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n. -immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-gamma production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.

  20. Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing. (United States)

    Mytych, Daniel T; Barger, Troy E; King, Chadwick; Grauer, Stephanie; Haldankar, Raj; Hsu, Eric; Wu, Michelle Min; Shiwalkar, Mukta; Sanchez, Sergio; Kuck, Andrew; Civoli, Francesca; Sun, Jilin; Swanson, Steven J


    Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance

  1. Zirconium pressure tube testing: Test procedures, Production Assurance Program (Project H-700)

    Energy Technology Data Exchange (ETDEWEB)

    Zaloudek, F.R.; Lewis, M. [Pacific Northwest Lab., Richland, WA (United States)


    UNC Nuclear Industries (UNC) has initiated a plan for the fabrication of zirconium alloy pressure tubes required for the future operation of N-Reactor. As part of this plan, UNC is establishing a program to qualify and develop a process capable of fabricating these pressure tubes to the requirements of UNC specification HWS 6502, REV. 4, Amendment 1. The objective of the Pressure Tube Testing Task is to support the UNC program-by performing physical, mechanical and chemical testing on prototype tube sections produced during FY-1986, 1987 and 1988 and to test samples from production runs after 1988 as may be required. The types of tests included in the Zirconium Pressure Tube Testing Program will be as follows: tensile tests; burst tests; fracture toughness tests; corrosion tests; chemical composition analyses; grain structure evaluations. The purpose of this document is to define the procedures that will be used in each type of test included in this task.

  2. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS


    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.

  3. [Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies]. (United States)

    Akgüç, Miray; Karatayli, Ersin; Çelik, Esra; Koyuncu, Duygu; Çelik, İnci; Karatayli, Senem Ceren; Özden, Ali; Bozdayi, A Mithat


    At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H

  4. Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.

    NARCIS (Netherlands)

    Gorlani, A.; Hulsik, D.L.; Adams, H.; Vriend, G.; Hermans, P.; Verrips, T.


    Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that


    Energy Technology Data Exchange (ETDEWEB)

    Jantzen, C; Ned Bibler, N


    The Product Consistency Test (PCT), American Society for Testing Materials (ASTM) Standard C1285, is currently used world wide for testing glass and glass-ceramic waste forms for high level waste (HLW), low level waste (LLW), and hazardous wastes. Development of the PCT was initiated in 1986 because HLW glass waste forms required extensive characterization before actual production began and required continued characterization during production ({ge}25 years). Non-radioactive startup was in 1994 and radioactive startup was in 1996. The PCT underwent extensive development from 1986-1994 and became an ASTM consensus standard in 1994. During the extensive laboratory testing and inter- and intra-laboratory round robins using non-radioactive and radioactive glasses, the PCT was shown to be very reproducible, to yield reliable results rapidly, to distinguish between glasses of different durability and homogeneity, and to easily be performed in shielded cell facilities with radioactive samples. In 1997, the scope was broadened to include hazardous and mixed (radioactive and hazardous) waste glasses. In 2002, the scope was broadened to include glass-ceramic waste forms which are currently being recommended for second generation nuclear wastes yet to be generated in the nuclear renaissance. Since the PCT has proven useful for glass-ceramics with up to 75% ceramic component and has been used to evaluate Pu ceramic waste forms, the use of this test for other ceramic/mineral waste forms such as geopolymers, hydroceramics, and fluidized bed steam reformer mineralized product is under investigation.

  6. Alternatives to animal testing in the safety evaluation of products. (United States)

    Knight, Derek J; Breheny, Damien


    The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.

  7. Ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells. (United States)

    Bancos, Simona; Bernard, Matthew P; Topham, David J; Phipps, Richard P


    The widely used non-steroidal anti-inflammatory drugs (NSAIDs) function mainly through inhibition of cyclooxygenases 1 and 2 (Cox-1 and Cox-2). Unlike Cox-1, Cox-2 is considered an inducible and pro-inflammatory enzyme. We previously reported that Cox-2 is upregulated in activated human B lymphocytes and using Cox-2 selective inhibitors that Cox-2 is required for optimal antibody synthesis. It is not known whether commonly used non-prescription and non-Cox-2 selective drugs also influence antibody synthesis. Herein, we tested a variety of Cox-1/Cox-2 non-selective NSAIDs, namely ibuprofen, tylenol, aspirin and naproxen and report that they blunt IgM and IgG synthesis in stimulated human peripheral blood mononuclear cells (PBMC). Ibuprofen had its most profound effects in inhibiting human PBMCs and purified B lymphocyte IgM and IgG synthesis when administered in the first few days after activation. As shown by viability assays, ibuprofen did not kill B cells. The implications of this research are that the use of widely available NSAIDs after infection or vaccination may lower host defense. This may be especially true for the elderly who respond poorly to vaccines and heavily use NSAIDs.

  8. Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Caroline A Menezes


    Full Text Available Strains of enterotoxigenic Escherichia coli (ETEC are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

  9. Comparison of serological tests for the detection of ovine and caprine antibody to Brucella melitensis. (United States)

    Nielsen, K; Gall, D; Smith, P; Balsevicius, S; Garrido, F; Ferrer, M Durán; Biancifiori, F; Dajer, A; Luna, E; Samartino, L; Bermudez, R; Moreno, F; Renteria, T; Corral, A


    The indirect enzyme-linked immunosorbent assay (IELISA), the competitive enzyme-linked immunosorbent assay (CELISA) and the fluorescence polarisation assay (FPA) were evaluated with sera from sheep experimentally infected with Brucella melitensis and negative Canadian sheep. The sensitivity and specificity of the assays were as follows: IELISA: 91.7% and 97.6%, CELISA: 75.0% and 99.8% and FPA: 91.7% and 89.5%. Sera from the same experimental population were divided according to serological reaction in the rose bengal agglutination test (RBT) and the complement fixation test (CFT). Reactivity relative to the RBT positive and CFT positive sera were as follows: IELISA: 99.7%, CELISA: 93.2% and FPA: 99.1%. Since sera from goats with proven B. melitensis infection were not available, 699 sera from goats judged positive in the buffered antigen plate agglutination test (BPAT) and CFT and 982 BPAT/CFT negative Canadian goats were used. The sensitivity and specificity of the assays relative to the BPAT and CFT positive sera were: IELISA: 99.4% and 98.0%, CELISA: 95.4% and 97.1% and FPA: 92.7% and 99.8%.

  10. Field evaluation of a rapid diagnostic test to detect antibodies in human toxocariasis. (United States)

    Lim, P K C; Yamasaki, H; Mak, J W; Wong, S F; Chong, C W; Yap, I K S; Ambu, S; Kumarasamy, V


    Human toxocariasis which is caused mainly by the larvae of Toxocara canis and Toxocara cati, is a worldwide zoonotic disease that can be a potentially serious human infection. The enzyme-linked immunosorbent assay (ELISA) using T. canis excretory-secretory (TES) antigens harvested from T. canis larvae is currently the serological test for confirming toxocariasis. An alternative to producing large amounts of Toxocara TES and improved diagnosis for toxocariasis is through the development of highly specific recombinant antigens such as the T. canis second stage larva excretory-secretory 30 kDa protein (recTES-30). The aim of this study was to evaluate the sensitivity and specificity of a rapid diagnostic kit (RDT, named as iToxocara kit) in comparison to recTES-30 ELISA in Serendah Orang Asli village in Selangor, Malaysia. A total of 133 subjects were included in the study. The overall prevalence rates by ELISA and RDT were 29.3% and 33.1%, respectively, with more positive cases detected in males than females. However, no association was found between toxocariasis and gender or age. The percentage sensitivity, specificity, positive predictive value and negative predictive value of RDT were 85.7%, 90.1%, 80% and 93.2%, respectively. The prevalence for toxocariasis in this population using both ELISA and RDT was 27.1% (36/133) and the K-concordance test suggested good agreement of the two tests with a Cohen's kappa of 0.722, Ptoxocariasis.

  11. Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    Jackson MARCONDES; Ana Beatriz GARCIA


    Among various physiological responses to salt stress,the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl.In order to better understand the role of the SALT protein in the physiological processes involving salinity,it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique.This variety turned out to be an excellent model for that purpose,since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer.This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity.Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition.Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization,its lectin-like activity seems to play an important role in the establishment of stable complexes,either to other proteins or to oligosaccharides that are translocated to the chloroplast.

  12. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp. (United States)

    Wongtangprasert, Tossapon; Natakuathung, Wirongrong; Pimpitak, Umaporn; Buakeaw, Anumart; Palaga, Tanapat; Komolpis, Kittinan; Khongchareonporn, Nanthika


    A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.

  13. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zheng

    Full Text Available Human FAM76B (hFAM76B is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s of FAM76B, murine monoclonal antibodies (MAbs against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s of FAM76B.

  14. Red Blood Cell Antibody Identification (United States)

    ... ID, RBC; RBC Ab ID Formal name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC ... I should know? How is it used? Red blood cell (RBC) antibody identification is used as a follow- ...

  15. Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test. (United States)


    encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika. Two Sandfly Fever viruses (213452 and Candiru) were...thaw at room temperature for 5 minutes,. washed with PBS and drained. The cells were first incubated at 35°C wit an appropriate dilution of hybridoma...include IB8, IB10, ICl0, 4G2, 2C4, 1B6, 4BI0, and ID7 which generally reacted with all of the flaviviruses tested and did not recognize the sandfly

  16. Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A2: A Biotechnological Tool to Improve the Production of Antibodies

    Directory of Open Access Journals (Sweden)

    C. L. S. Guimarães


    Full Text Available Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2 of this toxin (BthTX-I and BthTX-II were chemically modified (alkylation by p-bromophenacyl bromide (BPB in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated. BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2 diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

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

    Directory of Open Access Journals (Sweden)

    Konstantinos Tsiveriotis


    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.

  18. Antineutrophil cytoplasmic antibodies testing in a large cohort of unselected greek patients. (United States)

    Tsiveriotis, Konstantinos; Tsirogianni, Alexandra; Pipi, Elena; Soufleros, Konstantinos; Papasteriades, Chryssa


    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.

  19. Paired comparison product testing when individual preferences are stochastic

    NARCIS (Netherlands)

    B. Wierenga (Berend)


    textabstractThe preference of an individual in a paired comparison product test is stochastic, i.e. the probability p, that he will prefer one of the two alternatives, is not 0 or 1, but lies somewhere between these values. It is shown that the distribution of p, when approximated by a beta distribu

  20. Detection of antibody responses by using haemagglutination inhibiton test and the protection titer of avian influenza virus H5N1 subtype

    Directory of Open Access Journals (Sweden)

    Risa Indriani


    Full Text Available Study on the detection of antibody responses using haemagglutination inhibition (HI test and the protection titer to Avian influenza (AI virus H5N1 subtype local isolate has been conducted at the Research Institute for Veterinary Science (RIVS. A total number of 50 village chicken (10 chicken served as un-injected controls and 30 quail were injected intramuscularly with inactivated virus of AI H5N1 subtype local isolate. Serum samples were collected 3 weeks after injection and were tested using haemagglutination inhibition tests. The correlation between antibody titer and its protection to AI virus H5N1 local isolate were measured by challenging the birds with AI virus H5N1 local isolate The HI test was then used to determine field serum samples. A total number of 48 village chicken from three (3 Districts (Bekasi, Tangerang and Bogor and 96 quails from two (2 farms in District of Sukabumi which were all vaccinated with commercial AI adjuvant vaccine were sampled. The study revealed that village chicken and quails showed antibody responses after 3 weeks vaccination and that titer of ≥ 3 log 2 was able to protect chicken and quails when they were challenged with local isolate virus. Based on this result, village chicken field samples from Districts of Tangerang, Bekasi and Bogor showed antibody titer which will protect 50, 100 and 85% of the flocks respectively. While quail field samples from Farm I and Farm II in District of Sukabumi showed antibody titer which will protect 60-100% and 0-80% of the flocks respectively. It is concluded that the study has successfully measured antibody titer to AI virus H5N1 subtype which protect village chicken and quails from local isolate virus challenge so that the results will be used to analyze field serum samples after vaccination program to eradicate AI from Indonesia.

  1. Indirect Haemagglutination Test in Comparison with ELISA for Detection of Antibodies against Invasive Amoebiasis (United States)

    Dhanalakshmi, Sankaramoorthy; Meenachi, Chidambaram


    Introduction Diagnosis of amoebiasis is based on combination of tests like microscopy, imaging, serology and molecular methods. In absence of molecular techniques, serology can be used as an alternative aid. Various serological techniques were reported with different sensitivity and specificity. The diagnostic efficiency of these assays mainly depends on the characteristics of antigen that is being used and various conditions of performance. Aim To evaluate the efficiency of recombinant calcium binding domain containing protein by Indirect Haemagglutination Assay (IHA) against a commercial ELISA among amoebic liver abscess cases and control group. Materials and Methods The study was carried out during the period of 2011-2015 and blood samples were collected from suspected amoebiasis cases who were attending the clinics of Medicine and Paediatrics department, JIPMER. A total of 200 sera samples which included 100 Amoebic Liver Abscess (ALA), 50 cases of other parasitic infections and liver diseases and 50 presumed healthy controls were examined by IHA and commercial ELISA. In brief, chick cells were stabilized by Double Aldehyde Sensitization (DAS) method. Optimum Sensitizing Dose (OSD) of the antigen was determined. The test was performed in a U-bottomed microtiter plate with recombinant amoebic antigen (12.5μg/ml), incubated at Room Temperature (RT) for 2 hours. RIDASCREEN Entamoeba IgG ELISA kit which is commercially available was used to evaluate the samples as per manufacturer’s instruction. Results The overall sensitivity and specificity of the IHA was 62% and 96%, respectively when compared to ELISA having sensitivity and specificity of 69% and 90%, respectively. The positive predictive value of the IHA was 91% while negative predictive value was 79%. Similarly, the positive predictive value of the ELISA was 87% while negative predictive value was 74%. Conclusion As serology heavily suffers due to lack of a standardised test system employing the native

  2. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody. (United States)


    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic...

