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Sample records for polyclonal antibody libraries

  1. Recombinant Polyclonal Antibody Libraries for Breast Cancer Therapy

    National Research Council Canada - National Science Library

    Sharon, Jacqueline

    1999-01-01

    .... In the present study, V region genes for construction of a polyclonal Fab phage display library were obtained from the spleen, bone marrow, and intestine of a BALB/c mouse that had been immunized with BT-20 cells...

  2. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    Future research should be directed to epitope screening of synthetic HMCV peptides, which could help to understand HCMV infection and virus-neutralising antibodies more fully and to prepare HCMV vaccines and antiviral drugs. Key words: Human cytomegalovirus, AD169 strain, Towne strains, polyclonal antibody.

  3. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  4. Research Paper Polyclonal antibodies production against ...

    African Journals Online (AJOL)

    The main aim of this project is to produce polyclonal antibodies directed against the Staphylococcus aureus protein A and their use to appreciate bacteriological analysis of milk quality. In this context, an immunization produce was set up to test and detect in a batch of animals the convenient responder to the injected ...

  5. Polyclonal antibodies of Ganoderma boninense isolated from ...

    African Journals Online (AJOL)

    Polyclonal antibodies of Ganoderma boninense isolated from Malaysian oil palm for detection of basal stem rot disease. ... ELISA-PAb shows better detection as compared to cultural-based method, Ganoderma selective medium (GSM) with an improvement of 18% at nursery trial. The present study also demonstrates ...

  6. Polyclonal Antibody Therapies for Clostridium difficile Infection

    Directory of Open Access Journals (Sweden)

    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  7. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

    Directory of Open Access Journals (Sweden)

    Edith S Málaga-Machaca

    2017-11-01

    Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

  8. Comparative Immunologic Activities of Polyclonal Antibodies ...

    African Journals Online (AJOL)

    Three types of Vibrio cholerae (El tor, Ogawa) whole cell antigens constituted with liquid paraffin adjuvant (LPA), Freund's incomplete adjuvant (FIA) and physiological saline (SA) were used for polyclonal antisera production in adult rabbits. Antisera were compared for serologic reactivities with homologous and ...

  9. Preparation and characterization of the polyclonal antibody against ...

    African Journals Online (AJOL)

    To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR ...

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

    African Journals Online (AJOL)

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

  11. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... The identification of the synthetic peptide antibody was confirmed by ... cell virus transmission and fusion of infected cells, as well ..... Cytomegalovirus and Epstein-. Barr virus subtypes-The search for clinical significance.

  12. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

    Science.gov (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

    2014-01-25

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Synthesis of Polyclonal Antibodies against Aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Wiyogo Prio Wicaksono

    2015-09-01

    Full Text Available Polyclonal antibodies of aflatoxin B1 were successfully produced from New Zealand White female rabbits after immunization by the hapten of aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO conjugated with bovine serum albumin (BSA as the antigen. The hapten was synthesized using the carbodiimide method with CMO as a linker. Absorption peaks at 362, 264, and 218 nm were observed as a result of characterization with UV-Vis spectroscopy, while IR spectroscopy showed peaks at 3448 cm-1 and 1642 cm-1 attributable to the hydroxyl and nitrile groups, respectively. Furthermore, mass spectrometry showed fragmentation at the m/z of 386, 368.2, and 310, which confirms that the hapten of AFB1-CMO was successfully synthesized. The hapten was then conjugated with BSA to serve as an antigen of AFB1 when it was injected into the rabbits. The specificity of the antigen towards its antibody and the confirmation of hapten-BSA conjugation were characterized using the dot blot immunoassay, which showed a BSA concentration of 1.74 mg/mL. Two weeks after the primary immunization by its antigen, agar gel precipitation testing showed that the rabbit blood serum had positive results for polyclonal antibodiest against AFB1 with the highest concentration of antibodiest of 2.19 mg/mL.

  14. Polyclonal antibody to ovomucoid determination in gamma irradiated laying eggs

    International Nuclear Information System (INIS)

    Harder, Marcia N.C.; Arthur, Valter; Silva, Lucia C.A.S.; Lopes, Tatiana G.G.; Duarte, Keila M.R.; Canniatti-Brazaca, Solange G.; Savino, Vicente J.M.; Coelho, Antonio A.D.

    2009-01-01

    To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in female rabbits immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTA-ELISA (Plate trapped antigen). All procedures were according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs. So the samples were submitted to the gamma radiation coming from a source of Co 60 , type Multipurpose, under a dose rate of 19.4 and 31.8 Gy/hour, in the doses: 0 (control); 10 KGy; 20 KGy and 30 KGy, in all rates. By the ELISA.s test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples. (author)

  15. Polyclonal antibody to ovomucoid determination in gamma irradiated laying eggs

    Energy Technology Data Exchange (ETDEWEB)

    Harder, Marcia N.C.; Arthur, Valter; Silva, Lucia C.A.S.; Lopes, Tatiana G.G. [Centro de Energia Nuclear na Agricultura (CENA/USP, Piracicaba, SP. Dept. de Radiobiologia e Ambiente) (Brazil)], e-mail: mnharder@cena.usp.br, e-mail: arthur@cena.usp.br, e-mail: tgglopes@cena.usp.br; Duarte, Keila M.R. [Instituto de Zootecnia (IZ . Nova Odessa), Nova Odessa, SP (Brazil)], e-mail: keila@iz.sp.gov.br; Canniatti-Brazaca, Solange G.; Savino, Vicente J.M.; Coelho, Antonio A.D. [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil)], e-mail: sgcbraza@esalq.usp.br, e-mail: vjmsavin@esalq.usp.br, e-mail: aadcoelh@esalq.usp.br

    2009-07-01

    To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in female rabbits immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTA-ELISA (Plate trapped antigen). All procedures were according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs. So the samples were submitted to the gamma radiation coming from a source of Co{sup 60}, type Multipurpose, under a dose rate of 19.4 and 31.8 Gy/hour, in the doses: 0 (control); 10 KGy; 20 KGy and 30 KGy, in all rates. By the ELISA.s test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples. (author)

  16. Production and purification of polyclonal antibody against melatonin hormone

    Directory of Open Access Journals (Sweden)

    Fooladsaz K

    1999-09-01

    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.

  17. Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

    OpenAIRE

    Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

    2017-01-01

    Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme a...

  18. Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA.

    Science.gov (United States)

    Wang, Ting; Li, Peiwu; Zhang, Qi; Zhang, Wen; Zhang, Zhaowei; Wang, Tong; He, Ting

    2017-06-28

    Aspergillus and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of Aspergillus flavus to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to A. flavus was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both A. flavus and A. parasiticus. To construct a sandwich format immunoassay, polyclonal antibodies against Aspergillus were raised with rabbits. Finally, a highly selective nanobody-polyclonal antibody sandwich enzyme-linked immunosorbent assay was optimized and developed. The results revealed that the detection limits of the two fungi were as low as 1 μg mL -1 , and that it is able to detect fungal concentrations below to 2 μg mg -1 of peanut and maize grains in both artificially and naturally contaminated samples. Therefore, we here provided a rapid and simple method for monitoring Aspergillus spp. contamination in agricultural products.

  19. Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

    Science.gov (United States)

    Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

    2017-01-01

    The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

  20. [Preparation and characterization of mouse polyclonal antibody against conserved region of human FOXO3].

    Science.gov (United States)

    Li, Lei; Lyu, Dan

    2017-06-01

    Objective To purify the recombinant protein specific to conserved region of forkhead box O3 (FOXO3) and prepare mouse anti-human FOXO3 polyclonal antibody. Methods The DNA fragment (aa290-472) encoding conserved domain of FOXO3 was amplified by PCR, and subsequently cloned into pET28a vector. Following transformation into E.coli BL21, the soluble fusion protein His-FOXO3 was induced by IPTG and purified by Ni-NTA affinity chromatography. The purified protein was used to immunize BALB/c mice to generate polyclonal antibody. The characteristics of the polyclonal antibody were assessed by ELISA, Western blotting and immunoprecipitation assays. Results We successfully prepared the expression vector pET28a-FOXO3 (aa290-472) and expressed the purified fusion protein in a soluble form. By immunizing mice with the fusion protein, we obtained anti-human FOXO3 polyclonal antibody. ELISA and Western blotting showed that the mouse antibody could recognize specifically the endogenous FOXO3 protein. Conclusion The polyclonal antibody against conserved domain of FOXO3 can identify the endogenous FOXO3 protein. It can be used to analyze the endogenous FOXO3 expression level.

  1. Studies on Purification and Coatation of Polyclonal Antibody for Prolactin Solid Phase Radioimmunoassay in Human Serum

    International Nuclear Information System (INIS)

    El-Bayoumy, A.S.A.; Sallam, Kh.M.; Shafik, H.M.

    2012-01-01

    The objective of the present study was oriented to produce purified polyclonal antibody to prepare a prolactin solid phase coated tubes radioimmunoassay system. In the present study, production of polyclonal antibodies was carried out through immunization of three healthy white male mature New Zealand rabbits with a highly purified sheep prolactin antigen. The obtained anti-sera was purified using an anion exchange reactive group, diethylamino ethyle (DEAE) covalently linked to Sepharose. The purified polyclonal antibody was used for coating polystyrene tubes. The preparation of 125 I-prolactin tracer was carried out using chloramine-T method. The preparation of standards was performed using assay buffer to cover the range from 2 to 200 ng/ml. The optimization and validation tests of the assay were performed to evaluate the validity of the prepared system. In conclusion, this low cost assay would be used in diagnosis of pituitary dysfunction and diagnosis of infertility in males and females

  2. Studies on Purification and Coatation of Polyclonal Antibody for Prolactin Solid Phase Radioimmunoassay in Human Serum

    International Nuclear Information System (INIS)

    El-Bayoumy, A.S.A.; Sallam, Kh.M.; Shafik, H.M.

    2014-01-01

    The objective of the present study was oriented to produce purified polyclonal antibody to prepare a prolactin solid phase coated tubes radioimmunoassay system. In the present study, production of polyclonal antibodies was carried out through immunization of three healthy white male mature New Zealand rabbits with a highly purified sheep prolactin antigen. The obtained anti-sera was purified using an anion exchange reactive group, diethylamino ethyle (DEAE) covalently linked to Sepharose. The purified polyclonal antibody was used for coating polystyrene tubes. The preparation of 125 I-prolactin tracer was carried out using chloramine-T method. The preparation of standards was performed using assay buffer to cover the range from 2 to 200 ng/ml. The optimization and validation tests of the assay were performed to evaluate the validity of the prepared system. In conclusion, this low cost assay would be used in diagnosis of pituitary dysfunction and diagnosis of infertility in males and females.