  3. Comparison of local Salmonella pullorum antigen with imported product in whole blood agglutination test

    Directory of Open Access Journals (Sweden)

    Priyani Medewewa

    Full Text Available Background: Salmonellosis is considered as one of the most important diseases in poultry as it causes devastating losses in chicken industry. Proper identification of the infected and carrier birds is required to control the disease among chickens. In field situation whole blood agglutination test is performed in order to identify carriers of Pullorum and Fowl typhoid particularly, in breeder operations. In this test, serum antibodies are detected by using a specially made antigen for this purpose. In Sri Lanka, three antigen products are used commonly in whole blood agglutination test. Objectives: This study was carried out to compare these two locally available S. Pullorum antigen products and to determine any difference in the efficacy. Methods:“Shaver Brown” commercial layer birds (70 in number were used in the experiment. Birds were inoculated orally with 1.8X109cfu/ml of S. Pullorum at 16 weeks age. After Three weeks post inoculation, blood was collected from each bird and Whole blood agglutination test was performed using both antigen products. Fifteen (15 inoculated hens were selected randomly and cloacal swabs were cultured on cultured Agar on same day of serum collected. Results: In this study, there was no significant difference observed between two antigens to detect carrier birds by whole blood agglutination test. Salmonella was not isolated from cloacal swabs since no observed excretion of Salmonella Pullorum through faces. All cloacal swabs gave negative results, when cultured on artificial Agar. Conclusion: Both antigen can be used effectively to detect carrier birds under the control program in country. [Vet World 2012; 5(9.000: 546-548

  4. Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity (United States)

    Ramon, Sesquile; Bancos, Simona; Serhan, Charles N.; Phipps, Richard P.


    Summary Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPMs have been identified in important immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A4 (LXA4) and its receptor ALX/FPR2 on human B cells. LXA4 decreased IgM and IgG production on activated B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA4 also inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. Lastly, LXA4 decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the actions of lipoxins on human B cells, which shows a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B cell antibody production can be beneficial to threat inflammatory and autoimmune disorders. PMID:24166736


    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明


    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  6. Detection of filaria-specific IgG4 antibodies using Brugia Rapid test in individuals from an area highly endemic for Brugia timori. (United States)

    Supali, T; Rahmah, N; Djuardi, Y; Sartono, E; Rückert, Paul; Fischer, P


    The filarial parasite Brugia timori is of great public health importance in some islands of Eastern Indonesia. To establish a simple serological test for the identification and post-treatment monitoring of areas endemic for B. timori, a rapid immunochromatographic dipstick test (Brugia Rapid, BR) was evaluated on microfilaraemic and amicrofilaraemic individuals. This test is based on the detection of anti-filarial IgG4 antibodies that react with a recombinant Brugia malayi antigen (BmR1). In our study area on Alor island the prevalence of microfilaraemia was 26%. With the BR test, 100% of 196 sera from microfilaraemic persons and 76% of 563 sera from amicrofilaraemic persons, either symptomatic or asymptomatic, reacted positive. All 50 control sera from areas non-endemic for lymphatic filariasis gave negative BR test results. This study showed that the BR test can be also used to detect antibodies against B. timori. Due to the high prevalence of IgG4 antibodies as detected by the BR test (81%), no significant correlation with the prevalence of microfilaraemia could be detected within the endemic village. The BR test also shows great promise to be employed as a monitoring tool for B. timori in the framework of the Global Program to Eliminate Lymphatic Filariasis (GPELF).

  7. Thermal influences on antibody production and metabolism in chicken lines divergently selected for immune responsiveness.

    NARCIS (Netherlands)

    Donker, R.


    IntroductionThe international character of the poultry industry requires that poultry bred under temperate conditions should be able to perform under tropical conditions as well. However, abrupt changes in environmental temperature or continuous high temperatures may affect production traits, im

  8. Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site. (United States)

    Kawabe, Yoshinori; Inao, Takanori; Komatsu, Shodai; Huang, Guan; Ito, Akira; Omasa, Takeshi; Kamihira, Masamichi


    Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.

  9. Enhanced performance of an innovative dengue IgG/IgM rapid diagnostic test using an anti-dengue EDI monoclonal antibody and dengue virus antigen. (United States)

    Lee, Jihoo; Kim, Young-Eun; Kim, Hak-Yong; Sinniah, Mangalam; Chong, Chom-Kyu; Song, Hyun-Ok


    High levels of anti-dengue IgM or IgG can be detected using numerous rapid diagnostic tests (RDTs). However, the sensitivity and specificity of these tests are reduced by changes in envelope glycoprotein antigenicity that inevitably occur in limited expression systems. A novel RDT was designed to enhance diagnostic sensitivity. Dengue viruses cultured in animal cells were used as antigens to retain the native viral coat protein. Monoclonal antibodies (mAbs) were then developed, for the first time, against domain I of envelope glycoprotein (EDI). The anti-dengue EDI mAb was employed as a capturer, and EDII and EDIII, which are mainly involved in the induction of neutralizing antibodies in patients, were fully available to bind to anti-dengue IgM or IgG in patients. A one-way automatic blood separation device prevented reverse migration of plasma and maximize the capture of anti-dengue antibodies at the test lines. A clinical evaluation in the field proved that the novel RDT (sensitivities of 96.5% and 96.7% for anti-dengue IgM and IgG) is more effective in detecting anti-dengue antibodies than two major commercial tests (sensitivities of 54.8% and 82% for SD BIOLINE; 50.4% and 75.3% for PanBio). The innovative format of RDT can be applied to other infectious viral diseases.

  10. An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk (United States)

    A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

  11. Antimitochondrial antibody (United States)

    ... page: // Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  12. Seroepidemiological detection of antibodies against Leptospira spp using microscopic agglutination test in Urmia cows and sheep

    Directory of Open Access Journals (Sweden)

    Ramin Ag


    Full Text Available The study was designed to determine the level of incidence, titer and various serovars of leptospira in 203 cows and 166 sheep at Urmia abattoir in 2011. Blood samples were collected during the slaughter of animals and sera were separated to evaluate the serological reaction to Leptospira spp by Microscopic Agglutination Test (MAT using live antigens representing Leptospira interrogans serogroups: pomona, grippotyphosa, canicola, hardjo, icterrohaemoragiae, and ballum. Overall, 36% of cows and 19.3% of sheep including 33.8% of bulls, 40.5% of female cows, 18.3% of rams and 25% of ewes had a positive reaction to at least one of the leptospira serovars. The most prevalent serovars in cows were pomona (22.7%, grippotyphosa (13.8%, and hardjo (8.4%, and in sheep were grippotyphosa (66.7%, pomona (26.2% and canicola (7.1%. Other serovars were not detected in cows and sheep. The most prevalent serological titers of 1:100 and 1:200 in cows was 18.2% and 26.6%, and for sheep were 13.5% and 8%, respectively, and of 1:400 in sheep was 2.3%. Cows with a positive reaction to one, two and three serovars were 28.6%, 5.9%, and 1.5% and sheep positive to one and two serovars were 13.3% and 6%, respectively. Age comparison in seropositive cows and sheep showed a significantly increased infection (p<0.05 from young to adult ruminants, while no differences were seen regarding gender. The main mixed serovars were between grippotyphosa/pomona, grippotyphosa/canicola and canicola/pomona. The gender comparison of the serovars' distribution revealed that the pomona and grippotyphosa were predominant among other leptospiral serovars in cows and sheep, respectively. In conclusion, the rate of leptospirosis in Urmia cows was about 2 fold in sheep. The most current serovars in cows and sheep were pomona and grippotyphosa, respectively. The majority of animals was infected with one serovar, but polyserovars, are also possible. The highest titer (1:200 was observed in cows

  13. Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column. (United States)

    Zhang, Zhaowei; Hu, Xiaofeng; Zhang, Qi; Li, Peiwu


    Mycotoxins usually found in agricultural products such as peanut, corn, and wheat, are a serious threat to human health and their detection requires multiplexed and sensitive analysis methods. Herein, a simultaneous determination for aflatoxin B1, B2, G1, G2, ochratoxin A, zearalanone and T-2 toxin was investigated using high performance liquid chromatography coupled with tandem mass spectrometry in a single run via a home-made multiple immunoaffinity column. Four monoclonal antibodies were produced in our lab against aflatoxins, ochratoxin A, zearalanone and T-2 toxin, respectively, then combined as a pool and bound to Sepharose-4B for affinity chromatography. Seven mycotoxins were effectively extracted from the agricultural product samples by using acetonitrile/water/acetic acid (80:19:1, v/v/v) Then, the extraction was cleanup by multiple immunoaffinity column. This method demonstrated a considerable linear range of 0.30-25, 0.12-20, 0.30-20, 0.12-20, 0.60-30, 0.30-25, and 1.2-40μgkg(-1)and lower limits of detection at 0.1, 0.04, 0.1, 0.04, 0.2, 0.1 and 0.4μgkg(-1) for AFB1, AFB2, AFG1, AFG2, OTA, ZEN and T-2, respectively, in comparison with previously reported methods, as well as excellent recoveries. The mIAC capacity for AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and T-2 were 187, 181, 153, 151, 105, 130, 88ng, respectively. It was found that all of the 7 mycotoxins were present in 90 agricultural product samples. The proposed method meets the requirements for rapid sample preparation and highly sensitive identification of multiple mycotoxins in agricultural product and food safety. This method provides a promising alternative with high throughput and high sensitivity for rapid analysis of seven mycotoxins in the monitoring of food safety.

  14. Antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of IL-10 production in monocytes.

    Directory of Open Access Journals (Sweden)

    Tsung-Ting Tsai


    Full Text Available Interleukin (IL-10 levels are increased in dengue virus (DENV-infected patients with severe disorders. A hypothetical intrinsic pathway has been proposed for the IL-10 response during antibody-dependent enhancement (ADE of DENV infection; however, the mechanisms of IL-10 regulation remain unclear.We found that DENV infection and/or attachment was sufficient to induce increased expression of IL-10 and its downstream regulator suppressor of cytokine signaling 3 in human monocytic THP-1 cells and human peripheral blood monocytes. IL-10 production was controlled by activation of cyclic adenosine monophosphate response element-binding (CREB, primarily through protein kinase A (PKA- and phosphoinositide 3-kinase (PI3K/PKB-regulated pathways, with PKA activation acting upstream of PI3K/PKB. DENV infection also caused glycogen synthase kinase (GSK-3β inactivation in a PKA/PI3K/PKB-regulated manner, and inhibition of GSK-3β significantly increased DENV-induced IL-10 production following CREB activation. Pharmacological inhibition of spleen tyrosine kinase (Syk activity significantly decreased DENV-induced IL-10 production, whereas silencing Syk-associated C-type lectin domain family 5 member A caused a partial inhibition. ADE of DENV infection greatly increased IL-10 expression by enhancing Syk-regulated PI3K/PKB/GSK-3β/CREB signaling. We also found that viral load, but not serotype, affected the IL-10 response. Finally, modulation of IL-10 expression could affect DENV replication.These results demonstrate that, in monocytes, IL-10 production is regulated by ADE through both an extrinsic and an intrinsic pathway, all involving a Syk-regulated PI3K/PKB/GSK-3β/CREB pathway, and both of which impact viral replication.

  15. The Role of TLR4 on B Cell Activation and Anti-β2GPI Antibody Production in the Antiphospholipid Syndrome (United States)


    High titer of anti-β2-glycoprotein I antibodies (anti-β2GPI Ab) plays a pathogenic role in antiphospholipid syndrome (APS). Numerous studies have focused on the pathological mechanism in APS; however, little attention is paid to the immune mechanism of production of anti-β2GPI antibodies in APS. Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human β2-glycoprotein I (β2GPI). TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with β2GPI. However, TLR4 provides a third signal for B cell activation and then promotes B cells better receiving signals from both B cell antigen receptor (BCR) and CD40, thus promoting B cell activation, surface molecules expression, anti-β2GPI Ab production, and cytokines secretion and making B cell functioning like an antigen presenting cell (APC). At the same time, TLR4 also promotes B cells producing antibodies by upregulating the expression of B-cell activating factor (BAFF). In this paper, we aim to review the functions of TLR4 in B cell immune response and antibody production in autoimmune disease APS and try to find a new way for the prevention and treatment of APS. PMID:27868072

  16. Efficient production of a bioactive Bevacizumab monoclonal antibody using the 2A self-cleavage peptide in transgenic rice callus

    Directory of Open Access Journals (Sweden)

    Lei Chen


    Full Text Available Bevacizumab, a humanized monoclonal antibody (mAb targeting to the vascular endothelial growth factor (VEGF, has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC and heavy chain (BHC genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus (FMDV, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme linked immunosorbent assay (ELISA analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg kg-1FW in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target hVEGF antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

  17. Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus (United States)

    Chen, Lei; Yang, Xiaoyu; Luo, Da; Yu, Weichang


    Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7–242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment. PMID:27555853

  18. Dengue NS1 and prM antibodies increase the sensitivity of acute dengue diagnosis test and differentiate from Japanese encephalitis infection. (United States)

    Gowri Sankar, S; Balaji, T; Venkatasubramani, K; Thenmozhi, V; Dhananjeyan, K J; Paramasivan, R; Tyagi, B K; John Vennison, S


    Accurate and early diagnosis of dengue infection is essential for dengue case management. In outbreak conditions, it is essential to include two different tests to diagnose dengue and the choice depends on the number of days after the onset of illness in which the sample is collected. During the laboratory diagnosis of dengue in late acute and convalescent phase by MAC-ELISA, it is necessary to rule out possible cross reactions of closely related flavivirus, such as Japanese encephalitis virus which is commonly co-circulating. In the present investigation, the usefulness of dengue virus NS1 and prM antibodies in diagnosing and differentiating dengue from Japanese encephalitis infection was assessed using samples collected during out-breaks. It was shown here that, detection of antibodies against dengue NS1 and prM proteins increases the sensitivity of dengue diagnosis until 15days. Moreover, detection of antibodies against both proteins was able to differentiate dengue from Japanese encephalitis infection.

  19. Hepatitis C Test (United States)

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities Hepatitis C Testing Share this page: Was this page helpful? Also known as: Hepatitis C Antibody; Anti-HCV; HCV-PCR; HCV-RNA; ...

  20. Product consistency testing of West Valley Compositional Variation Glasses

    Energy Technology Data Exchange (ETDEWEB)

    Olson, K.M.; Marschman, S.C.; Piepel, G.F.; Whiting, G.K.


    Nuclear waste glass produced by the West Valley Demonstration Project (WVDP) must meet the requirements of the Waste Acceptance Preliminary Specification (WAPS) as developed by the US Department of Energy (DOE). To assist WVDP in complying with WAPS, the Materials Characterization Center (MCC) at Pacific Northwest Laboratory (PNL) used the Product Consistency Test (PCT) to evaluate 44 West Valley glasses that had previously been tested in FY 1987 and FY 1988. This report summarizes the results of the PCTs. The glasses tested, which were fabricated as sets of Compositional Variation Glasses for studies performed by the West Valley Support Task (WVST) at PNL during FY 1987 and FY 1988, were doped with Th and U and were variations of West Valley reference glasses. In addition, Approved Reference Material-1 (ARM-1) was used as a test standard (ARM-1 is supplied by the MCC). The PCT was originated at Westinghouse Savannah River Company (WSRC) by C. M. Jantzen and N. R. Bibler (Jantzen and Bibler 1989). The test is a seven-day modified MCC-3 test that uses crushed glass in the size range -100 +200 mesh with deionized water in a Teflon container. There is no agitation during the PCT, and no attempt to include CO{sub 2} from the test environment. Based on B and Li release, the glasses performed about the same as in previous modified MCC-3 testing performed in FY 1987 and FY 1988 (Reimus et al. 1988). The modified MCC-3 tests performed by Reimus et al. were similar to the PCT containers and the exclusion of CO{sub 2} from the tests.

  1. Frequencies and Specificities of “Enzyme-Only” Detected Erythrocyte Alloantibodies in Patients Hospitalized in Austria: Is an Enzyme Test Required for Routine Red Blood Cell Antibody Screening?

    Directory of Open Access Journals (Sweden)

    Dietmar Enko


    Full Text Available The aim of this study was to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS- indirect antiglobulin test (IAT and with an enzyme-pretreated (papain 3-cell panel fully automated on the ORTHO AutoVue Innova System. The antibody identification was carried out manually with an 11-cell panel in the LISS-IAT and with an enzyme-pretreated (papain 11-cell panel. In total 4.05% (n=98 of all patients (n=2420 had a positive RBC antibody screening result. Of them 25.51% (25/98 showed “enzyme-only” detected specific or nonspecific RBC alloantibodies. Rhesus and Lewis system antibodies were found the only specificities of “enzyme-only” RBC alloantibodies: all in all 4.8% (4/98 were detected with anti-E, 3.06% (3/98 with anti-Lea, 3.06% (3/98 with anti-D after anti-D prophylaxis and 1.02% (1/98 with anti-e. In total, 14.29% (14/98 showed a nonspecific RBC alloantibody result with the enzyme test. The results of the present study demonstrate that a high number of unwanted positive reactions with the enzyme technique overshadows the detection of “enzyme-only” RBC alloantibodies. (Trial Registration: K-37-13.

  2. Antithyroid microsomal antibody (United States)

    ... to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also used to find ... positive test may be due to: Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also ...

  3. Accuracy of a point-of-care ELISA test kit for predicting the presence of protective canine parvovirus and canine distemper virus antibody concentrations in dogs. (United States)

    Litster, A L; Pressler, B; Volpe, A; Dubovi, E


    Canine parvovirus (CPV) and canine distemper virus (CDV) are highly infectious and often fatal diseases with worldwide distributions, and are important population management considerations in animal shelters. A point-of-care ELISA test kit is available to detect serum antibodies to CPV and CDV, and presumptively to predict protective status. The aim of this study was to determine the diagnostic accuracy of the test compared to CPV hemagglutination inhibition titers and CDV serum neutralization titers determined by a reference laboratory, using sera collected from dogs housed at animal shelters. The ELISA test was used under both field and laboratory conditions and duplicate specimens were processed using an extra wash step. The test kit yielded accurate results (CPV: sensitivity 92.3%, specificity 93.5%; CDV: sensitivity 75.7%, specificity 91.8%) under field conditions. CDV sensitivity was improved by performing the test under laboratory conditions and using an optical density (OD) meter (laboratory performed 94.0%; OD 88.1%). Point-of-care ELISA testing for serum CPV and CDV antibody titers was demonstrated to be a useful tool for determining antibody status when making decisions regarding the need for CPV and/or CDV vaccination and also in animal shelters for population management.

  4. Transient productivity index for numerical well test simulations

    Energy Technology Data Exchange (ETDEWEB)

    Blanc, G.; Ding, D.Y.; Ene, A. [Institut Francais du Petrole, Pau (France)] [and others


    The most difficult aspect of numerical simulation of well tests is the treatment of the Bottom Hole Flowing (BHF) Pressure. In full field simulations, this pressure is derived from the Well-block Pressure (WBP) using a numerical productivity index which accounts for the grid size and permeability, and for the well completion. This productivity index is calculated assuming a pseudo-steady state flow regime in the vicinity of the well and is therefore constant during the well production period. Such a pseudo-steady state assumption is no longer valid for the early time of a well test simulation as long as the pressure perturbation has not reached several grid-blocks around the well. This paper offers two different solutions to this problem: (1) The first one is based on the derivation of a Numerical Transient Productivity Index (NTPI) to be applied to Cartesian grids; (2) The second one is based on the use of a Corrected Transmissibility and Accumulation Term (CTAT) in the flow equation. The representation of the pressure behavior given by both solutions is far more accurate than the conventional one as shown by several validation examples which are presented in the following pages.

  5. Production of LEU Fully Ceramic Microencapsulated Fuel for Irradiation Testing

    Energy Technology Data Exchange (ETDEWEB)

    Terrani, Kurt A [ORNL; Kiggans Jr, James O [ORNL; McMurray, Jake W [ORNL; Jolly, Brian C [ORNL; Hunt, Rodney Dale [ORNL; Trammell, Michael P [ORNL; Snead, Lance Lewis [ORNL


    Fully Ceramic Microencapsulated (FCM) fuel consists of tristructural isotropic (TRISO) fuel particles embedded inside a SiC matrix. This fuel inherently possesses multiple barriers to fission product release, namely the various coating layers in the TRISO fuel particle as well as the dense SiC matrix that hosts these particles. This coupled with the excellent oxidation resistance of the SiC matrix and the SiC coating layer in the TRISO particle designate this concept as an accident tolerant fuel (ATF). The FCM fuel takes advantage of uranium nitride kernels instead of oxide or oxide-carbide kernels used in high temperature gas reactors to enhance heavy metal loading in the highly moderated LWRs. Production of these kernels with appropriate density, coating layer development to produce UN TRISO particles, and consolidation of these particles inside a SiC matrix have been codified thanks to significant R&D supported by US DOE Fuel Cycle R&D program. Also, surrogate FCM pellets (pellets with zirconia instead of uranium-bearing kernels) have been neutron irradiated and the stability of the matrix and coating layer under LWR irradiation conditions have been established. Currently the focus is on production of LEU (7.3% U-235 enrichment) FCM pellets to be utilized for irradiation testing. The irradiation is planned at INL s Advanced Test Reactor (ATR). This is a critical step in development of this fuel concept to establish the ability of this fuel to retain fission products under prototypical irradiation conditions.

  6. Development, Production and Testing of 4500 Beam Loss Monitors

    CERN Document Server

    Holzer, E B; Dehning, B; Ferioli, G; Grishin, V; Jimenez, T M; Koshelev, A; Kramer, Daniel; Larionov, A; Taborelli, M; Seleznev, V; Sleptsov, M; Sytin, A; Wevers, I


    Beam-loss monitoring (BLM) [1] is a key element in the LHC machine protection. 4250 nitrogen filled ionization chambers (IC) and 350 secondary emission monitors (SEM) have been manufactured and tested at the Institute for High Energy Physics (IHEP) in Protvino, Russia, following their development at CERN. Signal speed and robustness against aging were the main design criteria. Each monitor is permanently sealed inside a stainless-steel cylinder. The quality of the welding was a critical aspect during production. The SEMs are requested to hold a vacuum of $10^{-7}$ bar. Impurity levels from thermal and radiationinduced desorption should remain in the range of parts per million in the ICs. To avoid radiation aging (up to $2·10^{8}$ Gy in 20 years) production of the chambers followed strict UHV requirements. IHEP designed and built the UHV production stand. Due to the required dynamic range of $10^{8}$, the leakage current of the monitors has to stay below 2 pA. Several tests during and after production were ...

  7. Development and Testing of the VAHIRR Radar Product (United States)

    Barrett, Joe III; Miller, Juli; Charnasky, Debbie; Gillen, Robert; Lafosse, Richard; Hoeth, Brian; Hood, Doris; McNamara, Todd


    Lightning Launch Commit Criteria (LLCC) and Flight Rules (FR) are used for launches and landings at government and commercial spaceports. They are designed to avoid natural and triggered lightning strikes to space vehicles, which can endanger the vehicle, payload, and general public. The previous LLCC and FR were shown to be overly restrictive, potentially leading to costly launch delays and scrubs. A radar algorithm called Volume Averaged Height Integrated Radar Reflectivity (VAHIRR), along with new LLCC and FR for anvil clouds, were developed using data collected by the Airborne Field Mill II research program. VAHIRR is calculated at every horizontal position in the coverage area of the radar and can be displayed similar to a two-dimensional derived reflectivity product, such as composite reflectivity or echo tops. It is the arithmetic product of two quantities not currently generated by the Weather Surveillance Radar 1988 Doppler (WSR-88D): a volume average of the reflectivity measured in dBZ and the average cloud thickness based on the average echo top height and base height. This presentation will describe the VAHIRR algorithm, and then explain how the VAHIRR radar product was implemented and tested on a clone of the National Weather Service's (NWS) Open Radar Product Generator (ORPG-clone). The VAHIRR radar product was then incorporated into the Advanced Weather Interactive Processing System (AWIPS), to make it more convenient for weather forecasters to utilize. Finally, the reliability of the VAHIRR radar product was tested with real-time level II radar data from the WSR-88D NWS Melbourne radar.

  8. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo


    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  9. Phase 1 pixel modules production and High Density Interconnect testing

    CERN Document Server

    Still, Joseph


    During the first run of the LHC, luminosity peaked at $1 \\times 10^{34} cm^{-2}s^{-1}$ with $ \\approx 50 ns$ bunch spacing a pile-up of about 25, or simultaneous inelastic collisions per crossing, occur in the CMS experiment. However after the upgrade of of the LHC during long shut down 1, luminosity, and therefore pile-up. Therefore the CMS pixel tracker has to be upgraded to be able to operate correctly under this news stronger constraints. That is how this CERN Summer Student project, which took place at the CERN Meyrin site, comes within the framework of the pixel detector upgrade in the CMS experiment with a work aimed on the phase 1 of pixel modules production and tests of the HDI. The production and tests of the HDI were held in cleanroom facilities. This included first hand as well as to work on pixel modules building and performing size and flatness tests on them, and on a other hand testing several HDIs. At first, prototypes modules were assembled before real modules building. Another aspect of work...