  3. Production of Monoclonal and Polyclonal Antibodies against a ...

    African Journals Online (AJOL)

    Phil Berger

    Banana streak virus is serologically and genomically heterogenous worldwide and there has been the need to produce antibodies that can detect all known serotypes of this virus. Antibody production requires purified virus, since BSV titre is low in Musa tissues, there was the need for an efficient method of purifying the virus ...

  4. Generation and Characterization of Polyclonal Antibody Against Part of Immunoglobulin Constant Heavy υ Chain of Goose

    Science.gov (United States)

    Zhao, Panpan; Guo, Yongli; Ma, Bo; Wang, Junwei

    2014-01-01

    Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin that is the major antibody in bird, reptile, and lungfish blood. IgY consists of two light (λ) and two heavy (υ) chains. In the present study, polyclonal antibody against IgYFc was generated and evaluated. rIgYCυ3/Cυ4 was expressed in Escherichia coli, purified and utilized to raise polyclonal antibody in rabbit. High affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:204,800 for ELISA assay. The antibody can specifically recognize both rIgYCυ3/Cυ4 and native IgY by Western bolt analysis. Furthermore, the serum of Grus japonensis or immunoglobulin of chicken, duck, turkey, and silkie samples and dynamic changes of serum GoIgY after immunogenicity with GPV-VP3-virus-like particles (GPV-VP3-VLPs) can be detected with the anti-GoIgYFc polyclonal antibody. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of GoIgY. PMID:25171010

  5. Apoptotic Effect of Anti myeloma Polyclonal Antibodies on The Growth of Myeloma Cells

    International Nuclear Information System (INIS)

    Abd El-Ghany, I.Y.; El-Kolaly, M.T.; Moustafa, K.A.; El-Shershaby, H.M.; Sayed, A.A.; Borai, I.H.; El-Lahloby, N.M.

    2013-01-01

    Multiple myeloma (MM) is a malignancy characterized by proliferation of plasma cells. Cancer immunotherapy is a major branch of biological therapy that utilizes living cells and their products. The aim of this study is to produce and evaluate the antiproliferative effect of anti myeloma polyclonal antibodies (with and without labelling with radioactive isotopes) against the growth of myeloma cells. The production of polyclonal antibodies (PAb) was generated by immunizing five healthy female mature white New-Zealand rabbits with myeloma cells (SP2/OR) through primary injection and five booster doses. The preparation of labelled anti myeloma antibodies was carried out using chloramine-T method and it was purified using PD-10 chromatographic column. The results obtained revealed that anti myeloma polyclonal antibodies inhibited proliferation and induced apoptosis of myeloma cell lines in vitro and induced apoptosis after serial intraperitoneal injection of PAb in ascites bearing mice in vivo. The present study suggested that the effect of labelled anti myeloma antibodies on myeloma cells growth inhibition was more effective than that of anti myeloma antibodies without labelling which is due to the cytotoxic effect of ionizing radiation. Apoptosis triggered by PAb was confirmed by flow cytometry, caspase -8 and -9 and β2-microglobulin.

  6. Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

    Science.gov (United States)

    Kotani, Osamu; Iwata-Yoshikawa, Naoko; Suzuki, Tadaki; Sato, Yuko; Nakajima, Noriko; Koike, Satoshi; Iwasaki, Takuya; Sata, Tetsutaro; Yamashita, Teruo; Minagawa, Hiroko; Taguchi, Fumihiro; Hasegawa, Hideki; Shimizu, Hiroyuki; Nagata, Noriyo

    2015-04-01

    The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different

  7. Use of polyclonal/monoclonal antibody therapies in transplantation.

    Science.gov (United States)

    Yeung, Melissa Y; Gabardi, Steven; Sayegh, Mohamed H

    2017-03-01

    For over thirty years, antibody (mAb)-based therapies have been a standard component of transplant immunosuppression, and yet much remains to be learned in order for us to truly harness their therapeutic capabilities. Current mAbs used in transplant directly target and destroy graft-destructive immune cells, interrupt cytokine and costimulation-dependent T and B cell activation, and prevent down-stream complement activation. Areas covered: This review summarizes our current approaches to using antibody-based therapies to prevent and treat allograft rejection. It also provides examples of promising novel mAb therapies, and discusses the potential for future mAb development in transplantation. Expert opinion: The broad capability of antibodies, in parallel with our growing ability to synthetically modulate them, offers exciting opportunities to develop better biologic therapeutics. In order to do so, we must further our understanding about the basic biology underlying allograft rejection, and gain better appreciation of how characteristics of therapeutic antibodies affect their efficacy.

  8. Iodine-131 labeled anti-CEA polyclonal antibody detection of gastrointestinal cancer

    International Nuclear Information System (INIS)

    Nabi, H.A.; Hinkle, G.H.; Olsen, J.O.; Haagensen, D.A.; Thurston, M.O.; Mojzisik, C.; Houchens, D.; Martin, E.W. Jr.

    1984-01-01

    To localize gastrointestinal tumor, 31 patients were injected with 1.7-2.1 mCi I-131 anti-CEA baboon polyclonal antibody. Whole body imaging at 48, 72, and occasionally 96 hrs was performed with a Signa Camera (Technicare) peaked at 364 keV with 20% window. Additional spot views were usually obtained. No subtraction methods were used. All patients had surgical and pathological confirmation of the nuclear medicine studies. Labeled antibody images were positive in 15 (8 recurrent or metastatic colorectal, 2 gastric, 1 pancreatic, 1 primary colon, and 1 breast metastatic to chest wall). In 1, antibody images were positive for metastatic deposits in para-aortic lymph nodes, but negative for primary rectal tumor. True negative images were observed in 6; false negative images in 9 (4 liver metastases, 2 rectal, 1 pancreatic, 1 mesenteric lymph node metastasis, 1 bone metastasis). In all cases, no correlation existed between preoperative CEA serum levels and imaging. I-131 labeled anti-CEA polyclonal antibody imaging proved highly efficient in detecting gastric cancer (2/2) and moderately efficient in detecting recurrent colorectal cancer (8/15). On the other hand, the I-131 labeled polyclonal anti-CEA antibody imaging was of limited value in detecting colon cancer (1/9), pancreatic cancer (1/4) and metastatic liver disease

  9. Preparation and Purification of Polyclonal Antibodies against Mycobacterium Avium Paratuberculosis Antigens in Rabbit

    Directory of Open Access Journals (Sweden)

    Hafezeh Alizadeh

    2012-12-01

    Full Text Available Background and Objective: Johne’s disease is the chronic granulomatous enteritis of ruminants, and a major health hazard worldwide. In recent years, researchers have focused on mycobacterium avium subsp. paratuberculosis (MAP antigens in diagnostic tests. Identification of antibodies against MAP antigens is, therefore, effective for the diagnosis or preparation of vaccine. The aim of this study was to prepare and purify polyclonal antibodies against MAP antigens. Materials and Methods: A New Zealand white rabbit was immunized at a certain time period with MAP antigens and Freund’s adjuvant. After the immunization of the animal, the rabbit was bled to obtain enriched serum. Immunoglobulins were obtained via sedimentation with ammonium sulfate 35% and then IgG was purified by ion exchange (DEAE-cellulose chromatography. Serologic test was used to evaluate the interaction of antigens and antibodies. Results: Ion exchange chromatography of IgG showed one peak, and SDS_PAGE of IgG showed a single band. Serologic test was applied and clear precipitation lines were appeared up to 1:16 dilution, which indicated the high quality of the product. Conclusion: In this study, the humoral immune response was induced well by immunization with MAP antigens in a New Zealand white rabbit and polyclonal antibodies were produced in high titers. Polyclonal antibodies are relatively inexpensive and easy to produce in large quantities and can connect to the more connective sites, resulting in better sensitivity. Identification of polyclonal antibodies via immunological tests can play a significant role in studying MAP disorders.

  10. Technetium-99m labeling anti-amastigote polyclonal antibodies of Leishmania amazonensis

    International Nuclear Information System (INIS)

    Araujo, J.G.V.C.; Toledo, V.P.C.P.; Guimaraes, T.M.P.D.; Bernardo-Filho, M.; Simal, C.J.R.; Mota, L.G.; Diniz, S.O.F.; Cardoso, V.N.

    2002-01-01

    Anti-amastigote polyclonal antibody (IgG) was incubated with solutions of stannous chloride and sodium borohidride. After that, 3.7 MBq of technetium-99m ( 99m Tc) was added. A labeling yield of the antibody about 84% was obtained. After filtration of 99m Tc-IgG, the radiochemical purity increased from 84 to 95%. The labeling of IgG with 99m Tc did not modify the immunoreactivity of the antibody, since it was able to identify in vitro and in vivo the specific antigen of Leishmania amazonensis

  11. A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

    International Nuclear Information System (INIS)

    Wakayama, Tomohiko; Kato, Yukio; Utsumi, Rie; Tsuji, Akira; Iseki, Shoichi

    2006-01-01

    Producing antibodies usually takes more than three months. In the present study, we introduce a faster way of producing polyclonal antibodies based on preparation of the recombinant oligopeptide as antigen followed by immunization of rats. Using this method, we produced antisera against two mouse proteins: ERGIC-53 and c-Kit. An expression vector ligated with a pair of complementary synthetic oligodeoxyribonucleotides encoding the protein was introduced into bacteria, and the recombinant oligopeptide fused with the carrier protein glutathione-S-transferase was purified. Wistar rats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for the antibody titer by immunohistochemistry. Antisera with 1600-fold titer at the maximum were obtained for both antigens and confirmed for their specificity by Western blotting. Anti-ERGIC-53 antisera recognized acinar cells in the sublingual gland, and anti-c-Kit antisera recognized spermatogenic and Leydig cells in the testis. These antisera were applicable to fluorescent double immunostaining with mouse monoclonal or rabbit polyclonal antibodies. Consequently, this method enabled us to produce specific rat polyclonal antisera available for immunohistochemistry in less than one month at a relatively low cost

  12. Characterization of a rabbit polyclonal antibody against threonine-AMPylation

    Science.gov (United States)

    Hao, Yi-Heng; Chuang, Trinette; Ball, Haydn L.; Luong, Phi; Li, Yan; Flores-Saaib, Ruben D.; Orth, Kim

    2014-01-01

    An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins. PMID:21185336

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

    Science.gov (United States)

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

    1989-01-01

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

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

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    Nazila Amini

    2014-06-01

    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.

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

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    Davoud Koolivand

    2016-10-01

    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.