  10. Endurance test on IR rig for RI production

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Heung June; Youn, Y. J.; Han, H. S.; Hong, S. B.; Cho, Y. G.; Ryu, J. S


    This report presents the pressure drop, vibration and endurance test results for IR rig for RI production which were desigened and fabricated by KAERI. From the pressure drop test results, it is noted that the flow rate through the IR rig corresponding to the pressure drop of 200 kPa is measured to be about 3.12 kg/sec. Vibration frequency for the IR rig ranges from 13 to 17 Hz. RMS(Root Mean Square) displacement for the IR rig is less than 30 {mu}m, and the maximum displacement is less than 110{mu}m. These experimental results show that the design criteria of IR rig meet the HANARO limit conditions. Endurance test results show that the appreciable fretting wear for the IR rig does not occur, however tiny trace of wear between contact points is observed.

  11. Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A. (United States)

    de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Al Saati, Talal; Boyes, Jeannine; Delsol, Georges; Allal, Cuider; Marsili, Sabrina; Silvente-Poirot, Sandrine; Poirot, Marc


    We have recently discovered the existence of 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-o-hemisuccinate with a carboxylic spacer arm attached to the 3β-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

  12. Production of anti-fullerene C{sub 60} polyclonal antibodies and study of their interaction with a conjugated form of fullerene

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickson, O. D., E-mail:; Fedyunina, N. S. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation); Martianov, A. A. [Moscow State University (Russian Federation); Zherdev, A. V.; Dzantiev, B. B. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation)


    The aim of this study was to produce anti-fullerene C{sub 60} antibodies for the development of detection systems for fullerene C{sub 60} derivatives. To produce anti-fullerene C{sub 60} antibodies, conjugates of the fullerene C{sub 60} carboxylic derivative with thyroglobulin, soybean trypsin inhibitor, and bovine serum albumin were synthesized by carbodiimide activation and characterized. Immunization of rabbits by the conjugates led to the production of polyclonal anti-fullerene antibodies. The specificity of the immune response to fullerene was investigated. Indirect competitive immunoenzyme assay was developed for the determination of conjugated fullerene with detection limits of 0.04 ng/mL (calculated for coupled C{sub 60}) and 0.4 ng/mL (accordingly to total fullerene-protein concentration).

  13. An alternative chemical redox method for the production of bispecific antibodies: implication in rapid detection of food borne pathogens.

    Directory of Open Access Journals (Sweden)

    Mohammad Owais

    Full Text Available Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC and the food borne pathogen Listeria monocytogenes (L. monocytogenes were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.

  14. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae. (United States)

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji


    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  15. Anti-smooth muscle antibody (United States)

    ... gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the presence ...

  16. Comparison of Buffered, Acidified Plate Antigen to Standard Serologic Tests for the Detection of Serum Antibodies to Brucella abortus in Elk (Cervus canadensis). (United States)

    Clarke, P Ryan; Edwards, William H; Hennager, Steven G; Block, Jean F; Yates, Angela M; Ebel, Eric; Knopp, Douglas J; Fuentes-Sanchez, Antonio; Jennings-Gaines, Jessica; Kientz, Rebecca L; Simunich, Marilyn


    Brucellosis (caused by the bacterium Brucella abortus) is a zoonotic disease endemic in wild elk (Cervus canadensis) of the Greater Yellowstone Ecosystem, US. Because livestock and humans working with elk or livestock are at risk, validated tests to detect the B. abortus antibody in elk are needed. Using the κ-statistic, we evaluated the buffered, acidified plate antigen (BAPA) assay for agreement with the results of the four serologic tests (card test [card], complement fixation test [CF], rivanol precipitation plate agglutination test [RIV], standard plate agglutination test [SPT]) that are approved by the US Department of Agriculture for the detection of the B. abortus antibody in elk. From 2006 to 2010, serum samples collected from elk within B. abortus-endemic areas (n = 604) and nonendemic areas (n = 707) and from elk culture-positive for B. abortus (n = 36) were split and blind tested by four elk serum diagnostic laboratories. κ-Values showed a high degree of agreement for the card (0.876), RIV (0.84), and CF (0.774) test pairings and moderate agreement for the SPT (0.578). Sensitivities for the BAPA, card, RIV, CF, and SPT were 0.859, 0.839, 0.899, 1.00, and 0.813, whereas specificities were 0.986, 0.993, 0.986, 0.98, and 0.968, respectively. The positive predictive values and the negative predictive values were calculated for 2.6%, 8.8%, and 16.2% prevalence levels. These findings suggest the BAPA test is a suitable screening test for the B. abortus antibodies in elk.

  17. Production of Recombinant Echinococcus granulosus Antigen B Subunits, In Order to Using Of Them in Serodiagnostic Tests of Hydatidosis

    Directory of Open Access Journals (Sweden)

    R Pazoki


    Full Text Available Background: Hydatidosis is one of the most important helminthiasis, and is a public health problem in many regions of the world. Methods: With the aim of production of recombinant subunits of antigen B, two different sequences of Echinococcus granulosus Antigen B, acquired from Gene Bank and amplified with specific primers via RT-PCR reaction. The amplified fragments (HI, HII cloned into pTZ57R T.vector, and then subcloned into pGEMEX-1 expression vector. Resaults: The SDS-PAGE performed after induction of cloned genes, and production of about 35 K.Da recombinant fusion proteins were confirmed for either two cloned genes. The immunogenicity of the recombinant fusion proteins were tested using double diffusion and immunoblotting. Both recombinant fusion proteins derived from lysate of transformed bacteria, were reactive for antibodies in serum of cystic hydatid patient. Conclusion: The produced recombinant antigen B subunits can be use in seroldiagnostic tests of hydatidosis, after purification.

  18. Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors. (United States)

    van der Geest, Kornelis S M; Lorencetti, Pedro G; Abdulahad, Wayel H; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H


    Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So far, few studies have directly assessed the effect of aging on the latter B cell function. Here, we determined if and how human aging influences the production of cytokines by B cells. In a cross-sectional study, we found that absolute numbers of circulating B cells were similar in 31 young (ages 19-39) and 73 old (age ≥ 60) individuals. Numbers of transitional B cells (CD19(+)CD27(-)CD38(High)CD24(High)) were decreased in old individuals, whereas numbers of naive and memory B cell subsets were comparable in young and old individuals. Short-term in vitro stimulation of whole blood samples revealed that numbers of B cells capable of producing TNF-α were similar in young and old individuals. In contrast, B cells capable of IL-10 production were decreased in old subjects. This decline of IL-10(+) B cells was observed in old individuals that were ANA positive, and in those that were negative for both ANAs and RFs. However, IL-10(+) B cells were remarkably well retained in the circulation of old subjects that were RF positive. Thus, pro-inflammatory TNF-α(+) B cells are retained in the elderly, whereas IL-10(+) B cells generally decline. In addition, our findings indicate that IL-10(+) B cells may differentially impact the development of ANAs and RFs in the elderly.

  19. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Anju Mohan


    Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

  20. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  1. MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica. (United States)

    Valero, M Adela; Ubeira, Florencio M; Khoubbane, Messaoud; Artigas, Patricio; Muiño, Laura; Mezo, Mercedes; Pérez-Crespo, Ignacio; Periago, M Victoria; Mas-Coma, Santiago


    During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.

  2. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

    Full Text Available Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs in transchromosomic (Tc cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin (Ig heavy-chain (hIGH, kappa-chain (hIGK, and lambda-chain (hIGL germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO. However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK and lambda-chain (bIGL genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ. To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J and constant (C gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5 by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ.

  3. Prokaryotic expression, purification, and production of polyclonal antibody against novel human serum inhibited related protein I (SI1). (United States)

    Ma, Mingxing; Ma, Jie; Shi, Yinghui; Wu, Hong; Zhao, Wenxiu; Huang, Weiwei; Jiao, Yang; Tan, Deyong


    A novel serum inhibited related gene (SI1) has been cloned in our lab by using mRNA differential display analysis of U251 cells in the presence or absence of serum, the expression of SI1 was dramatically inhibited by the addition of serum to serum starved cells. Previous reports suggested the potential significance of SI1 in regulating the cell cycle. In this study, the plasmid construction, protein expression and purification, as well as the generation of anti-SI1 polyclonal antibody are described. A full-length cDNA of Si1 was inserted in a prokaryotic expression plasmid pET28-b(+) and efficiently expressed in E. coli Rosetta (DE3) strain after induction by isopropyl-b-D: -thiogalactoside. The expressed 6His-tagged SI1 fusion protein was purified by Ni(+) affinity column and then used to immunize Balb/C mice, and the anti-SI1 polyclonal antibody was purified by protein A column. To determine the sensitivity and specificity of the antibody against SI1, a cell lysate of pEGFP-N2-SI1 plasmid transiently transfected Hela cell was identified by anti-GFP monoclonal antibody and anti-SI1 polyclonal antibody. Both the GFP-SI1 fusion protein and endogenous SI1 protein in Hela cell can be recognized by the anti-SI1 polyclonal antibody. The anti-SI1 polyclonal antibody will provide a useful tool for further characterization of SI1.

  4. Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies. (United States)

    Justiniano, Steven E; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P; Byrd, John C; Tridandapani, Susheela


    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

  5. Fcγ Receptor-induced Soluble Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Production Inhibits Angiogenesis and Enhances Efficacy of Anti-tumor Antibodies* (United States)

    Justiniano, Steven E.; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M.; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D.; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P.; Byrd, John C.; Tridandapani, Susheela


    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. PMID:23902770

  6. Production of cement requiring low energy expenditure. An industrial test

    Energy Technology Data Exchange (ETDEWEB)

    Gimenez, S.; Blanco, M.T.; Palomo, A.; Puertas, F. (Instituto de Ciencias de la Construccion, Madrid (Spain))


    A new method for making cement is proposed. It is based on the use of CaF{sub 2} and CaSO{sub 4} for partial replacement of the usual raw materials in cement manufacturing. This paper shows the feasibility of the proposed method on an industrial scale. A test carried out in a Spanish cement factory (1500 t yield of the new cement) has revealed that the mehtod can not only be adapted to the current technology but also requires a much lower energy expenditure. The final product is shown to have excellent properties in comparison with OPC. (orig.).

  7. Phase Retrieval Algorithm for Form Testing Metrology in Production Environment

    Directory of Open Access Journals (Sweden)

    Stephan Stuerwald


    Full Text Available Form testing interferometry permits a fast, non-tactile and full- field quantitative phase imaging of components in ultra precise manufacturing. To reduce the influence of vibrations under manufacturing conditions, it is most common to use the FT- based spatial carrier phase measurement technique (SCPM which requires only a single interferogram recording. The utilization of a generalized, relatively new spatial phase-shifting method operating in the position space opens up prospects for reduced phase noise and less reconstruction errors of the calculated phase-map under production conditions. Therefore this phase-shifting technique is investigated for applicability in machine integrated interferometric form testing of optical lenses. A characterization of the algorithm and a comparison with the commonly used FT-based algorithm is performed. As a reference, measurements are carried out with a coordinate measuring machine with nanometre accuracy.

  8. Low Speed PSP Testing in Production Wind Tunnels (United States)

    Bell, James; Mehta, Rabi; Schairer, Ed; Hand, Larry; Nixon, David (Technical Monitor)


    The brightness signal from a pressure-sensitive paint varies inversely with absolute pressure. Consequently high signal-to-noise ratios are required to resolve aerodynamic pressure fields at low speeds, where the pressure variation around an object might only be a few percent of the mean pressure. This requirement is unavoidable, and implies that care must be taken to minimize noise sources present in the measurement. This paper discusses and compares the main noise sources in low speed PSP testing using the "classical" intensity-based single-luminophore technique. These are: temperature variation, model deformation, and lamp drift/paint degradation. Minimization of these error sources from the point of view of operation in production wind tunnels is discussed, with some examples from recent tests in NASA Ames facilities.

  9. Skin autoreactivity in Hashimoto's thyroiditis patients without urticaria: autologous serum skin test positivity correlation with thyroid antibodies, sonographical volume and grading. (United States)

    Turkoglu, Zafer; Zindanci, Ilkin; Turkoglu, Ozlem; Can, Burce; Kavala, Mukaddes; Tamer, Gonca; Ulucay, Vasfiye; Akyer, Erdal


    Recent studies have shown an association between anti-thyroid antibodies and autologous serum skin test (ASST) positive urticaria patients. However, a connection between thyroid and this reliable skin test for mast cell autoreactivity, ASST, has not been reported yet. We investigated ASST in patients with Hashimoto's thyroiditis (HT) without urticaria and compared the results with laboratory and sonographical findings of HT. 154 HT patients, 100 healthy volunteers without HT as a first control group and 46 patients with multinodular goitre but without autoimmune thyroid disease as a second control group underwent testing with ASST. ASST was applied to these groups according to two criteria, first as ASST(new): autologous serum red wheal response 1.5 mm bigger than negative control; second as ASST(old): serum red wheal response 5 mm bigger than negative control accepted as positive. Free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), thyroid peroxidase antibody (anti-TPO) and thyroglobulin antibody (anti-Tg) levels were measured. ASST(old), ASST(new) scored positive in 51.3-60.4% of HT patients, with statistically significant differences. Thyroid volume grades were inversely proportional with ASST(old) and (new) positivity. Moderate (+) titers of anti-Tg in ASST(old) and (new) (+) cases were significantly higher than the same titers of anti-Tg in ASST(old) and (new) (-) cases. The prevalence of ASST positivity in HT patients was not affected by the following factors: gender, age at screening, laboratory measurements of thyroid function tests, anti-TPO antibodies and thyroid ultrasound (US) echogenicity. Positivity of ASST in HT has shown that there is a skin mast cell autoreactivity in HT patients independent of autoreactive chronic urticaria (ACU).