  16. Anti-IL-39 (IL-23p19/Ebi3) polyclonal antibodies ameliorate autoimmune symptoms in lupus-like mice

    Science.gov (United States)

    Wang, Xiaoqian; Zhang, Yu; Wang, Zhiding; Liu, Xiaoling; Zhu, Gaizhi; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Wang, Tianxiao; Shen, Beifen; Li, Yan; Xiao, He; Ma, Ning; Wang, Renxi

    2018-01-01

    The interleukin (IL)-12 family cytokines have been examined as therapeutic targets in the treatment of several autoimmune diseases. Our previous study showed that a novel IL-12 family cytokine, IL-39 (IL-23p19/Ebi3) mediates inflammation in lupus-like mice. In the present study, the effect of anti-mouse IL-39 polyclonal antibodies on autoimmune symptoms in lupus-like mice was investigated. Rabbit anti-mouse IL-39 polyclonal antibodies were produced by immunization with recombinant mouse IL-39, and purified using protein A chromatography. These antibodies were subsequently used to treat lupus-like mice. Flow cytometry, captured images, ELISA and H&E staining were used to determine the effect of anti-IL-39 polyclonal antibodies on inflammatory cells, autoantibody titers, proteinuria, infiltrating inflammatory cells and the structure of the glomerular region. The anti-IL-39 polyclonal antibodies effectively reduced the numbers of inflammatory cells, splenomegaly, autoantibody titers, proteinuria, infiltrating inflammatory cells, and restored the structure of the glomerular region in MRL/lpr mice. Taken together, these results suggested that anti-IL-39 polyclonal antibodies ameliorated autoimmune symptoms in lupus-like mice. Therefore, IL-39 may be used as a possible target for the treatment of systemic lupus erythematosus. PMID:29138852

  17. A methodological approach for production and purification of polyclonal antibody against dog IgG.

    Science.gov (United States)

    Sadeghi, Somayeh; Aghebati-Maleki, Leili; Nozari, Samira; Majidi, Jafar

    2018-01-01

    Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens. In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

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

    OpenAIRE

    Fanning, Lorna M.

    2002-01-01

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

  19. THE USE OF THE SPECIFIC ANTI-SALMONELLA POLYCLONAL ANTIBODIES ISOLATED FROM HEN EGGS, IN SALMONELLOSIS PROPHYLAXIS

    Directory of Open Access Journals (Sweden)

    CRISTE ADRIANA

    2007-01-01

    Full Text Available The administration of increased doses of antibodies in groupsexperimentally infected with Salmonella gallinarum, in order torecord the efficiency of their administration in salmonellosisprophylaxis was the aim of our research. When a low infectiondose, 1x107 CFU Salmonella gallinarum, was used theadministration of IgY polyclonal antibodies as immunoglobulinextract, or even yolk administration had a protective effect againstgerms invasion. This effect was not recorded when a 10 folds higherdose was administered (1x108 CFU. The prophylactic effect of theadministration of polyclonal antibodies is demonstrated.

  20. THE USE OF THE SPECIFIC ANTI-SALMONELLA POLYCLONAL ANTIBODIES ISOLATED FROM HEN EGGS, IN SALMONELLOSIS PROPHYLAXIS

    Directory of Open Access Journals (Sweden)

    ADRIANA CRISTE

    2007-05-01

    Full Text Available The administration of increased doses of antibodies in groups experimentallyinfected with Salmonella gallinarum, in order to record the efficiency of theiradministration in salmonellosis prophylaxis was the aim of our research. When alow infection dose, 1x107 CFU Salmonella gallinarum, was used theadministration of IgY polyclonal antibodies as immunoglobulin extract, or evenyolk administration had a protective effect against germs invasion. This effect wasnot recorded when a 10 folds higher dose was administered (1x108 CFU. Theprophylactic effect of the administration of polyclonal antibodies is demonstrated.

  1. The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation

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    Mei Liu

    2013-04-01

    Full Text Available The enolase2 gene is usually expressed in mature neurons and also named neuron specific enolase (NSE. In the present study, we first obtained the NSE gene cDNA sequence by using the RACE method based on the expressed sequence tag (EST fragment from the cDNA library of Gekko japonicus and identified one transcript of about 2.2 kb in central nervous system of Gekko japonicus by Northern blotting. The open reading frame of NSE is 1305 bp, which encodes a 435 amino-acid protein. We further investigated the multi-tissue expression pattern of NSE by RT-PCR and found that the expression of NSE mRNA was very high in brain, spinal cord and low in heart, while it was not detectable in other tissues. The real-time quantitative PCR was used to investigate the time-dependent change in the expression of the NSE mRNA level after gecko spinal cord transection and found it significantly increased at one day, reaching its highest level three days post-injury and then decreasing at the seventh day of the experiment. The recombinant plasmid of pET-32a-NSE was constructed and induced to express His fused NSE protein. The purified NSE protein was used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65536 determined by ELISA. Western blotting showed that the prepared antibody could specifically recognize the recombinant and endogenous NSE protein. The result of immunohistochemistry revealed that positive signals were present in neurons of the brain and the spinal cord. This study provided the tools of cDNA and polyclonal antibody for studying NSE function in Gekko japonicus.

  2. PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

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    Nurhadi Nurhadi

    2016-10-01

    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.

  3. Cross-Reactivity of Polyclonal Antibodies against Canavalia ensiformis (Jack Bean) Urease and Helicobacter pylori Urease Subunit A Fragments.

    Science.gov (United States)

    Kaminski, Zbigniew Jerzy; Relich, Inga; Konieczna, Iwona; Kaca, Wieslaw; Kolesinska, Beata

    2018-01-01

    Overlapping decapeptide fragments of H. pylori urease subunit A (UreA) were synthesized and tested with polyclonal antibodies against Canavalia ensiformis (Jack bean) urease. The linear epitopes of UreA identified using the dot blot method were then examined using epitope mapping. For this purpose, series of overlapping fragments of UreA, frameshifted ± four amino acid residues were synthesized. Most of the UreA epitopes which reacted with the Jack bean urease polyclonal antibodies had been recognized in previous studies by monoclonal antibodies against H. pylori urease. Fragments 11 - 24, 21 - 33, and 31 - 42 were able to interact with the Jack bean urease antibodies, giving stable immunological complexes. However, the lack of recognition by these antibodies of all the components in the peptide map strongly suggests that a non-continuous (nonlinear) epitope is located on the N-terminal domain of UreA. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  4. Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

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    Anne-Sophie Dugast

    Full Text Available Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC, are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC, we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

  5. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

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    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG

    2016-06-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  6. Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

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    Datis Kharrazian

    2017-01-01

    Full Text Available Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw and modified (cooked and roasted foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease.

  7. Solid phase radioimmunoassay for detection of malaria antigen. Comparison of monoclonal and polyclonal antibodies

    International Nuclear Information System (INIS)

    Khusmith, S.; Tharavanij, S.; Patarapotikul, J.; Kasemsuth, R.; Bunnag, D.

    1986-01-01

    A solid phase competitive binding radioimmunoassay (RIA) was developed for the detection of Plasmodium falciparum in infected blood. A suspension of NP40 treated red blood cells was mixed with labelled antimalarial IgG, incubated and then added to malarial antigen coated microtitre plate. Antimalarial IgGs were purified either from high titre sera from individuals living in a malaria endemic area in Thailand or from a locally produced monoclonal antibody (MAB) which showed a bright generalized immunofluorescent staining pattern against all blood stages of P. falciparum, including gametocytes. This MAB reacted with 27 of 31 P. falciparum isolates from Thailand. Using dilution of red blood cells from in vitro cultures of P. falciparum, the test was found to detect parasites at levels equivalent to 13 and 2.2 parasites/10 6 red blood cells with labelled polyclonal IgG (PIgG) and labelled monoclonal IgG (MIgG), respectively. No false positive results were obtained among samples from non-malarial subjects. Of the samples that gave negative results upon microscopic examination, 50 and 35% were still positive with RIA using MIgG and PIgG, respectively. There was a correlation between RIA and the number of parasites, especially when MIgG was used. The results indicate that the IgG fraction of sera from individuals with natural acquired immunity to malaria showed a lower degree of sensitivity in parasite detection than the IgG from monoclonal antibody. (author)

  8. Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

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    Ana Teresa B.F. Antonangelo

    2012-09-01

    Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

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

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    Zhang Shunchuan

    2010-09-01

    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. Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

    Science.gov (United States)

    Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

    2015-12-01

    Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

  11. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

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    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  12. A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies

    International Nuclear Information System (INIS)

    Sheng Jianwu; He Miao; Shi Hanchang; Qian Yi

    2006-01-01

    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae), and microcystin-leucine-arginine (MC-LR) is among the most frequent and most toxic microcystin congeners. In this study, a free amino group was introduced to MC-LR at its seventh amino acid residue with 2-mercaptoethylamine, and the product aminoethyl-MC-LR was coupled to bovine serum albumin (BSA) and horseradish peroxidise (HRP) by glutaraldehyde to be complete antigen (MC-LR-BSA) and labelled hapten (MC-LR-HRP), respectively. Polyclonal antibodies against MC-LR were generated by immunization with MC-LR-BSA. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect the MCs in waters, which showed a good cross-reactivity with MC-LR, MC-RR, MC-YR, MC-LF, MC-LW and nodularin, and have a detection limit for MC-LR 0.12 μg L -1 , the 50% inhibition concentration (IC 50 ) for MC-LR was 0.63 ± 0.06 μg L -1 and the quantitative detection range was from 0.17 to 2.32 μg L -1 , the analysis result of water samples showed good recovery and reliability. So the comprehensive and reliable dc-ELISA will well potentially suit for sensitive analysis for total MCs in drinking as well as resource water samples

  13. Production and Characterization of Anti-LH Polyclonal Antibodies for Establishment of Sepharose Solid Phase Radioimmunoassay

    International Nuclear Information System (INIS)

    Ebeid, N.H.; Mehany, N.L.

    2016-01-01

    The present study was designed to achieve the production and characterization of polyclonal antibody of luteinizing hormone (Anti-LH) as a basic component of LH radioimmunoassay (RIA). The main objective was to improve the immunogenicity of LH by conjugation of LH with bovine serum albumin (LH: BSA) as a protein carrier using Ethyl dimethylaminopropyl Carbodiimide (ECDI). Production of Anti-LH was described where LH : BSA immunogen was immunized into three male mature white New-Zealand rabbits through primary immunization and four boosters. The criteria for selecting LH antiserum for liquid phase RIA system were mainly titer, displacement and immuno response profile and followed by partial purification of IgG-LH. The radioiodinated 125 I-LH tracer was carried out using Chloramine-T as an oxidizing agent and the tracer was purified through PD-10 column. The preparation of LH standards was carried out. Coupling of purified IgG-LH to activated Sepharose particles CL-4B was carried out after activation of Sepharose particles with 1,1-carbonyldiimidazole. Extensive studies were carried out to obtain the optimum conditions of using solid phase Sepharose particles to reach the optimum separation efficiency. The results of validation tests revealed that the local solid phase RIA system is precise and accurate to detect LH concentration in human serum to be used as a diagnostic tool in investigating the infertility in the hypothalamic pituitary gonadal disorders

  14. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

    International Nuclear Information System (INIS)

    Smith, J.W.; Dey, B.; Knauer, D.J.