  10. An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model

    Energy Technology Data Exchange (ETDEWEB)

    Ma Jun [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Tian Weiming [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Hou Shaoping [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing 100054 (China); Xu Qunyuan [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing 100054 (China); Spector, Myron [Tissue Engineering, VA Boston Healthcare System, Harvard Medical School, Boston, MA (United States); Cui Fuzhai [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)


    The objective of the study was to determine the effects of a hyaluronic-acid-based (HA-based) hydrogel implant, carrying a polyclonal antibody to the Nogo-66 receptor (NgR), on adult rats that underwent middle cerebral artery occlusion (MCAO). Behavioral tests of a forelimb-reaching task suggested that the disabled function of the impaired forelimb in this stroke model was ameliorated by the implant to a certain extent. These behavioral findings were correlated with immunohistochemical results of investigating the distribution of NgR antibody, neurofilaments (NF) and neuron-specific class III {beta}-tubulin (TuJ1) in the brain sections. The porous hydrogel functioned as a scaffold to deliver the NgR antibody, support cell migration and development. In addition, it was found NF-positive and TuJ1-positive expressions were distributed in the implanted hydrogel. Collectively, the results demonstrate the promise of the HA hydrogel as a scaffold material and the delivery vehicle of the NgR antibody for the repair of defects and the support of neural regeneration in the brain.

  11. A latex agglutination test for the field determination of abnormal vitellogenin production in male fishes contaminated by estrogen mimics

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, Ilizabete [Laboratoire d' Immunologie-Microbiologie (LIM/ESE-CNRS, unite FRE2635), IUT de Thionville-Yutz, 1, Impasse A Kastler, F-57970 Yutz (France); Pihan, Jean-Claude [Laboratoire de Production des Ecosystemes et Ecotoxicologie (LBFE), UFR SciFa, campus Bridoux, rue du G Delestraint, F-57070 Metz (France); Falla, Jairo [Laboratoire d' Immunologie-Microbiologie (LIM/ESE-CNRS, unite FRE2635), IUT de Thionville-Yutz, 1, Impasse A Kastler, F-57970 Yutz (France)


    Estrogen mimics are pollutants present in the aquatic environment. These compounds induce abnormalities in the reproductive system of male fishes, which lead to a total or partial male feminization, or to their demasculinization. Ultimately, these alterations could lead to a disappearance of the total contaminated fish population. Moreover, these toxic substances possess the capacity to mimic endogenous estrogens and to induce the abnormal production of vitellogenin (VTG) in male and immature fishes. The purpose of this research was to develop an easy, specific, cheap and fast method for diagnosing the contamination of male fishes by estrogen mimics, using VTG as biomarker. The selected method is based on a reverse latex agglutination test (rLAT), developed with monoclonal antibodies specific of this biomarker. The development of this VTG-rLAT has involved, firstly, the purification of carp VTG to produce monoclonal antibodies, specifics of this protein. One of these antibodies was selected to recover latex particles (diameter: 1 {mu}m). Finally, the immunoreactivity of the VTG-rLAT was verified with different fish plasma samples from males treated with 17{beta}-estradiol and non-treated males or females in vitellogenesis.

  12. Modified Hodge Test: A Simple and Effective Test for Detection of Carbapenemase Production

    Directory of Open Access Journals (Sweden)

    N Malik


    Full Text Available Background and Objectives: Resistance among bacterial isolates is the leading cause of increased mortality and morbidity worldwide. Carbapenems once thought to be effective are becoming ineffective mostly due to the emergence of carbapenemase. This study was designed to determine in vitro efficacy of Modified Hodge test for detection of carbapenemase production in Gram negative rods.Material and Methods: The study was done in the Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi Pakistan from January 2010 to December 2010. A total of 200 Gram negative rods from different clinical samples were taken. Those isolates which showed intermediate or susceptible zones i.e 16mm-21mm on disc diffusion were included in the study. These isolates were then subjected to Modified Hodge test.Result: Out of 200 isolates, 138 (69% were positive for carbapenemase production by Modified Hodge test. Out of 138 MHT positive organisms, the frequency of E. coli was 38%, followed by Pseudomonas aeruginosa (30%, Klebsiella pneumoniae (17%, Acinetobacter baumannii (12%, Citrobacter diversus (2% and Enterobacter agglomerans (1.4%.Conclusion: Modified Hodge test is a simple test which can be performed in the routine lab for detection of carbapenemases in isolates showing intermediate or sensitive zone diameter on disc diffusion.

  13. A high-yielding, generic fed-batch process for recombinant antibody production of GS-engineered cell lines

    DEFF Research Database (Denmark)

    Fan, Li; Zhao, Liang; Sun, Yating;


    An animal component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of byproducts....... This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines....

  14. Comparison of serological tests for antibody to hepatitis A antigen, using coded specimens from individuals infected with the MS-1 strain of hepatitis A virus. (United States)

    Dienstag, J L; Krugman, S; Wong, D C; Purcell, R H


    To compare serological tests for antibody to hepatitis A antigen (anti-HA), we tested 15 paired serum specimens, submitted under code, from individuals infected with the MS-1 strain of hepatitis A virus. Immune electron microscopy (IEM), immune adherence hemagglutination (IAHA), and solid-phase radioimmunoassay (RIA) tests for anti-HA were performed with hepatitis A antigen (HA Ag) derived from human stool; results were also compared with previously reported titers determined by IAHA with HA Ag derived from marmoset liver. Antibody titers (IAHA and RIA) and ratings (IEM) determined with stool-derived HA Ag compared favorably, and a seroresponse to HA Ag was detected by all three methods for every serum pair tested. Differences in titers were noted between IAHA tests with liver-derived and with stool-derived HA Ag, but the discrepancies could be accounted for by differences in test technique. The agreement found in this study among the three techniques was quite good and confirms the specificity and sensitivity of tests for anti-HA that are done with stool-derived HA-Ag. PMID:186409

  15. Bone marrow fibrosis and production of anti—rhEPO antibody induced by long—term abministration of rhEPO

    Institute of Scientific and Technical Information of China (English)

    LuQJ; WenLQ


    Recombinant human erythropoietin(rhEPO) is used extensively in anemic patients on dialysis and other patients,and is considered as very safety and effective in the correction of anemia in these patients.In a safety evaluation recently done in Beagles dogs,mild or moderate bone marrow fibrosis and neutralizing anti-rhEPO IgG production were found in the high dosage group of 1800 U·kg-1 after 13-week subcutaneous administration of rhEPO.The results suggest that long-term administration of rhEPO may result in the occurrence of bone marrow fibrosis and the development of neutralizing antibodies to rhEPO.In clinical practice,it is important to choose an appropriate dose regimen to reduce the risk of antibody production and adverse effects associated with long-term administration of high dose of rhEPO while achieving target hematocrit hevel.

  16. Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009 hemagglutinin conserved domain (HA2: brief report

    Directory of Open Access Journals (Sweden)

    Somayeh Zamani


    Full Text Available Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2 for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID in both forms, Single RID (SRID and Double RID (DRID. Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

  17. Project Rio Blanco: additional production testing and reservoir analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ballou, L.B.


    Additional subsurface investigations of the Rio Blanco detonation region and adjacent reservoir have been underway since the last technical meeting at IAEA. The lowermost explosion cavity has been reentered and a production test from it was performed. A dry gas volume of 7.6 x 10/sup 5/ m/sup 3/ (27 Mmscf) was withdrawn. Chemical and radiochemical analyses of this gas show that (1) the yield of the bottom explosive was 31 +- 2 kt; (2) the cavity/chimney volume was 2.4 x 10/sup 4/ m/sup 3/ (8.4 x 10/sup 5/ ft/sup 3/); (3) about 7 percent of the tritium produced is associated with the gas; and (4) a slight (approximately 0.1 percent) gas contribution from the middle explosion region was noted. The reservoir/chimney model implies an unstimulated reservoir flow capacity of 0.15 mdarcy-m (0.50 md-ft) connected to the bottom chimney region. A cavity radius of 21 +- 3 m (70 +- 10 ft) was deduced. Unstimulated reservoir production parameters were investigated in a well offset 190 m (625 ft) from the emplacement hole. Insufficient productivity was obtained in the Mesaverde formation (in which the bottom explosive was detonated) to evaluate reservoir properties. The productive sandstones in the Fort Union formation adjoining the top detonation region were individually evaluated. Their aggregate flow capacity was determined to be 0.14 +- 0.2 mdarcy-m (0.45 +- 0.08 md-ft). A numerical simulation model which incorporates these data is described. The lack of a high-permeability connection between the three explosion regions remains unexplained. The two chimney reentry wells have been cemented to the surface and abandoned. The offset well has been plugged in a way which preserves the option for additional subsurface investigation in the future. Project facilities have been removed and the site restored to conditions which minimize environmental impact.

  18. Influence of Exogenous Reproductive Hormones on Specific Antibody Production in Genital Secretions after Vaginal Vaccination with Recombinant Cholera Toxin B Subunit in Humans


    Wassen, Lotta; Jertborn, Marianne


    The objective of this study was to investigate the influence of exogenous reproductive hormones on the local and systemic production of specific immunoglobulin A (IgA) and IgG antibodies after vaginal vaccination with recombinant cholera toxin subunit B (CTB). Three groups of women using either progesterone-containing intrauterine devices (n = 9), oral contraceptives (n = 8), or no hormonal contraceptive methods (n = 9) were vaginally immunized twice, 2 weeks apart. Cervical secretions, vagin...

  19. Serotype-specific anti-Dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production. (United States)

    Masrinoul, Promsin; Omokoko, Magot Diata; Pambudi, Sabar; Ikuta, Kazuyoshi; Kurosu, Takeshi


    Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

  20. Clinical Utility of Random Anti–Tumor Necrosis Factor Drug–Level Testing and Measurement of Antidrug Antibodies on the Long‐Term Treatment Response in Rheumatoid Arthritis (United States)

    Jani, Meghna; Chinoy, Hector; Warren, Richard B.; Griffiths, Christopher E. M.; Plant, Darren; Fu, Bo; Morgan, Ann W.; Wilson, Anthony G.; Isaacs, John D.; Hyrich, KimmeL.; Prouse, P. J.; Moitra, R. K.; Shawe, D. J.; Nisar, M.; Fairburn, K.; Nixon, J.; Barnes, T.; Hui, M.; Coady, D.; Wright, D.; Morley, C.; Raftery, G.; Bracewell, C.; Bridges, M.; Armstrong, D.; Chuck, A. J.; Hailwood, S.; Kumar, N.; Ashok, D.; Reece, R.; O'Reilly, S. C.; Ding, T.; Badcock, L. J.; Deighton, C. M.; Raj, N.; Regan, M. R.; Summers, G. D.; Williams, R. A.; Lambert, J. R.; Stevens, R.; Wilkinson, C.; Kelly, C. A.; Hamilton, J.; Heycock, C. R.; Saravanan, V.; Cope, A.; Garrood, T.; Ng, N.; Kirkham, B.; Green, M.; Gough, A.; Lawson, C.; Das, D.; Borbas, E.; Wazir, T.; Emery, P.; Bingham, S.; Bird, H. A.; Conaghan, P.G.; Pease, C. T.; Wakefield, R. J.; Buch, M.; Bruce, I.; Gorodkin, R.; Ho, P.; Parker, B.; Smith, W.; Jenkins, E.; Mukhtyar, C.; Gaffney, K.; Macgregor, A. J.; Marshall, T.; Merry, P.; DeSilva, C.; Birrell, F. N.; Crook, P. R.; Szebenyi, B.; Bates, D.; James, D.; Gillott, T.; Alvi, A.; Grey, C.; Browning, J.; McHale, J. F.; Gaywood, I.C.; Jones, A. C.; Lanyon, P.; Pande, I.; Doherty, M.; Gupta, A.; Courtney, P. A.; Srikanth, A.; Abhishek, A.; Das, L.; Pattrick, M.; Snowden, H. N.; Bowden, A. P.; Smith, E. E.; Klimiuk, P.; Speden, D. J.; Naz, S.; Ledingham, J. M.; Hull, R. G.; McCrae, F.; Cooper, A.; Young‐Min, S. A.; Wong, E.; Shaban, R.; Woolf, A. D.; Davis, M.; Hutchinson, D.; Endean, A.; Mewar, D.; Tunn, E. J.; Nelson, K.; Kennedy, T. D.; Dubois, C.; Pauling, J.; Korendowych, E.; Jenkinson, T.; Sengupta, R.; Bhalla, A.; McHugh, N.; O'Neil, T.; Herrick, A. L.; Jones, A. K.; Cooper, R. G.; Dixon, W. G.; Harrison, B.; Buckley, C. D.; Carruthers, D. C.; Elamanchi, R.; Gordon, P. C.; Grindulis, K. A.; Khattak, F.; Raza, K.; Situnayake, K.; Akil, M.; Till, S.; Dunkley, L.; Tattersall, R.; Kilding, R.; Tait, T.; Maxwell, J.; Till, S.; Kuet, K.-P.; Plant, M. J.; Clarke, F.; Fordham, J. N.; Tuck, S.; Pathare, S. K.; Paul, A.; Marguerie, C. P.; Rigby, S. P.; Dunn, N.; Abbas, I.; Filer, C.; Abernethy, V. E.; Clewes, A. R.; Dawson, J. K.; Kitas, G.; Erb, N.; Klocke, R.; Whallett, A. J.; Douglas, K.; Pace, A.; Sandhu, R.; John, H.; Shand, L.; Lane, S.; Foster, H.; Griffiths, B.; Griffiths, I.; Kay, L.; Ng, W.-F.; Platt, P. N.; Walker, D. J.; Peterson, P.; Lorenzi, A.; Friswell, M.; Thompson, B.; Lee, M.; Pratt, A.; Hopkinson, N. D.; Dunne, C. A.; Quilty, B.; Marks, J.; Mukherjee, S.; Mulherin, D.; Chalam, S. V.; Price, T.; Sheeran, T.; Venkatachalam, S.; Baskar, S.; Al- Allaf, W.; McKenna, F.; Shah, P.; Filer, A.; Bowman, S. J.; Jobanputra, P.; Rankin, E. C.; Allen, M.; Chaudhuri, K.; Dubey, S.; Price‐Forbes, A.; Ravindran, J.; Samanta, A.; Sheldon, P.; Hassan, W.; Francis, J.; Kinder, A.; Neame, R.; Moorthy, A.; Bukhari, M.; Ottewell, L.; Palkonyai, E.; Hider, S.; Hassell, A.; Menon, A.; Dowson, C.; Kamath, S.; Packham, J.; Dutta, S.; Price, S.; Roddy, E.; Paskins, Z.; O'Reilly, D. T.; Rajagopal, V.; Bhagat, S.; Chattopadhyay, C. B.; Green, M.; Quinn, D.; Isdale, A.; Brown, A.; Saleem, B.; Foo, B.; Al Saffar, Z.; Koduri, G.


    . A body mass index of ≥30 kg/m2 and poor adherence were associated with lower drug levels. Conclusion Pharmacologic testing in anti–tumor necrosis factor–treated patients is clinically useful even in the absence of trough levels. At 3 months, antidrug antibodies and low adalimumab levels are significant predictors of no response according to the EULAR criteria at 12 months. PMID:26109489

  1. Serum herpes simplex antibodies (United States)

    ... 2. HSV-1 most often causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test ... whether a person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  2. 抗磷脂抗体检测的现状及展望%Current status and prospects of antiphospholipid antibodies tests

    Institute of Scientific and Technical Information of China (English)

    张蜀澜; 李永哲


    抗磷脂抗体(APLs)是辅助诊断抗磷脂综合征(APS)的重要实验室指标,是血栓形成和合并妊娠时的主要危险因素之一.但是由于APLs存在显著异质性,检测方法标准化程度较低,不同实验室间检测结果一致性较差,一定程度上限制了APLs检测的临床应用和对于APS诊治结果的进一步认识.因此,亟须建立APLs检测的标准化体系,提高检测方法的特异性、敏感性和重复性,实施设计合理的APLs检测结果大规模的临床评估.%Antiphospholipid antibodies (APLs) are important for the diagnosis of antiphospholipid syndrome (APS),especially for predicting the risk of thrombosis and pathological pregnancy.However,the heterogeneity of antiphospholipid antibodies,lacking of standardization and significant interlaboratory variation binder the clinical application of APLs and better understanding of APS diagnosis and treatment.Therefore,it is urgent to establish a standardize system for antiphospholipid antibodies test and to improve the performance of the test and perform well-designed clinical evaluation.

  3. New product testing and the utilization of user expertise: Evidence from the Pharmaceutical industry


    Smed, Marie; Salomo, Søren; Schultz, Carsten; Getz, Kenneth A.


    A significant and often neglected obstacle in new product development is the testing and approval process in the late stages of development. The testing process has primarily been observed as an in-house decision process, however, in many industries products undergo extensive testing before market launch where external stakeholders play a key role. Users are often integrated in testing of new product candidates, and supply valuable knowledge to developers by testing the new product in natural...

  4. Power production experiments at the Test Beam Line in the CLIC Test Facility 3

    CERN Document Server

    Lillestøl, Reidar Lunde; Adli, Erik; Lundheim, Lars Magne


    CLIC is an international study of a future multi-TeV electron-positron linear collider, where the energy of a high-intensity drive beam is extracted and transferred to the main beam via Power Extraction and Transfer Structures (PETS) in the form of rf power. The study of power production is therefore essential for the feasibility of CLIC. Power production in PETS has been studied, and ex- periments have been performed in the decelerator Test Beam Line in the CLIC Test Facility 3. In particular, the correlation of the power production and the beam position inside the structure has been studied. It is shown that the total produced power is constant when the beam has a position offset through the PETS. In addition, the difference between the measured phases from each side is independent of the beam position, which allows for efficient combination of the fields. However, the ratio of the power on each side of the PETS unexpectedly shows a linear dependence on the horizontal offset, with a correlation value of 0.8...

  5. Improving Quality of Seal Leak Test Product using Six Sigma (United States)

    Luthfi Malik, Abdullah; Akbar, Muhammad; Irianto, Dradjad


    Seal leak test part is a polyurethane material-based product. Based on past data, defect level of this product was 8%, higher than the target of 5%. Quality improvement effort was done using six sigma method that included phases of define, measure, analyse, improve, and control. In the design phase, a Delphi method was used to identify factors that were critical to quality. In the measure phase, stability and process capability was measured. Fault tree analysis (FTA) and failure mode and effect analysis (FMEA) were used in the next phase to analize the root cause and to determine the priority issues. Improve phase was done by compiling, selecting, and designing alternative repair. Some improvement efforts were identified, i.e. (i) making a checklist for maintenance schedules, (ii) making written reminder form, (iii) modifying the SOP more detail, and (iv) performing a major service to the vacuum machine. To ensure the continuity of improvement efforts, some control activities were executed, i.e. (i) controlling, monitoring, documenting, and setting target frequently, (ii) implementing reward and punishment system, (iii) adding cleaning tool, and (iv) building six sigma organizational structure.

  6. Iodox process tests in a transuranium element production campaign

    Energy Technology Data Exchange (ETDEWEB)

    Collins, E. D.; Benker, D. E.


    The Iodox process, an advanced method for removing gaseous radioiodine from airstreams, was tested at high activity levels during transuranium element production operations. Special equipment was used to remove radioiodine from 3-5M HNO/sub 3/ dissolver solutions by volatilization and to absorb the iodine from the dissolver off-gas into hyperazeotropic HNO/sub 3/. More than 96% of the /sup 131/I in the dissolver solutions was removed by heeating to 100/sup 0/C and air-sparging. Treatments such as boiling, sparging with 5% N/sub 2/O/sub 3/, and adjustment to 10/sup -4/ M KI or 1% H/sub 2/O/sub 2/ effected the additional removal of /sup 131/I by factors of 2 to 5. Other than /sup 131/I, no fission products were volatilized during the treatments. A six-stage bubble-cap column was used for the abosrpttion. Off-gas decontamination factors were in the range of 1 x 10/sup 4/ to 8 x 10/sub 4/, or 5 to 7 per stage; these values are similar to those obtained during previous low-activity studies.

  7. The effect of prophylaxis with chloroquine and proguanil on delayed-type hypersensitivity and antibody production following vaccination with diphtheria, tetanus, polio, and pneumococcal vaccines

    DEFF Research Database (Denmark)

    Gyhrs, A; Pedersen, B K; Bygbjerg, I;


    (1,000 mg/week), or 4) proguanil hydrochloride (200 mg/day) for six weeks. Skin testing was performed on days 0 and 28. Vaccinations with diphtheria, tetanus, polio, and pneumococcal polysaccharide antigen vaccines were performed on day 28, and the presence of specific antibodies was determined...... dosages, does not induce any detectable suppression of delayed-type hypersensitivity or vaccination responses to diphtheria, tetanus, polio, or pneumococcal polysaccharide antigens....

  8. [Anti-alpha-gliadin antibodies. Sensitivity, specificity and correlation with blood xylose test in the 3 diagnostic stages of celiac disease in children]. (United States)

    Ventura, A; Florean, P; Trevisan, M; Not, T; Perticarari, S; Quadrifoglio, F; Torre, G; Cauci, S


    The specificity and sensibility of IgA and IgG alfagliadin antibody test (AaGA) for screening, diagnosis and follow-up of childhood coeliac disease has been evaluated. We have compared AaGA test to D-xylose blood test and at last we have examined the false positive and negative results given by the test. Two groups of subjects were considered: 1) 90 children with untreated coeliac disease (21 newly diagnosed (I stage), 50 in gluten withdrawal (II stage), 19 in challenge (III stage); 2) 255 disease controls including: 157 healthy controls; 31 children with gastroenterological disorders other than coeliac disease; 31 children with food allergy and atopic dermatitis; 36 children with "constitutional" short stature (without GH deficiency and with normal intestinal mucosa). The sensibility of AaGA test in the first stage of coeliac disease has been of 95.2% for the IgG class antibody and 90.4% for the IgA class; on the other hand the showed a specificity of 83.6% for IgG class antibody and 96.9% for IgA class. In only two newly diagnosed coeliac children we have found false negative results: in the first case the patient was IgA-deficient, in the second the age was above 3 years. AaGA IgA resulted positive only in the 12.9% of the group of gastroenterological and atopic controls; particularly most cases were affected by multiple food allergies and two patients by chronic autoimmune disease of small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics. (United States)

    Porwancher, Richard B; Hagerty, C Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J B; Kopnitsky, Mark; Steere, Allen C; Kulas, Karen; Wong, Susan J


    The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥ 95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted.

  10. Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics ▿ (United States)

    Porwancher, Richard B.; Hagerty, C. Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J. B.; Kopnitsky, Mark; Steere, Allen C.; Kulas, Karen; Wong, Susan J.


    The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

  11. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii. (United States)

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M


    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  12. Antibody Response to Plague Vaccination in Humans as Assayed by Staphylococcal Radioimmune Precipitation (St-RIP) Test (United States)


    Medical Microbiology and Immunology 163, 25-35. Cavanaugh, D. C., Elisberg, B. L., Llewellyn, C. H., Marshall, J. D., Jr., Rust, J. H.,I: Williams J. E...Schaeg, W. (1977). A radioimmunoassay for tetanus antibodies using protein A-containing Staphylococcus aureus. Medical Microbiology and Immunology 163...Putenherpesvirus. Medical Microbiology and Immunology 163, 14l-156. Kessler, S. W. (1975) Rapid isolation of antigens from tells with a staphylococa! protein

  13. Test elements of direct sums and free products of free Lie algebras

    Indian Academy of Sciences (India)

    Cennet Eskal; Naime Ekici


    We give a characterization of test elements of a direct sum of free Lie algebras in terms of test elements of the factors. In addition, we construct certain types of test elements and we prove that in a free product of free Lie algebras, product of the homogeneous test elements of the factors is also a test element.

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

    Suzuki, Lisandra Akemi; Rossi, Cláudio Lúcio


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

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

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

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

  16. RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection in response to antibody-enhanced dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Michael G Brown

    Full Text Available Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of

  17. CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies.

    Directory of Open Access Journals (Sweden)

    Dennis Wolf

    Full Text Available BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/- mice consumed a high fat diet (HFD for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/- mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/- mice. However, CD40L(-/- mice consuming HFD were not protected from the onset of diet-induced obesity (DIO, insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/- mice consuming a low fat diet (LFD showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

  18. Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

    Directory of Open Access Journals (Sweden)

    Umarphorn Chadseesuwan


    Full Text Available Aflatoxin M1 (AFM1 is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17 were obtained that showed cross-reactivity with aflatoxin B1 (AFB1 and aflatoxin G1 (AFG1 but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50 and the limit of detection (LOD values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV and % recovery of the detection was investigated for both intra- (n = 6 and inter- (n = 12 variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.

  19. Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia. (United States)

    Cañavate, Carmen; Herrero, Merce; Nieto, Javier; Cruz, Israel; Chicharro, Carmen; Aparicio, Pilar; Mulugeta, Abate; Argaw, Daniel; Blackstock, Anna J; Alvar, Jorge; Bern, Caryn


    We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect(®), DiaMed-IT Leish(®), DAT, and IFAT in 35 polymerase chain reaction-confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect(®), DiaMed-IT Leish(®), DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia.