    1990-01-01

    Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation

  15. Neutralization of feline immunodeficiency virus by polyclonal cat antibody: Simultaneous involvement of hypervariable regions 4 and 5 of the surface glycoprotein.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); W. Huisman (Willem); J.A. Karlas (Jos); G.F. Rimmelzwaan (Guus); M.L. Bosch (Marnix); A.D.M.E. Osterhaus (Albert)

    1995-01-01

    textabstractSites involved in antibody-mediated neutralization of feline immunodeficiency virus were mapped by reciprocal exchange of envelope fragments or amino acids between molecular clones of feline immunodeficiency virus with different susceptibilities to neutralization by a polyclonal cat

  16. A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

    Science.gov (United States)

    Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

    2017-03-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

  17. Antiparasitic effects induced by polyclonal IgY antibodies anti-phospholipase A2 from Bothrops pauloensis venom.

    Science.gov (United States)

    Borges, Isabela Pacheco; Silva, Mariana Ferreira; Santiago, Fernanda Maria; de Faria, Lucas Silva; Júnior, Álvaro Ferreira; da Silva, Rafaela José; Costa, Mônica Soares; de Freitas, Vitor; Yoneyama, Kelly Aparecida Geraldo; Ferro, Eloísa Amália Vieira; Lopes, Daiana Silva; Rodrigues, Renata Santos; de Melo Rodrigues, Veridiana

    2018-06-01

    Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A 2 (PLA 2 ) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLA s. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA 2 s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

    Directory of Open Access Journals (Sweden)

    Nadya V. Ilicheva

    2018-03-01

    Full Text Available Background: TRF2 (telomeric repeat binding factor 2 is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2 between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3. The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina. Keywords: Chromosomes, Molecular cloning, Nuclear lamina, Nucleoprotein complexes, Polyclonal antibodies, Recombinant polypeptide, Shelterin, Telomere-binding protein TRF2, Telomeres, Telomeric DNA, TTAGGG repeats

  19. Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use.

    Science.gov (United States)

    Frandsen, Torben P; Naested, Henrik; Rasmussen, Søren K; Hauptig, Peter; Wiberg, Finn C; Rasmussen, Lone Kjaer; Jensen, Anne Marie Valentin; Persson, Pia; Wikén, Margareta; Engström, Anders; Jiang, Yun; Thorpe, Susan J; Förberg, Cecilia; Tolstrup, Anne B

    2011-09-01

    The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition. Copyright © 2011 Wiley Periodicals, Inc.

  20. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis

    1992-01-01

    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  1. Recent progress of diagnostic and therapeutic approach to cancers using polyclonal or monoclonal antibodies

    International Nuclear Information System (INIS)

    Koji, Toshihiko

    1982-01-01

    Among the major topics of interest in cancer immunology, immunodiagnosis and immunotherapy with the antibodies are summarized historically and prospectively. The concept of injecting anti-tumor cell antibodies to localize tumors was first introduced in experimental systems by Pressman (1957). Since then, various trials have been achieved with human tumors using specific or nonspecific tumor-localizing antibodies diagnostically or therapeutically. In 1970's, successes in immunodiagnosis with the antibodies to oncofetal proteins also have been reported. Recently, there are numerous papers dealed with a series of external scanning or serotherapeutic trials by the use of monoclonal antibodies that bind selectively to tumor cells. Various relevant problems with them are discussed. (author)

  2. Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

    Science.gov (United States)

    Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

    2017-05-25

    Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

  3. Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies

    Science.gov (United States)

    da Silva-Froufe, Lúcia Gracinda; Boddey, Robert Michael; Reis, Veronica Massena

    2009-01-01

    The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 105 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques. PMID:24031435

  4. Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies.

    Science.gov (United States)

    da Silva-Froufe, Lúcia Gracinda; Boddey, Robert Michael; Reis, Veronica Massena

    2009-10-01

    The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

  5. Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp. Using differente polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Lúcia Gracinda da Silva-Froufe

    2009-12-01

    Full Text Available The species Gluconacetobacterdiazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

  6. Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception

    Directory of Open Access Journals (Sweden)

    Asmarinah Asmarinah

    2011-02-01

    Full Text Available Background: Voltage dependent anion channels (VDAC mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility.Methods: Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides.  Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance and the number of unmoved sperm (million per ml which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software.Results: VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control. We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control.Conclusion: VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future. (Med J Indones 2011; 20:5-10Keywords: VDAC3 antiserum, sperm, motility, contraception

  7. Evaluation of the inflammatory activity in chronic osteomyelitis. Contribution of the scintigraphy with polyclonal antibodies

    International Nuclear Information System (INIS)

    Sapienza, Marcelo Tatit

    1996-01-01

    Active chronic osteomyelitis or complicating osteomyelitis (superimposed on diseases that changes the normal bone structure fractures, post-surgery, prosthesis) can be difficult to diagnose by anatomic radiological imaging modalities, like plain radiograph and CT. These diseases frequently cause also increased bone remodeling, leading to nonspecific uptake of Tc-99m-bone scan agents and gallium-67. New radiopharmaceuticals with greater inflammation/infection avidity and specificity are being developed, including the nonspecific polyclonal immunoglobulin (IgG) labeled with technetium-99. Tc-99m-IgG may be available as a ready to use kit, with no reported side effects, low patient absorbed radiation dose and low cost. The mechanism of IgG uptake at the inflammation site has not been fully elucidated yet. Specific (receptor linking, physico-chemical immunoglobulin properties) and nonspecific mechanisms (enhanced vascular permeability and macromolecular exudate) has been suggested. IgG scintigraphy results are affected by the isotope, labeling procedure adopted and characteristics of the inflammatory focus. Nineteen patients with suspected osteomyelitis (active chronic osteomyelitis or violated bone osteomyelitis) were studied by Tc-99m-IgG scintigraphy (directly labeled polyclonal immunoglobulin, Sandoglobuilina - Sandoz). All patients also underwent standard three-phase bone scintigraphy using methylene diphosphonate (Tc-99m-MDP), gallium-67 scintigraphy and plain radiographs. Infection was found in 8 sites. Sensitivity and specificity for Tc-99m-MDP, gallium-67 and Tc 99m-IgG scintigraphy were, respectively, 88 and 36%, 75 and 73%,88 and 82%. All patients with false positive IgG scintigraphies had previous surgery. Other current scintigraphic procedures used in the diagnosis of osteomyelitis are also reviewed. (author)

  8. A polyclonal antibody against extracellular loops 1 of chNHE1 blocks avian leukosis virus subgroup J infection.

    Science.gov (United States)

    Meng, Wei; Zhou, Defang; Li, Chengui; Wang, Guihua; Huang, Libo; Cheng, Ziqiang

    2018-05-02

    Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytomas and other various tumors, leading to great economical losses in poultry industry. It is a great challenge to develop effective preventive methods for ALV-J control due to its antigenic variations in the variable regions of envelope. In present study, we generated a mouse polyclonal antibody targeting the first extracellular loop (ECL1) of chicken Na + /H + exchanger isoform 1 (chNHE1), the receptor of ALV-J, to block ALV-J infection in vitro and in vivo. In ALV-J infected DF-1 cells, chNHE1 expression and the intracellular pH (pHi) were up-regulated with "wave" pattern, indicating that the disequilibrium of ALV-J infected cells associated with chNHE1. Next, we validated that ALV-J infection was significantly blocked with time dependent after treating with anti-ECL1 antibody and accordingly the pHi value were recovered, indicating the blockage of ALV-J infection did not affect Na + /H + exchange. Furthermore, in anti-ECL1 antibody treatment chickens that infected by ALV-J, weight gain and immune organs were recovered, and viral loads were significantly decreased, and the tissue injury and inflammation were reduced significantly from 21 to 35 days of age. The study demonstrated that anti-ECL1 antibody effectively blocks ALV-J infection without affecting Na + /H + exchange, and sheds light on a novel strategy for retroviruses control. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. DNA vaccine-generated duck polyclonal antibodies as a postexposure prophylactic to prevent hantavirus pulmonary syndrome (HPS.

    Directory of Open Access Journals (Sweden)

    Rebecca Brocato

    Full Text Available Andes virus (ANDV is the predominant cause of hantavirus pulmonary syndrome (HPS in South America and the only hantavirus known to be transmitted person-to-person. There are no vaccines, prophylactics, or therapeutics to prevent or treat this highly pathogenic disease (case-fatality 35-40%. Infection of Syrian hamsters with ANDV results in a disease that closely mimics human HPS in incubation time, symptoms of respiratory distress, and disease pathology. Here, we evaluated the feasibility of two postexposure prophylaxis strategies in the ANDV/hamster lethal disease model. First, we evaluated a natural product, human polyclonal antibody, obtained as fresh frozen plasma (FFP from a HPS survivor. Second, we used DNA vaccine technology to manufacture a polyclonal immunoglobulin-based product that could be purified from the eggs of vaccinated ducks (Anas platyrhynchos. The natural "despeciation" of the duck IgY (i.e., Fc removed results in an immunoglobulin predicted to be minimally reactogenic in humans. Administration of ≥ 5,000 neutralizing antibody units (NAU/kg of FFP-protected hamsters from lethal disease when given up to 8 days after intranasal ANDV challenge. IgY/IgYΔFc antibodies purified from the eggs of DNA-vaccinated ducks effectively neutralized ANDV in vitro as measured by plaque reduction neutralization tests (PRNT. Administration of 12,000 NAU/kg of duck egg-derived IgY/IgYΔFc protected hamsters when administered up to 8 days after intranasal challenge and 5 days after intramuscular challenge. These experiments demonstrate that convalescent FFP shows promise as a postexposure HPS prophylactic. Moreover, these data demonstrate the feasibility of using DNA vaccine technology coupled with the duck/egg system to manufacture a product that could supplement or replace FFP. The DNA vaccine-duck/egg system can be scaled as needed and obviates the necessity of using limited blood products obtained from a small number of HPS survivors. This

  10. Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

    Science.gov (United States)

    Lim, Theam Soon; Chan, Soo Khim

    2016-01-01

    Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Production of Polyclonal Antibodies to Potato virus X Using Recombinant Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Kmoníčková, Jitka; Dědič, P.