  20. Evaluation of Two rK39 Dipstick Tests, Direct Agglutination Test, and Indirect Fluorescent Antibody Test for Diagnosis of Visceral Leishmaniasis in a New Epidemic Site in Highland Ethiopia (United States)

    Cañavate, Carmen; Herrero, Merce; Nieto, Javier; Cruz, Israel; Chicharro, Carmen; Aparicio, Pilar; Mulugeta, Abate; Argaw, Daniel; Blackstock, Anna J.; Alvar, Jorge; Bern, Caryn


    We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect®, DiaMed-IT Leish®, DAT, and IFAT in 35 polymerase chain reaction–confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect®, DiaMed-IT Leish®, DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia. PMID:21212210

  1. Fabrication and testing history prototypes and production units

    Energy Technology Data Exchange (ETDEWEB)


    From April, 1951 to Aug, 1954, New York Shipbuilding Corp. carried out a subcontract with E.I. du Pont de Nemours & Company that was without parallel in the shipyard`s history. The work, designated the NYX Project for reasons of security, was vital to the operations of the Savannah River Plant, Aiken, S.C., which was then being designed and constructed by du Pont for the Atomic Energy Commission. It consisted of three broad parts: developmental and experimental work; fabrication and testing of a prototype unit; fabrication of production units. Five production units were ultimately built, one of them converted from the prototype. All were fabricated from stainless steel, and involved welding techniques, control of thermal distortion and tolerances never previously attempted on assemblies of comparable size. Du Pont`s technical experience and the background of New York Ship in heavy construction, particularly in the fabrication of naval gun turrets, were combined from the outset to resolve the difficult fabrication problems that occurred almost daily. Representatives of both companies worked together as a team in the shops and at supervisory levels to an unprecedented extent. The report is intended primarily to summarize New York Ship`s part in the project, but also includes some of du Pont`s activities since the work of the two organizations was so interrelated. Because of the scope of the program, it will not always be possible to provide detailed information, but rather to record what happened in general terms. Where the reader desires more specific data, he should refer to original plans and records, including various reports compiled during the course of the project.

  2. Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer. (United States)

    Tian, Baomin; Wong, Wah Yau; Hegmann, Elda; Gaspar, Kim; Kumar, Praveen; Chao, Heman


    A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

  3. Kinetics of the avian influenza-specific humoral responses in lung are indicative of local antibody production

    NARCIS (Netherlands)

    Geus, de E.D.; Rebel, J.M.J.; Vervelde, L.


    The role and kinetics of respiratory immunoglobulins in AIV infection has not been investigated. In this study we determined the numbers of both total antibody secreting cells (ASC) and virus-specific ASC in lung, spleen, blood and bone marrow (BM) following low-pathogenic AIV infection. Antiviral h

  4. New product testing and the utilization of user expertise: Evidence from the Pharmaceutical industry

    DEFF Research Database (Denmark)

    Smed, Marie; Salomo, Søren; Schultz, Carsten


    A significant and often neglected obstacle in new product development is the testing and approval process in the late stages of development. The testing process has primarily been observed as an in-house decision process, however, in many industries products undergo extensive testing before market...... launch where external stakeholders play a key role. Users are often integrated in testing of new product candidates, and supply valuable knowledge to developers by testing the new product in natural business settings. As especially high-tech products are closely linked to related services and usage...... generated by users in product testing. Further, the important user network in high-tech product testing may likely be globally dispersed, and the mean of communicating between user and producer in the testing process are therefore tested. A dataset of 395 site-representatives are applied to study...

  5. Rh(D)阴性献血者不规则抗体检测的结果分析%Analysis the result of irregular antibody test in Rh (D) negative donors

    Institute of Scientific and Technical Information of China (English)

    李健; 周英; 吕文彬; 陈雪; 迭敏; 龚希


    目的 分析Rh(D)阴性献血者不规则抗体检测的结果,探讨在献血者血液Rh(D)阴性确认试验时,进行不规则抗体检测的必要性.方法 用间接抗人球蛋白法进行Rh(D)阴性确认试验及抗体筛选试验、抗体鉴定试验,盐水法进行Rh表现型的检测.结果 1205份Rh(D)阴性献血员标本中,抗体筛选试验阳性的标本有20份,经过抗体鉴定,其中抗-D 10份,抗-M 6份,非特异性抗体2份,抗-Lea 1份,冷自身抗体1份.结论 Rh(D)阴性献血者抗体筛选阳性率高达1.66%,较以往报道的正常人群抗体筛选阳性率高.在临床供血上,其高检出率对临床用血安全有较为重要的意义,对Rh(D)阴性献血者更应该进行不规则抗体检测.%Objective To analyze the result of irregular antibody test in Rh (D) negatie donors, discuss in the necessity of irregular antibody test in Rh (D) negative confirmation experiment among the blood testing with Rh ( D) negative donors. Methods Rh (D) negative confirmation experiment antibody screening and antibody Identification test using indirect antiglobulin test(IAT) , The Rh phenotype were determined by saline method. Results among 120S samples of Rh (D) negative donors, 20 samples antibody screening test is positive. And after antibody Identification test, Among them 10 samples were anti- 0 , 6 samples were anti- M, 2 samples were Nonspecific antibody, 1 sample was anti- Le,l sample was cold antibody. Conclusion The positive rate of Antibody screening in Rh ( D) negative donors is 1. 66% . It is greater than Previous reports of the normal population positive antibody screening. The higher positive rate of Antibody screening has Important clinical significance in blood supply, therefore Rh ( D) negative donors more should do irregular antibody test.

  6. Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Daohong [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Li Peiwu [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Quality Inspection and Test Center for Oilseeds and Products of Ministry of Agriculture, Wuhan 430062 (China)], E-mail:; Zhang Qi; Zhang Wen; Huang Yanling; Ding Xiaoxia; Jiang Jun [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Quality Inspection and Test Center for Oilseeds and Products of Ministry of Agriculture, Wuhan 430062 (China)


    Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B{sub 1}, B{sub 2}, G{sub 1} and G{sub 2} were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B{sub 1}, B{sub 2} and G{sub 1} but showed a weak cross-reaction to G{sub 2}. Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B{sub 1}, B{sub 2}, G{sub 1} and G{sub 2} at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 pg mL{sup -1} respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.

  7. Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility. (United States)

    Kapoor, P; Compton, M M; Howarth, B


    The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

  8. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)


    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  9. 40 CFR 90.703 - Production line testing by the manufacturer. (United States)


    ... manufacturer. 90.703 Section 90.703 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Manufacturer Production Line Testing Program § 90.703 Production line testing by the manufacturer. (a) Manufacturers of small SI engines shall test production line engines from each engine family according to...

  10. 40 CFR 1048.301 - When must I test my production-line engines? (United States)


    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false When must I test my production-line...) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM NEW, LARGE NONROAD SPARK-IGNITION ENGINES Testing Production-line Engines § 1048.301 When must I test my production-line engines? (a) If you produce...

  11. 40 CFR 1054.301 - When must I test my production-line engines? (United States)


    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false When must I test my production-line...) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM NEW, SMALL NONROAD SPARK-IGNITION ENGINES AND EQUIPMENT Production-line Testing § 1054.301 When must I test my production-line engines? (a) If you...

  12. Tests of Environmental Effects on Type Ia Supernova Production (United States)

    Sadler, Suzanna M.; Strolger, L.; Wolff, S.


    The host galaxy environments of type Ia supernovae (SNe Ia) provide our best opportunity for constraining the mechanism(s) of the SN Ia progenitor system, i.e., the stars involved, the incubation times, and the sensitivity of SNe Ia to changes in the local gas-phase metallicity. The latter can affect the luminosity of the resultant event, and possibly the success in ultimately yielding a SN Ia. We seek to solidify possible environmental trends in SN Ia rates from direct measures of host galaxy properties, using the sample collected by the Nearby Galaxies Supernova Search project. This study will uncover which has the greatest influence on SN Ia production efficiency: parent population age, rate of star-formation, or metallicity. Here, we will show some preliminary results from SSP model fitting (of age and [Fe/H]) to a selection of hosts obtained thus far from this study. The complete sample will provide a validity test of the mostly indirect trends being established for SNe Ia from the LOSS, SDSS, SNfactory and other surveys, and may ultimately steer future investigations towards more precise SN Ia cosmology.

  13. Tests of Environmental Effects on SN Ia Production (United States)

    Strolger, Louis-Gregory; van Dyk, Schuyler; Wolff, Schuyler; Campbell, Lachlan; Sadler, Suzanna; Pease, April


    The largely unknown type Ia supernova mechanism remains one of the largest sources of possible systematic uncertainty in achieving precise measures of dark energy. The host galaxy environments of SNe Ia provide our best opportunity for constraining the mechanism(s) of the SN Ia progenitor system, i.e., the stars involved, the incubation times, and the sensitivity of SNe Ia to changes in the local gas-phase metallicity. The latter can affect the luminosity of the resultant Ia event, and possibly the success in ultimately yielding a SN Ia event. We seek to solidify possible environmental trends in SN Ia rates from direct measures of host galaxy properties, using the sample collected by the Nearby Galaxies Supernova Search project. This study will uncover which has the greatest influence on SN Ia production efficiency: parent population age, rate of star-formation, or metallicity. Here, we propose to continue our analysis using the Mayall 4m + RCSP to obtain broad SEDs, R_23 metallicities, and star formation rates for 18 of the 25 remaining host galaxies (all z≤0.15) from the NGSS sample (20 targets were obtained in the 2010 semesters). These data will be combined with the remaining sample to be completed in the 2011B semester. The sample will provide a validity test of the mostly indirect trends being established for SNe Ia from the LOSS, SDSS, SNfactory and other surveys, and ultimately steer future investigations towards more precise SN Ia cosmology.

  14. Titers of ABO antibodies in group O blood donors

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    Natalia Dallaval Galvão de França


    Full Text Available BACKGROUND: Plasma components of group O blood donations are rarely submitted to ABO antibody titrations even though it is well known that passively acquired antibodies may destroy the recipient's own red cells and tissue grafts. OBJECTIVE: Thus, group O donations stratified by gender and age were randomly titrated to identify the best source of products for apheresis and exsanguinous transfusion. METHODS: Samples from 603 blood donors were tested by ABO antibody titration using the conventional tube technique at room temperature. ABO antibody levels higher than 64 were considered high. After correction for gender, statistical analyses were performed using the Fisher exact and Kruskal-Wallis tests. RESULTS: Most donors in the blood bank were male (65.7%. ABO antibody titers ranged from 1 to 2048. The estimations of prevalence for the titers were: anti-A,B 128 = 2.16%; Anti-A > 128 = 9.29% and anti-B > 128 = 4.81%. Low mean titers for both anti-A and anti-B antibodies were found in over 50-year-old men (p-value = 0.040. High anti-B antibody levels were found in young women (p-value = 0.002. CONCLUSION: This study confirms that over 50-year-old O group men should be selected as blood donors in non-identical ABO transfusion situations. Also, titration of ABO antibodies in blood banks will increase safety in non-identical ABO transfusions.

  15. 'Criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010. (United States)

    Pierangeli, S S; de Groot, P G; Dlott, J; Favaloro, E; Harris, E N; Lakos, G; Ortel, T; Meroni, P L; Otomo, K; Pengo, V; Tincani, A; Wong, R; Roubey, R


    Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive 'standardized' laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-β(2)glycoprotein I [anti-β(2)GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these 'criteria' aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-β(2)GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to 'criteria' aPL tests in an evidence-based manner, during the 13(th) International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-β(2)GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-β(2)GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of 'criteria' aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.

  16. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology (United States)

    Kingston, Hugh W. F.; Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P. J.


    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively. PMID:26291089

  17. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens. (United States)

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A


    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  18. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants. (United States)

    Sack, Markus; Rademacher, Thomas; Spiegel, Holger; Boes, Alexander; Hellwig, Stephan; Drossard, Juergen; Stoger, Eva; Fischer, Rainer


    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.

  19. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells. (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill


    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  20. Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors. (United States)

    Mallaney, Mary; Wang, Szu-Han; Sreedhara, Alavattam


    During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

  1. Prospective assessment of rapid diagnostic tests for the detection of antibodies to hepatitis C virus, a tool for improving access to care. (United States)

    Chevaliez, S; Poiteau, L; Rosa, I; Soulier, A; Roudot-Thoraval, F; Laperche, S; Hézode, C; Pawlotsky, J-M


    Large-scale hepatitis C screening is required to prevent further spread of the infection, improve access to care in the context of new hepatitis C virus (HCV) drug regimens without interferon-alpha and subsequently reduce the risk of long-term complications of chronic liver disease. Rapid diagnostic tests (RDTs) represent an attractive alternative to enzyme immunoassay using blood from venepuncture. The aim of the present study was to prospectively assess the clinical performance of CE-marked RDTs detecting anti-HCV antibodies in fingerstick capillary whole blood and/or oral fluid. A total of 513 individuals, including 318 patients with chronic HCV infection, 25 patients with resolved HCV infection and 170 HCV-seronegative individuals, were prospectively enrolled. The specificity of RDTs with fingerstick whole blood varied from 98.8% to 100%. The clinical sensitivity was high for the OraQuick(®) and Toyo(®) tests (99.4% and 95.8%, respectively), but low for the Labmen(®) test (63.1%). The specificity and clinical sensitivity in crevicular fluid were both satisfactory for the OraQuick(®) test (100% and 97.6%, respectively). HCV antibody RDTs were easy and rapid to perform in the context of patient care. They were highly specific. Both the OraQuick(®) and Toyo(®) tests reached the expected level of performance for wide-scale use, with a performance advantage for the OraQuick(®) HCV test. RDTs appear to be a promising new tool for wide-scale screening of HCV infection in high-risk to medium-risk populations. Hence, careful assessment of the performance of HCV RDTs must be recommended before they can be implemented in clinical practice.