    2010-01-01

    Roč. 158, č. 1 (2010), s. 66-68 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus X * recombinant viral antigen * antibodies Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.937, year: 2010

  12. Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

    DEFF Research Database (Denmark)

    Bak, Hanne; Thomas, O.R.T.

    2007-01-01

    Protein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent AlP, Mab...... evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made....

  13. Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos

    Czech Academy of Sciences Publication Activity Database

    Bozzo, M.; Pergner, Jiří; Kozmik, Zbyněk; Kozmiková, Iryna

    2017-01-01

    Roč. 61, č. 10-12 (2017), s. 793-800 ISSN 0214-6282 R&D Projects: GA ČR GC15-21285J; GA MŠk(CZ) LM2015062; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : Branchiostoma * amphioxus * antibody * espression pattern Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 1.981, year: 2016

  14. Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

    Directory of Open Access Journals (Sweden)

    Haddad Muhammad

    2016-01-01

    Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

  15. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    International Nuclear Information System (INIS)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

  16. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  17. Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

    OpenAIRE

    Bonvin, Pauline; Venet, Sophie; Kosco-Vilbois, Marie; Fischer, Nicolas

    2015-01-01

    The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in nat...

  18. Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

    Science.gov (United States)

    Matsushita, Takahiko; Arai, Hidenao; Koyama, Tetsuo; Hatano, Ken; Nemoto, Naoto; Matsuoka, Koji

    2017-11-01

    Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

    Science.gov (United States)

    Ferrari, A; Weill, F S; Paz, M L; Cela, E M; González Maglio, D H; Leoni, J

    2012-03-01

    Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

  20. Development of 111In-DTPA-human polyclonal antibody complex for long-term inflammation/infection detection

    International Nuclear Information System (INIS)

    Jalilian, A.R.; Rowshanfarzad, P.; Kamali-Dehghan, M.; Moafian, J.; Akhlaghi, M.; Rajabifar, S.; Mirzaii, M.; Shafaii, K.; Babaii, M.

    2005-01-01

    Human polyclonal antibody (HIgG) was successively labeled with 111-indium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of solid DTPA-dianhydride (0.1-0.3 mg) to 100 μl of the HIgG solution (0.2-0.4 mg/ml) at pH = 6 in phosphate buffer media at 25 o C with continuous stirring for 30 min. Radio-thin-layer chromatography showed an overall radiochemical yield of 96-99% at optimized conditions (specific activity = 300-500 MBq/mg, radiochemical purity >98%). The final isotonic 111 In-DTPA-HIgG complex was checked by radio-TLC to ensure the formation of only one species followed by filtration through a 0.22 μ filter. Preliminary long-term in vivo studies in turpentine-oil induced inflammation in rat model was performed to determine late complex distribution of the radioimmunoconjugate. The target/skin and target/blood ratios were 27 and 51 after 24 h, and 23 and 51 after 110 h, showing a high selectivity of the radiopharmaceutical for inflammatory lesions. (author)

  1. Sensitive detection of capsaicinoids using a surface plasmon resonance sensor with anti-homovanillic Acid polyclonal antibodies.

    Science.gov (United States)

    Nakamura, Shingo; Yatabe, Rui; Onodera, Takeshi; Toko, Kiyoshi

    2013-11-13

    Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR) immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH) for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes.

  2. Sensitive Detection of Capsaicinoids Using a Surface Plasmon Resonance Sensor with Anti-Homovanillic Acid Polyclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Kiyoshi Toko

    2013-11-01

    Full Text Available Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes.

  3. Combination of Weight-Bearing Training and Anti-MSTN Polyclonal Antibody Improve Bone Quality In Rats.

    Science.gov (United States)

    Tang, Liang; Gao, Xiaohang; Yang, Xiaoying; Zhang, Didi; Zhang, Xiaojun; Du, Haiping; Han, Yanqi; Sun, Lijun

    2016-12-01

    Weight-bearing exercise is beneficial to bone health. Myostatin (MSTN) deficiency has a positive effect on bone formation. We wondered if a combination of weight-bearing training and polyclonal antibody for MSTN (MsAb) would augment bone formation to a greater degree than single treatment. In this study, rats were randomly assigned to four groups: Control, weight-bearing training (WT), MsAb, and WT+MsAb. The trained rats ran at 15 m/min bearing with 35% of their body weight, 40 min/day (2 min of running followed by 2 min of rest), 6 days/week, for 8 weeks. The rats with MsAb were injected once a week with MsAb for 8 weeks. MicroCT analysis showed that compared with the MsAb group, WT+MsAb significantly enhanced cortical bone mineral density (BMD) (p .05), weight-bearing training significantly increased energy absorption (p weight-bearing training and MsAb have a greater positive effect on bone than treatment with either MsAb or weight-bearing training alone, suggesting that resistance training in combination with MSTN antagonists could be an effective approach for improving bone health and reducing osteoporosis risk.

  4. Produção de anticorpos policlonais anti-ricina Production of polyclonal anti-ricin antibodies

    Directory of Open Access Journals (Sweden)

    Roselayne Ferro Furtado

    2011-02-01

    Full Text Available A ricina é uma proteína bastante tóxica presente nas sementes de mamona que impossibilita o uso da torta de mamona "in natura", como ração. A torta de mamona destoxificada necessita ainda de métodos de análise que garantam a ausência de traços dessa proteína. Objetivou-se, neste trabalho, produzir e avaliar a sensibilidade e especificidade de anticorpos policlonais anti-ricina, para serem empregados como possíveis componentes de métodos sorológicos na detecção de ricina em torta de mamona destoxificada. Foram avaliadas três doses da proteína: 400, 180 e 100 µg cada uma dividida em duas aplicações em coelhos. A primeira dose foi injetada no animal no início do experimento e a segunda após 21 dias. O método de ELISA indicou que as duas doses menores (100 e 180 µg induziram respostas imunológicas primária e secundária com produção de anticorpos específicos. Enquanto a dose maior (400 µg de ricina apresentou uma resposta primária com elevação dos títulos de anticorpos, seguida de uma supressão da resposta. Esse perfil é sugestivo de tolerância imunológica. Pela técnica de Western blotting verificou-se que os anticorpos policlonais produzidos são bastante específicos para a ricina, no entanto, por detectarem ricina na forma nativa e desnaturada não são recomendados para o monitoramento de ricina em torta de mamona destoxificada por tratamento térmico.Ricin is a very toxic protein found in castor bean plants, making it impossible to use natural castor cake as animal food. The detoxificated castor cake needs to be analyzed by methods that ensure the absence of traces of this protein. This work had the objective to produce and to evaluate the sensitivity and specificity of anti-ricin polyclonal antibodies, to be employed as component of sorologic methods as the ELISA in the detection of ricin in detoxificated castor cake. Three doses of protein, 400, 180 and 100 µg were evaluated each one injected twice into

  5. Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

    Directory of Open Access Journals (Sweden)

    Reda Salem

    2018-05-01

    Full Text Available Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV, the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp was cloned, sequenced and sub-cloned into pET-30(+ expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting

  6. Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

    Science.gov (United States)

    Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

    2013-06-01

    Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  7. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Highly sensitive determination of diclofenac based on resin beads and a novel polyclonal antibody by using flow injection chemiluminescence competitive immunoassay

    Science.gov (United States)

    Shi, Jing; Xu, Mingxia; Tang, Qinghui; Zhao, Kang; Deng, Anping; Li, Jianguo

    2018-02-01

    A novel flow injection chemiluminescence immunoassay for simple, sensitive and low-cost detection of diclofenac was established based on specific binding of antigen and antibody. Carboxylic resin beads used as solid phase carrier materials provided good biocompatibility and large surface-to-volume ratio for modifying more coating antigen. There was a competitive process between the diclofenac in solution and the immobilized coating antigen to react with the limited binding sites of the polyclonal antibody to form the immunocomplex. The second antibody labelled with horseradish peroxidase was introduced into the immunosensor and trapped by captured polyclonal antibody against diclofenac, which could effectively amplify chemiluminescence signals of luminol-PIP-H2O2. Under optimal conditions, the diclofenac could be detected quantitatively. The chemiluminescence intensity decreased linearly with the logarithm of the diclofenac concentration in the range of 0.1-100 ng mL- 1 with a detection limit of 0.05 ng mL- 1 at a signal-to-noise ratio of 3. The immunosensor exhibited high sensitivity, specificity and acceptable stability. This easy-operated and cost-effective analytical method could be valuable for the diclofenac determination in real water samples.

  9. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  10. 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: odhendrick@gmail.com; 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)

    2011-09-15

    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).

  11. Marrow Derived Antibody Library for the Treatment of Neuroblastoma

    Science.gov (United States)

    2015-12-01

    Award Number: W81XWH-12-1-0332 TITLE: Marrow-Derived Antibody Library for the Treatment of Neuroblastoma PRINCIPAL INVESTIGATOR: Giselle...Marrow-Derived Antibody Library for Treatment of Neuroblastoma 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...to Spectrum Health. 14. ABSTRACT Neuroblastoma (NB) is the most common solid tumor in children, which accounts for 15% of all pediatric cancer deaths

  12. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  13. Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

    Science.gov (United States)

    McElhiney, J; Lawton, L A; Porter, A J

    2000-12-01

    Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

  14. 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

    2015-10-01

    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.

  15. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2014-01-01

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  16. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  17. Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

    KAUST Repository

    Domina, Maria

    2014-12-04

    There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

  18. Arrayed antibody library technology for therapeutic biologic discovery.

    Science.gov (United States)

    Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

    2013-03-15

    Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

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

    Directory of Open Access Journals (Sweden)

    Al-Malaq Hamoud A

    2011-07-01

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

  20. Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

    Directory of Open Access Journals (Sweden)

    EL-Fakharany Esmail M

    2012-09-01

    Full Text Available Abstract Purpose To extend the study of the camel milk proteins which have antiviral activity against HCV, camel naïve polyclonal IgGs, α-lactalbumin were purified from camel milk and their anti-HCV effect was examined using PBMCs and Huh7.5 cell-lines. They were compared with the activity of human polyclonal IgGs and camel lactoferrin and casein. Material and methods Three types of experiments were performed on PBMCs and HuH7.5 cell. HCV was directly incubated with the purified proteins and then mixed with both cell types, or the proteins were incubated with the cells and then exposed to HCV, or the HCV pre-infected cells were treated with the proteins to inhibit intracellular replication. The proteins were added to cells or virus at different concentrations and time intervals. Results Pretreated PBMCs and Huh7.5 cells with milk proteins were not protected when exposed to HCV infection. The direct interaction between HCV and camel IgGs and camel lactoferrin (cLf led to a complete inhibition of HCV entry into cells, while casein, α-lactalbumin and human IgGs failed to inhibit HCV entry at any tested concentration. Camel IgGs showed ability to recognize HCV peptides with a significant titer (12 × 103 in comparison with human IgGs which failed to do it. Camel lactoferrin was capable of inhibiting the intracellular HCV replication at concentrations of 0.25-1.25 mg/ml. Conclusion Camel milk naïve polyclonal IgGs isolated from camel milk could inhibit the HCV infectivity and demonstrated strong signal against its synthetic peptides. Lactoferrin inhibit the HCV infectivity started from 0.25 mg/ml. However, α-lactalbumin, human IgGs and casein failed to demonstrate any activity against HCV infectivity.

  1. High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

    DEFF Research Database (Denmark)

    Bak, Hanne; Kyhse-Andersen, J.; Thomas, O.R.T.

    2007-01-01

    We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery. ...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

  2. A binary plasmid system for shuffling combinatorial antibody libraries.

    OpenAIRE

    Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

    1992-01-01

    We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind a...

  3. Construction of High-Quality Camel Immune Antibody Libraries.

    Science.gov (United States)

    Romão, Ema; Poignavent, Vianney; Vincke, Cécile; Ritzenthaler, Christophe; Muyldermans, Serge; Monsion, Baptiste

    2018-01-01

    Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 10 7 -10 8 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 10 8 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.

  4. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

    Science.gov (United States)

    Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

    2018-04-01

    Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

  5. In-Vivo Neutralization of Botulinum Neurotoxin Serotype E Using Rabbit Polyclonal Antibody Developed against BoNT/E Light Chain.

    Science.gov (United States)

    Rani, Sarita; Ponmariappan, S; Sharma, Arti; Kamboj, D V; Jain, A K

    2017-01-01

    Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment. In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours. The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F. The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population.

    Science.gov (United States)

    Wong, Arthur; Lanyon, Janet M; McKee, Sara J; Linedale, Richard; Woolford, Lucy; Long, Trevor; Leggatt, Graham R

    2018-06-01

    Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. In vivo evaluation of the cross-genotype neutralizing activity of polyclonal antibodies against hepatitis C virus

    DEFF Research Database (Denmark)

    Meuleman, Philip; Bukh, Jens; Verhoye, Lieven

    2011-01-01

    Control of hepatitis C virus (HCV) infection remains a huge challenge of global medical importance. Using a variety of in vitro approaches, neutralizing antibodies (nAbs) have been identified in patients with acute and chronic hepatitis C. The exact role these nAbs play in the resolution of acute...

  8. Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

    NARCIS (Netherlands)

    Goudsmit, Jaap; Marissen, Wilfred E.; Weldon, William C.; Niezgoda, Michael; Hanlon, Cathleen A.; Rice, Amy B.; Kruif, John de; Dietzschold, Bernhard; Bakker, Alexander B. H.; Rupprecht, Charles E.

    2006-01-01

    The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal

  9. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

    Directory of Open Access Journals (Sweden)

    Savinska L. O.

    2015-08-01

    Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

  10. [Research on medical speciality of traditional Chinese medicines using dot-immunoblotting method based on polyclonal antibody prepared from traditional Chinese medicines with hot/cold nature].

    Science.gov (United States)

    Wang, Houwei; Dou, Yanling; Tian, Jingzhen; Li, Feng; Wang, Shijun; Wang, Zhenguo

    2009-02-01

    To research on the substantial foundation of the medical speciality of Chinese traditional medicines from immunogenicity. Control antigen with hot nature was prepared from the mixture of the aqueous extracts of three Chinese traditional medicines with three typical hot nature of Alpinia officinarum, Cinnamomum cassia and Curculigo orchioides, while that with cold nature prepared with Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, and polyclonal antibody was prepared by immunizing rabbit with control antigen. Dot blotting was performed between the polyclonal antibody of control antigen and the aqueous extracts of nine Chinese traditional medicines on a piece of PVDF membrane, and the blotting signals were analyzed by the software of Quantity One. Blotting signals with hot control antigen of nine Chinese traditional medicines in descending were Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum and Phellodendron chinense, which degree of similarity to control antigen in peak value were 57.33%, 43.56 %, 34.16%, 30.2%, 28.81%, 26.53%, 21.68%, 17.62% and 14.85%, respectively. Blotting signals with cold control antigen were Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, Phellodendron chinense, Zingiber officinale, Lonicera japonica, Fraxinus rhynchophylla, Eucommia ulmoides and Aconitum carmichaeli in descending, of which degree of similarity to cold control antigen in peak value were 55.22%, 54.23%, 46.72%, 34.08%, 30.3%, 24.48%, 24.33%, 20.35% and 15.17%, respectively. Results of cluster analysis with Wistar's method showed that nine medicines were classified into two groups, one group included Phellodendron chinense, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum, another was Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica. Blotting signals of nine medicines

  11. A binary plasmid system for shuffling combinatorial antibody libraries.

    Science.gov (United States)

    Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

    1992-11-01

    We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

  12. Human Polyclonal Antibodies Produced through DNA Vaccination of Transchromosomal Cattle Provide Mice with Post-Exposure Protection against Lethal Zaire and Sudan Ebolaviruses.

    Directory of Open Access Journals (Sweden)

    Callie E Bounds

    Full Text Available DNA vaccination of transchromosomal bovines (TcBs with DNA vaccines expressing the codon-optimized (co glycoprotein (GP genes of Ebola virus (EBOV and Sudan virus (SUDV produce fully human polyclonal antibodies (pAbs that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000 were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000 were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA. Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT after four vaccinations. Neutralizing activity of human immunoglobulins (IgG purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg to groups of BALB/c mice one day after IP challenge with mouse adapted (ma EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/- mice receiving the purified IgG (100 mg/kg by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe

  13. [Levels and molecular heterogeneity of serotonin transporter protein in platelets of patients with different mental diseases: a comparative analysis with the use of monoclonal and polyclonal antibodies].

    Science.gov (United States)

    Brusov, O S; Faktor, M I; Zlobina, G P; Bologov, P V; Kaleda, V G; Oleĭchik, I V; Korenev, A N; Piatnitskiĭ, A N; Dupin, A M; Katasonov, A B; Morozova, M A; Beniashvili, A G; Lozier, R Kh; Pavlova, E V; Segal, O L; Massino, Iu S; Dmitriev, A D

    2001-01-01

    Polyclonal (PAb) and monoclonal (MAb) antibodies to CT2-epitope of the C-terminal fragment of serotonin transporter (SERT) protein were used to study the levels and molecular heterogeneity of platelet SERT in healthy donors and patients with affective (AD) and somatoform (SD) disorders, schizoaffective disorder (SAD) and schizophrenia. SERT was found to exist as high molecular wight (HMW) and low molecular weight (LMW) forms separated after electrophoresis. The levels of HMW and LMW forms of SERT were significantly, decreased in mentally ill patients as compared to healthy individuals. Unlike PAb, horse radish peroxidase (HRP)-conjugated MAbs were more sensitive and specific to SERT and could detect the LMW form of SERT as a duplet protein form with MW about 40 and 43 kDa. The MAb to CT2 C-terminal fragment of SERT conjugated with HRP is considered to be a new valuable tool for further investigation of SERT expression, properties, and posttranslation modification in the controls and in patients with different psychopathology.

  14. Production of polyclonal antibodies for lectin from Anticarsia gemmatalis hemolymph Produção de anticorpos policlonais para lectina de hemolinfa de Anticarsia gemmatalis

    Directory of Open Access Journals (Sweden)

    Mario Augusto Ono

    2005-10-01

    Full Text Available The velvet bean caterpillar Anticarsia gemmatalis promotes extensive damage to soybean and is controlled frequently by chemical insecticides. Due to risks to human and animal health as well as the environment, new approaches were developed to A. gemmatalis control such as the bioinsectide Baculoviru anticarsia. The development of resistance in A. gemmatalis populations treated along several generations with B. anticarsia was reported under laboratory conditions. The insects show complex mechanisms against microorganism infection, such as the lectins, that work as recognition molecules. The aim of this work was to develop polyclonal antibodies to A. gemmatalis lectin. The lectin activity in A. gemmatalis caterpillar hemolymph was evaluated using erythrocytes from human, rabbit, mouse, sheep and cow. Only bovine erythrocytes were not agglutinated by lectin. The rabbit erythrocytes showed stronger reactivity (1:512. Therefore the polyclonal antibodies were raised in rabbit immunized with autologous erythrocytes sensitized with lectin. The antibody to lectin showed a titer of 1:8 in immunodiffusion test. In this study we described a simple method for antibody production against hemolymph lectin without expensive purification techniques. A lagarta de Anticarsia gemmatalis promove extensos danos na cultura da soja e seu controle é geralmente baseado na aplicação de inseticidas químicos. Devido aos riscos à saúde humana, animal e ao meio ambiente, métodos alternativos de controle tem sido desenvolvidos como o bioinseticida Baculovirus anticarsia. Há relatos de desenvolvimento de resistência em populações de A. gemmatalis submetidas, em laboratório, ao tratamento com baculovirus durante várias gerações. Os insetos apresentam mecanismos elaborados de proteção contra agentes infecciosos, como as lectinas, que atuam como moléculas de reconhecimento. Assim, o objetivo deste estudo foi desenvolver anticorpos policlonais para lectina de A

  15. Library of monoclonal antibodies against brush border membrane epithelial antigens

    International Nuclear Information System (INIS)

    Behar, M.; Katz, A.; Silverman, M.

    1986-01-01

    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A 1 , C 7 , D 3 , D 7 and H 4 . As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D 3 exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK 1 cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells

  16. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

    Directory of Open Access Journals (Sweden)

    Xiaohua He

    Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

  17. [Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

    Science.gov (United States)

    Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

    2004-05-01

    U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong

  18. Characterization of a Syrian Chickpea chlorotic stunt virus strain and production of polyclonal antibodies for its detection

    Directory of Open Access Journals (Sweden)

    Yaseen ALNAASAN

    2013-05-01

    Full Text Available Reverse transcription-polymerase chain reaction analysis with two primer sets of luteoviruses was used to characterize an isolate of Chickpea chlorotic stunt virus (CpCSv, genus Polerovirus, family Luteoviridae (SC402-08 collected from Lattakia, Syria, during the 2007‒2008 chickpea growing season. Sequence analysis revealed that the coat protein gene of the isolate shared nucleotide sequence identities ranging from 97 to 98% with the CpCSv isolates from Egypt, morocco and Syria. The capsid protein was separated as a protein of approximately 20 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis, and was visually detected by its reaction with CpCSV monoclonal antibody in Western blot. SC402-08 isolate of CpCSV was purified from faba bean-infected plants, and yielded 112–182 μg of purified virions kg-1 of infected tissue. The purified preparation was injected into a white rabbit, and an antiserum was obtained and used to detect CpCSv in infected tissues by tissue-blot immunoassay. The antiserum obtained was able to detect CpCSv by the immunoassay up to a dilution of 1:1,024,000.