  2. A Dual Point-of-Care Test Shows Good Performance in Simultaneously Detecting Nontreponemal and Treponemal Antibodies in Patients With Syphilis: A Multisite Evaluation Study in China (United States)

    Yin, Yue-Ping; Chen, Xiang-Sheng; Wei, Wan-Hui; Gong, Kuang-Long; Cao, Wen-Ling; Yong, Gang; Feng, Liang; Huang, Shu-Jie; Wang, Dong-Mei; Han, Yan; Chen, Shao-Chun; Mabey, David; Peeling, Rosanna W.


    Background. Rapid point-of-care (POC) syphilis tests based on simultaneous detection of treponemal and nontreponemal antibodies (dual POC tests) offer the opportunity to increase coverage of syphilis screening and treatment. This study aimed to conduct a multisite performance evaluation of a dual POC syphilis test in China. Methods. Participants were recruited from patients at sexually transmitted infection clinics and high-risk groups in outreach settings in 6 sites in China. Three kinds of specimens (whole blood [WB], fingerprick blood [FB], and blood plasma [BP]) were used for evaluating sensitivity and specificity of the Dual Path Platform (DPP) Syphilis Screen and Confirm test using its treponemal and nontreponemal lines to compare Treponema pallidum particle agglutination (TPPA) assay and toluidine red unheated serum test (TRUST) as reference standards. Results. A total of 3134 specimens (WB 1323, FB 488, and BP 1323) from 1323 individuals were collected. The sensitivities as compared with TPPA were 96.7% for WB, 96.4% for FB, and 94.6% for BP, and the specificities were 99.3%, 99.1%, and 99.6%, respectively. The sensitivities as compared with TRUST were 87.2% for WB, 85.8% for FB, and 88.4% for BP, and the specificities were 94.4%, 96.1%, and 95.0%, respectively. For specimens with a TRUST titer of 1:4 or higher, the sensitivities were 100.0% for WB, 97.8% for FB, and 99.6% for BP. Conclusions. DPP test shows good sensitivity and specificity in detecting treponemal and nontreponemal antibodies in 3 kinds of specimens. It is hoped that this assay can be considered as an alternative in the diagnosis of syphilis, particularly in resource-limited areas. PMID:23132172

  3. Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens

    Energy Technology Data Exchange (ETDEWEB)

    Spring, B.; Pawelec, G.; Ziegler, A.


    To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray-induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA=A2, and TU161 for HLA-B13 molecules, respectively. Both MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells.

  4. Review for the generalist: The antinuclear antibody test in children - When to use it and what to do with a positive titer

    Directory of Open Access Journals (Sweden)

    Sailer-Hoeck Michaela


    Full Text Available Abstract The antinuclear antibody test (ANA is a much overused test in pediatrics. The ANA does have a role in serologic testing but it should be a very limited one. It is often ordered as a screening test for rheumatic illnesses in a primary care setting. However, since it has low specificity and sensitivity for most rheumatic and musculoskeletal illnesses in children, it should not be ordered as a screening test for non-specific complaints such as musculoskeletal pain. It should only be used as a diagnostic test for children with probable Systemic Lupus Erythematosus (SLE or Mixed Connective Tissue Disease, (MCTD and other possible overlap-like illnesses. Such children should have developed definite signs and symptoms of a disease before the ANA is ordered. This review presents data supporting these conclusions and a review of the ANA literature in adults and children. By limiting ANA testing, primary care providers can avoid needless venipuncture pain, unnecessary referrals, extra medical expenses, and most importantly, significant parental anxieties. It is best not to do the ANA test in most children but if it ordered and is positive in a low titer (

  5. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

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    Joanna Majewska


    Full Text Available A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79; the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

  6. Testing of the new Streptomyces strains for production of phenoloxidases

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    Full Text Available Thirty wild new strains of filamentous bacteria belonging to the genus Streptomyces isolated from different Romanian soil samples and ten strains from Collection of microorganisms of Bioaliment Research Platform (acronym MIUG were tested and screened for their ability to produce extracellular tyrosinase and laccase. Based on preliminary qualitative screening assays for the extracellular phenoloxidases production carried out by stationary cultivation on Gause agar medium (GMA supplemented with 1% (w/w L-tyrosine, nineteen strains were selected as active producers. Furthermore, a quantitative selection based on active strains ability to produce tyrosinase and laccase by submerged cultivation in liquid Gause salts basal medium supplemented with 1 g L-1 L-tyrosine and 0.001 g L-1 CuSO4, during 168 hours was performed. Results showed that 70% of the Streptomyces strains have a good potential for producing tyrosinase and 30% of strains were remarked for their ability to produce laccase. Moreover, it was observed that, Streptomyces strains coded MIUG 4.89 and MIUG 4.88 from MIUG Collection have the ability to simultaneously produce both enzymes. The effect of temperature and pH on enzymes activity was also investigated. The optimum temperature for both activity (tyrosinase and laccase was found to be 30°C. Laccase was found active over a pH range of 4.0 to 6.0 with maximum activity at pH 5.0. The optimum pH for tyrosinases activity was observed to be around 7.0. The obtained results are important for future applications of Streptomyces phenoloxidases in different areas.

  7. Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus. (United States)

    Wang, Changbing; You, Aiping; Tian, Xingui; Zhao, Mingqi; Chen, Yi; Lin, Tao; Zheng, Jianbin; Xiao, Misi; Zhang, Yingying; Kuang, Lu; Zhou, Zhenwen; Zhu, Bing


    Hand, foot and mouth disease (HFMD) in humans is caused mainly by Enterovirus 71(EV71) and Coxsackievirus A16 (CVA16). EV71 is associated with severe HFMD cases but not CVA16. Use of IgM-capture enzyme-linked immunosorbent assay (ELISA) is important for the early diagnosis of EV71 infection, but cross-reactivity of the anti-CVA16 IgM antibody with EV71 produces false-positive results. In this report, we designed a new EV71 IgM-capture ELISA method using the EV71 VP1 peptide instead of the EV71 virion as the detectable antigen, and tested sera from patients infected with EV71 or CVA16. The results showed that acute sera from 76 EV71-infected patients had similar sensitivity for virus detection (98.68%) or VP1 detection (97.37%). When acute sera from patients infected with CVA16 were used, significant differences between the two methods were observed. The cross-reactivity rate of the virus detection method was 29.4% (5/17), but no cross-reactivity was observed using the VP1 detection method. Western immunoblotting demonstrated that EV71 VP3 cross-reacted with part of the CVA16 IgM antibody. The results demonstrate that EV71 VP3 is the cross-reactive antigen in the EV71 IgM-capture ELISA when testing CVA16 sera using the virus-antibody detection method. The problem of false-positive results was resolved by using the VP1 peptide as the detectable antigen.

  8. Influence of single nucleotide polymorphisms of cytokine genes on anti-HBs antibody production after hepatitis B vaccination in a Japanese young adult population. (United States)

    Yukimasa, Nobuyasu; Sato, Shoichi; Oboshi, Wataru; Watanabe, Toru; Uzawa, Ryuichi


    Hepatitis B (HB) vaccination is one of the most efficient tools to prevent the transmission of the virus. Considerable variability exists in HB vaccine responses, with 5-10% of healthy Japanese adults demonstrating no response following a standard vaccination. Recently, polymorphisms of immune-regulatory genes, such as cytokine genes, have been reported to influence the immune response to HB vaccine. The aim of this study was to investigate the underlying mechanisms of the genetic association between several cytokine gene polymorphisms and the immune response to HB vaccination in a Japanese population. One hundred and twenty three vaccinated young adults were classified according to the level of antibody-titer (anti-HBs). Single nucleotide polymorphism typing for IFN-γ (+874, 3'-UTR), IL-10 (-591, -819, -1082), and TNF-α (-308, -857), was accomplished using the PCR-RFLP or SSP-PCR method. The TNF-α (-857) CC type and the IL-10 (-1082) AG type were present more frequently in the low titer group than in the high titer group. The TNF-α (-857) CC type was found to be significantly associated with low response of serum anti-HBs. The anti-HBs antibody was not readily produced in the IL-10 (-1082) AG and TNF-α (-857) CC haplotype. Conversely, the antibody was readily produced in the IL-10 (-1082) AA and TNF-α (-857) CC haplotype, and the IL-10 (-1082) AA and TNF-α (-857) CT haplotype, suggesting a high likelihood of the IL-10 (-1082) AG type to be included in the low anti-HBs group, and high anti-HBs antibody production in those with the TNF-α (-857) CT type. These SNPs may produce ethnically-specific differences in the immune response to HB vaccine in the Japanese population. J. Med. Invest. 63: 256-261, August, 2016.

  9. The influence of beryllium on cell survival rates in theIn-vitro culture system, on intracellular DNA synthesis and on SRBC-IgM antibody production responses


    Yoshida, Tsutomu; Shima , Syogo; Kurita , Hideki; Nagaoka, Kaoru; Taniwaki, Hiroshige; Asada, Yasuki; Shai , Kai-ping; Koike, Mitsumasa; Morita, Kunihiko


    Immunocytotoxicity of beryllium (Be) was evaluated by studying cell viability, intracellular DNA synthesis and SRBC-IgM response in an in-vitro culture system using non-sensitized spleen cells of a C57BL mouse. Be addition showed a suppressive effect on cell viability, an enhancing effect on DNA synthesis and on IgM antibody production. The suppressive effect on cell viability manifested itself markedly as the concentration of Be was increased or the culture time was prolonged. The DNA synthe...

  10. Validation of a test for dams carrying foetuses persistently infected with bovine viral-diarrhoea virus based on determination of antibody levels in late pregnancy. (United States)

    Lindberg, A; Groenendaal, H; Alenius, S; Emanuelson, U


    Our objective was to estimate, using a generalised linear mixed-model approach, the sensitivity and specificity of an indirect ELISA when used to identify dams pregnant with persistently bovine viral-diarrhoea virus (BVDV)-infected foetuses. Cows that had been tested for antibodies to BVDV with a positive result during their pregnancy and where the offspring had been tested for both antibody and virus were identified by accessing the Swedish BVD database and the official pedigree records. The resulting data set consisted of 2162 cow-calf pairs in 126 herds, of which 281 included virus-positive calves. The sensitivities and specificities at 12 different decision thresholds (corresponding to optical densities (ODs) between 0.5 and 1.6) were estimated using generalised linear mixed models (binomial error, logit link), in which the gold standard (the BVDV status of the calf) was included as a covariate. In each model, the dependent variable was the dichotomous test result at the decision threshold in question. There was a significant positive interaction between the calf's status and gestational stage in all 12 models--indicating that the sensitivity and specificity at any given decision threshold was improved when the the test was performed later in pregnancy. The test should be applied only when samples have been taken in late gestation--not before the seventh month in pregnancy. If applied during the last months of pregnancy, the point estimate of the sensitivity ranges between 0.94 and 1.0 as the decision threshold is moved from 1.0 and downwards to 0.7. Similarly, the specificity ranges between 0.39 and 0.67 as the decision threshold is moved from 0.8 and upwards to 1.1.

  11. Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery (United States)

    Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher


    Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

  12. 76 FR 69481 - Testing and Labeling Pertaining to Product Certification (United States)


    ... do not apply to ``ordinary books or ordinary paper-based printed materials.'' The Commission has... Electrotechnical Commission (ISO/ IEC) Guide 65, General requirements for bodies operating product...

  13. Effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis vaccines: value of laboratory tests as predictors of clinical performance. (United States)

    Miller, E; Ashworth, L A; Redhead, K; Thornton, C; Waight, P A; Coleman, T


    The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtheria/tetanus/pertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 3/5/9 month and 2/3/4 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were significantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. Differences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 2/3/4 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 2/3/4 month schedule but was unaffected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 2/3/4 month schedule. PT antibody persistence until 4-5 years of age was significantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 3/5/9 month schedule. However, diphtheria antitoxin levels were reduced in

  14. Problems of Automatic Test of Insulation in Cable Production (United States)

    Red'ko, V. V.; Leonov, A. P.; Red'ko, L. A.; Bolgova, V. A.


    The article presents a qualitative and quantitative assessment of cable products insulation defects that can be reliably detected by means of the electrosparking control during the cable production process. The performance potential of technological control is evaluated: the limit of reliable detection of defective places in insulation taking into account the technical capabilities of modern control devices is marked.

  15. A Test of Color-Octet Heavy Quarkonium Production Mechanism

    Institute of Scientific and Technical Information of China (English)

    DUAN Chun-Gui; YAN Zhan-Yuan; HE Zhen-Min


    Using the scale evolution of nuclear parton distributions,the contribution of color-octet heavy quarkonium production mechanism to the P + Fe → J/ψ + γ + X process in calculated and discussed.Comparing our theoretical results with the future experimental data,the color-octet heavy quarkonium production mechanism can be examined.``

  16. Development and application of an indirect ELISA test for the detection of antibodies to Mycoplasma crocodyli infection in crocodiles (Crocodylus niloticus). (United States)

    Dawo, Fufa; Mohan, Krishna


    Non-availability of a standardized rapid serodiagnostic test for quick and accurate diagnosis of Mycoplasma crocodyli (M. crocodyli) infection in crocodiles was the underlining reason for conducting the present study. An indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies (Ab) to M. crocodyli infection in crocodile sera was developed using sonicated antigen (Ag) and anti-crocodile conjugate. The