  19. Cross-reactivity of a polyclonal antibody against Chinemys reevesii vitellogenin with the vitellogenins of other turtle species: Chelydra serpentina , Macrochelys temminckii , and Pelodiscus sinensis.

    Science.gov (United States)

    Saka, Masahiro; Tada, Noriko; Kamata, Yoichi

    2008-09-01

    Vitellogenin (VTG), a yolk-precursor protein in oviparous vertebrates, is a useful biomarker for reproductive physiology and environmental estrogenic pollution. To examine interspecific applicability of an enzyme-linked immunosorbent assay (ELISA) for quantifying Chinemys reevesii VTG, we observed cross-reactivity between a polyclonal antibody against Chinemys reevesii VTG and the VTGs from other turtle species: Chelydra serpentina (Chelydridae), Macrochelys temminckii (Chelydridae), and Pelodiscus sinensis (Trionychidae), which are phylogenetically distant from Chinemys reevesii (Geoemydidae). The VTGs of the three species were induced by injecting estradiol 17beta into the turtles and purified by using the EDTA-MgCl(2) precipitation method. The purified VTG appeared as a 200-kDa protein in sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating that the molecular mass of the VTGs of the three species was similar to that of Chinemys reevesii VTG. The purified VTGs were serially diluted (0.004-2 mug/ml) and applied to the ELISA. Although the VTGs of the two chelydrid turtles showed cross-reactivity in a concentration-dependent manner, the degree of cross-reactivity was only 22.8-41.2% (mean=30.0%) and 19.7-53.0% (mean=33.2%) for Chelydra serpentina VTG and Macrochelys temminckii VTG, respectively. The ELISA may therefore be theoretically applicable to measure relative levels of the VTGs of these two species, but the absolute concentration values may be inaccurate. Pelodiscus sinensis VTG showed almost no cross-reactivity (8.0-9.7%, mean=8.9%) at any concentration tested, thus indicating the inapplicability of the ELISA to quantify Pelodiscus sinensis VTG. There are thus limitations in extending the applicability of the ELISA across species, even within the order Testudines.

  20. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  1. An improved yeast transformation method for the generation of very large human antibody libraries.

    Science.gov (United States)

    Benatuil, Lorenzo; Perez, Jennifer M; Belk, Jonathan; Hsieh, Chung-Ming

    2010-04-01

    Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

  2. Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

    Science.gov (United States)

    Menzel, Ulrike; Greiff, Victor; Khan, Tarik A; Haessler, Ulrike; Hellmann, Ina; Friedensohn, Simon; Cook, Skylar C; Pogson, Mark; Reddy, Sai T

    2014-01-01

    High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

  3. Modular and aggregation resistant Vh antibodies from a phage display library

    DEFF Research Database (Denmark)

    Friis, Niels Anton; Mandrup, Ole Aalund; Lykkemark, Simon

    2012-01-01

    through immunisation of sharks or camels, or alternatively from recombinant libraries1. The domain antibodies have certain advantages, both pharmacologically and technically. Here we report the construction of a semi-synthetic and highly modular antibody library, based on a human framework (V3-23/D47......Directed evolution of antibodies through phage display is a powerful technique for producing binders of various biological targets. One of the recent innovations in the fi eld is the domain antibody, an antibody consisting only of a single variable domain. These anti bodies can be obtained either......). The antibody scaffold has been codon optimised to improve expression, and the CDR’s have been created using trinucleotide synthesis. These methods give a strict control over the randomisations, and the ability to design a library with minimal aggregation propensity. To facilitate further manipulation, unique...

  4. A novel heavy domain antibody library with functionally optimized complementarity determining regions.

    Directory of Open Access Journals (Sweden)

    Ole Aalund Mandrup

    Full Text Available Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

  5. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  6. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer

    2016-01-01

    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  7. Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

    Science.gov (United States)

    Welschof, M; Little, M; Dörsam, H

    1998-01-01

    Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

  8. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    Directory of Open Access Journals (Sweden)

    Kathrin Tissot

    2011-05-01

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

  11. Evaluation of the inflammatory activity in chronic osteomyelitis. Contribution of the scintigraphy with polyclonal antibodies; Avaliacao de atividade inflamatoria em osteomielite cronica. Contribuicao da cintilografia com anticorpos policlonais

    Energy Technology Data Exchange (ETDEWEB)

    Sapienza, Marcelo Tatit

    1996-07-01

    Active chronic osteomyelitis or complicating osteomyelitis (superimposed on diseases that changes the normal bone structure fractures, post-surgery, prosthesis) can be difficult to diagnose by anatomic radiological imaging modalities, like plain radiograph and CT. These diseases frequently cause also increased bone remodeling, leading to nonspecific uptake of Tc-99m-bone scan agents and gallium-67. New radiopharmaceuticals with greater inflammation/infection avidity and specificity are being developed, including the nonspecific polyclonal immunoglobulin (IgG) labeled with technetium-99. Tc-99m-IgG may be available as a ready to use kit, with no reported side effects, low patient absorbed radiation dose and low cost. The mechanism of IgG uptake at the inflammation site has not been fully elucidated yet. Specific (receptor linking, physico-chemical immunoglobulin properties) and nonspecific mechanisms (enhanced vascular permeability and macromolecular exudate) has been suggested. IgG scintigraphy results are affected by the isotope, labeling procedure adopted and characteristics of the inflammatory focus. Nineteen patients with suspected osteomyelitis (active chronic osteomyelitis or violated bone osteomyelitis) were studied by Tc-99m-IgG scintigraphy (directly labeled polyclonal immunoglobulin, Sandoglobuilina - Sandoz). All patients also underwent standard three-phase bone scintigraphy using methylene diphosphonate (Tc-99m-MDP), gallium-67 scintigraphy and plain radiographs. Infection was found in 8 sites. Sensitivity and specificity for Tc-99m-MDP, gallium-67 and Tc 99m-IgG scintigraphy were, respectively, 88 and 36%, 75 and 73%,88 and 82%. All patients with false positive IgG scintigraphies had previous surgery. Other current scintigraphic procedures used in the diagnosis of osteomyelitis are also reviewed. (author)

  12. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  13. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  14. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    Science.gov (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  15. Effects of interferon gamma and specific polyclonal antibody on the infection of murine peritoneal macrophages and murine macrophage cell line PMJ2-R with Encephalitozoon cuniculi

    Czech Academy of Sciences Publication Activity Database

    Jelínek, Jiří; Salát, Jiří; Sak, Bohumil; Kopecký, Jan

    2007-01-01

    Roč. 54, č. 3 (2007), s. 172-176 ISSN 0015-5683 R&D Projects: GA ČR GP524/03/D167 Institutional research plan: CEZ:AV0Z60220518 Keywords : microsporidia * Encephalitozoon cuniculi * antibody * macrophage s * interferon gamma (IFN-gamma) Subject RIV: EC - Immunology Impact factor: 1.000, year: 2007

  16. Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

    Science.gov (United States)

    Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

    2018-03-01

    B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 9 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

  17. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  18. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  19. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

    Science.gov (United States)

    Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

    2012-10-26

    For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  20. POLYCLONAL ANTIBODIES TO DETECT THE CP-RT PROTEIN OF Potato Mop-Top VIRUS ANTICUERPOS POLICLONALES PARA LA DETECCIÓN DE LA PROTEÍNA CP-RT DEL Potato Mop-Top VIRUS

    Directory of Open Access Journals (Sweden)

    Derly Andrade Molina

    2012-06-01

    Full Text Available Abstract. Potato mop-top virus (PMTV causes an important re-emerging disease in potato crops in Colombia due to the increased incidence of its protozoan vector Spongospora subterranea f.sp. subterranea (Sss, the causing agent of Powdery scab disease. For an accurate detection of PMTV it is recommended to combine different diagnostic tests and evaluate multiple samples per plant or tissue. In order to increase the number of available tools for detection of PMTV, antibodies targeting the RT domain of the CP-RT protein were developed in this work. Sequencing of the RT domain from the colombian strain R25 was achieved and using bioinformatic analysis, a potential antigenic region was identified. A peptide mimicking the antigenic region was inoculated in rabbits for the production of polyclonal antibodies. The antibodies were tested by ELISA using Nicotiana benthamiana bait-plants infected with Sss cystosori and potato plants collected in La Unión (Antioquia. The validity of serological tests was confirmed by RT-PCR. A complete sequence of the RT domain and 441 nt of the CP gene were obtained. Phylogenetic analysis identified strain R25 as closely associated to the PMTV lineage distributed worldwide. A total of 19.26 mg of anti-CP-RT polyclonal antibodies useful in detecting PMTV in infected plants were obtained. As CP-RT is involved in transmission of PMTV by Sss, these antibodies will be useful for supporting not only diagnostic programs but also basic and epidemiologic studies aimed at understanding interactions between PMTV and Sss.Resumen. El Potato mop-top virus (PMTV es uno de los virus re-emergentes en los cultivos de papa de Colombia, como resultado del aumento de la incidencia de su vector natural, el protozoo Spongospora subterranea f.sp. mamarinm@unal.edu.co (Sss, agente causal de la Sarna polvosa de la papa. Para la detección del virus se recomienda la realización simultánea de diferentes pruebas, así como la evaluación de m

  1. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Antibodies to Placental Immunoregulatory Ferritin with Transfer of Polyclonal Lymphocytes Arrest MCF-7 Human Breast Cancer Growth in a Nude Mouse Model

    Directory of Open Access Journals (Sweden)

    Marisa Halpern

    2007-06-01

    Full Text Available The recently cloned human gene named “placental immunoregulatory ferritin” (PLIF is a pregnancyrelated immunomodulator. Recombinant PLIF and its bioactive domain C48 are immune-suppressive and induce pronounced IL-10 production by immune cells. PLIF is expressed in the placenta and breast cancer cells. Blocking PLIF in pregnant mice by anti-C48 antibodies inhibited placental and fetal growth and modulated the cytokine network. It has been revealed that anti-C48 treatment inhibited MCF-7 tumor growth in nude mice. However, this significant effect was observed only in those transfused with human peripheral blood mononuclear cells. Blocking PLIF in tumor-engrafted human immune cell transfused mice resulted in massive infiltration of human CD45+ cells (mainly CD8+ T cells, both intratumorally and in the tumor periphery, and a significant number of caspase-3+ cells. In vitro, antiC48 treatment of MCF-7 tumor cells cocultured with human lymphocytes induced a significant increase in interferon-γ secretion. We conclude that blocking PLIF inhibits breast cancer growth, possibly by an effect on the cytokine network in immune cells and on breakdown of immunosuppression.

  3. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

  4. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  5. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  6. Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

    Science.gov (United States)

    Henry, Kevin A; Tanha, Jamshid

    2018-05-01

    Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Detection of PMTV Using Polyclonal Antibodies Raised Against a Capsid-Specific Peptide Antigen / Detección de PMTV Utilizando Anticuerpos Policlonales Contra un Péptido Antigénico Derivado de la Cápside Viral

    Directory of Open Access Journals (Sweden)

    Yuliana Gallo García

    2013-12-01

    Full Text Available Potato mop-top virus (PMTV; genus Pomovirus;family Virgaviridae is the causing agent of the spraing disease in potato (Solanum tuberosum. PMTV is transmitted by Spongospora subterranea f. sp. subterranea (Sss. This disease has a widespread distribution in potato growing regions around the world. The possibility of obtaining strain specific antibodies at low cost can greatly increase the sensitivity and use of serological tests in seed certification programs, plant breeding and quarantine regulations to avoid dissemination of this injurious virus. This work presents an alternative procedure for the production of PMTV specific antibodies useful in serological test such as ELISAand lateral flow. In contrast to standard methods requiring theisolation of viral particles or expression of recombinant capsid, this method uses peptides mimicking the N-terminal region of PMTV capsid protein as antigen for the production of specific polyclonal antibodies. The antibodies were tested against bait plants grown in soil infested with viruliferous Sss, as well as potato plants obtained from naturally Sss infested fields in Colombia. PMTV was detected in 9/14 and 24/28 foliage samples of N. benthamiana and S. phureja, respectively. In the case of field plants, the virus wasdetected in eight out of 12 root tissues evaluated. The minimumpeptide concentration detected by ELISA was of the order of 0.1 nM. / Potato mop-top virus (PMTV; género Pomovirus; familia Virgaviridae es transmitido por Spongospora subterranea f. sp. subterranea (Sss, agente causal de la sarna polvosa de la papa. Esta enfermedad tiene una amplia distribución en las regiones cultivadoras de papa alrededor del mundo. La posibilidad de obtener anticuerpos específicos contra cepas de este virus, puede incrementar la sensibilidad y la utilización de pruebas serológicas en programas de certificación de semilla, mejoramiento genético y regulaciones cuarentenarias que eviten su diseminaci

  8. Polyclonal antibodies production against Staphylococcus aureus ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-02-01

    Feb 1, 2010 ... ... and the enterotoxins staphylococcies dried dosage remains the unique means to define the food innocuousness. The standard methods of cultivation used to detect .... A second sample of pasteurized and packaged milk.

  9. Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Bødtger, Uffe; Kristensen, Peter

    2004-01-01

    Allergen-specific Fab fragments isolated from combinatorial IgE and IgG libraries are useful tools for studying allergen-antibody interactions. To characterise the interaction between different allergens and antibodies we have created recombinant human phage antibody libraries in the Fab format...

  10. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. Copyright © 2015 Elsevier B

  11. Development and application of an enzyme-linked immunosorbent assay for specific detection of mangiferin content in various cultivars of Mangifera indica leaves using anti-mangiferin polyclonal antibody.

    Science.gov (United States)

    Yusakul, Gorawit; Kitirattrakarn, Wongsathorn; Tanwanichkul, Narunat; Tanaka, Hiroyuki; Putalun, Waraporn

    2012-04-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for determining mangiferin content in plant samples using a polyclonal antibody (PAb) against mangiferin. The developed ELISA showed a full measurement range from 0.12 to 31.25 μg/mL mangiferin with a relative standard deviation (RSD) less than 6% for both intra- and interassay precision levels. The accuracy was determined by a percent recovery experiment at three concentration levels and it showed 97.8%-103.7% recovery in Mangifera indica leaf samples. The developed ELISA exhibited a high correlation value (R² = 0.992) with the standard high-performance liquid chromatography (HPLC) method in various mangiferin-containing plant samples. Our results suggest that the validated ELISA methodology using a PAb against mangiferin can be applied to determine mangiferin content with high specificity, rapidity and simplicity in various mangiferin-containing plant samples. The mangiferin content in the mature leaves of fifty M. indica cultivars were determined using the developed ELISA. The mangiferin contents ranged from 1.94 ± 0.13% to 13.79 ± 0.84% dry wt. The Thawai cultivar leaves contained the highest level of mangiferin (13.79 ± 0.84% dry wt), but it is a rare cultivar. The Namdokmai, which is more commonly cultivated in Thailand, contain 12.41 ± 0.60% dry wt mangiferin; therefore, this cultivar leaf was recommended as the source of raw material for the pharmaceutical, nutraceutical and cosmetic product industries. Currently, natural heath products are accepted worldwide for healthcare. Mangiferin-containing plants and products exhibit health benefits against oxidative stress-related diseases, such as cardiovascular diseases, cancer, dyslipidemia and diabetes. We have developed an ELISA with high specificity, rapidity and simplicity for the quality control of mangiferin-derived product production. Moreover, we found that the Namdokmai leaf, a Thai M. indica cultivar, was recommended as the source of raw

  12. Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Laura Frigotto

    2015-05-01

    Full Text Available We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR in antibody fragment libraries and next generation sequencing (NGS analysis of their quality and diversity.

  13. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  14. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    Directory of Open Access Journals (Sweden)

    Kristensen Peter

    2009-01-01

    Full Text Available Abstract Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

  15. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  16. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

    Science.gov (United States)

    Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

    2018-04-01

    Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

  17. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  18. Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients.

    Science.gov (United States)

    Ayat, Hoda; Burrone, Oscar R; Sadghizadeh, Majid; Jahanzad, Eissa; Rastgou, Nasrin; Moghadasi, Sarrira; Arbabi, Mehdi

    2013-11-01

    Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning. Copyright © 2013 The International Alliance for Biological Standardization. All rights reserved.

  19. Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

    Science.gov (United States)

    Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

    2018-05-01

    Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  20. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

  1. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  2. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  3. Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

    Science.gov (United States)

    Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

    2002-01-01

    AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

  4. Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

    Science.gov (United States)

    Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2018-01-01

    Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

  5. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Another milestone in the history of antibodies was the work of Porter and Edelman ... transgenic animals (Lonberg et al., 1994; Green et al.,. 1994) or .... create and to screen human recombinant antibodies libraries, that is ...

  6. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  7. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    Science.gov (United States)

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Protection from the 2009 H1N1 pandemic influenza by an antibody from combinatorial survivor-based libraries.

    Directory of Open Access Journals (Sweden)

    Arun K Kashyap

    2010-07-01

    Full Text Available Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06 against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 "Swine" H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.

  9. Antibody Engineering and Therapeutics

    Science.gov (United States)

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  10. Upregulation of Mrps18a in breast cancer identified by selecting phage antibody libraries on breast tissue sections

    DEFF Research Database (Denmark)

    Sørensen, Karen Marie Juul; Meldgaard, Theresa; Melchjorsen, Connie Jenning

    2017-01-01

    BACKGROUND: One of the hallmarks of cancer is an altered energy metabolism, and here, mitochondria play a central role. Previous studies have indicated that some mitochondrial ribosomal proteins change their expression patterns upon transformation. METHOD: In this study, we have used the selection...... of recombinant antibody libraries displayed on the surface of filamentous bacteriophage as a proteomics discovery tool for the identification of breast cancer biomarkers. A small subpopulation of breast cells expressing both cytokeratin 19 and cytokeratin 14 was targeted using a novel selection procedure....... RESULTS: We identified the mitochondrial ribosomal protein s18a (Mrps18a) as a protein which is upregulated in breast cancer. However, Mrps18a was not homogeneously upregulated in all cancer cells, suggesting the existence of sub-populations within the tumor. The upregulation was not confined...

  11. Library

    OpenAIRE

    Dulaney, Ronald E. Jr.

    1997-01-01

    This study began with the desire to design a public town library of the future and became a search for an inkling of what is essential to Architecture. It is murky and full of contradictions. It asks more than it proposes, and the traces of its windings are better ordered through collage than logical synthesis. This study is neither a thesis nor a synthesis. When drawing out the measure of this study it may be beneficial to state what it attempts to place at the ...

  12. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  13. Producción de anticuerpos policlonales IgG contra una proteína con actividad de óxido nítrico sintetasa de Toxoplasma gondii recombinante (NOS-Tg-r y marcación inmunológica en taquizoítoso Production of Polyclonal Antibodies against Toxoplasma gondii Recombinant protein with Nitric Oxide Synthase activity and immunologic marking in Tachyzoites

    Directory of Open Access Journals (Sweden)

    Jonathan Mauricio Padilla-Londoño

    2008-06-01

    Full Text Available La enzima óxido nítrico sintetasa ha sido estudiada en mamíferos; en los últimos años se ha descrito que existe también en protozoos, pero se desconocen aspectos importantes de su función. Se logró producir anticuerpos policlonales contra la proteína recombinante con actividad de óxido nítrico sintetasa (NOS-Tg-r de Toxoplasma gondii y realizar marcación inmunológica en taquizoítos. Se usaron dos conejos Nueva Zelanda (Oryctolagus cuniculusque se inmunizaron por vía intramuscular con NOS-Tg-r, y dos tipos de adyuvantes, hidróxido de aluminio y adyuvante de Freund. Se comprobó la presencia de anticuerpos policlonales con la técnica de ensayo inmunoenzimático indirecto. Los resultados obtenidos mostraron que a NOS-Tg-r con adyuvante de Freund indujo mayor respuesta inmune que la de la NOS-Tg-r con hidróxido aluminio p 0,005. Para verificar si había reacción cruzada, se realizó una prueba ELISA utilizando como antígenos: metaloproteasa de T. gondii recombinante, cisteína proteasa 5 de Entamoeba histolytica recombinante, albúmina al 2%, hidróxido de aluminio y adyuvante de Freund. Los valores obtenidos con sueros preinmunes y contra proteínas alternas no superaron el punto de corte (0,069, lo cual indica que los anticuerpos policlonales obtenidos son específicos para NOS-Tg-r. Se realizó marcación inmunológica en taquizoítos de Toxoplasma gondii con inmunofluorescencia indirecta que mostró una marcación difusa a nivel de citoplasma y confirmó la presencia de esta proteína en los taquizoítos.The nitric oxide synthase (NOSis an enzyme well described on mammals but little is known about the role of these enzymes on pathogenic parasites. We produced polyclonal antibodies against a recombinant NOS enzyme from Toxoplasma gondii nd e lso er formed n mmunol locali zation of the enzyme on tachyzoites. We used two New Zealand rabbits (Oryctolagus cuniculus to perform intramuscular immunization and we used two types of

  14. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

    Science.gov (United States)

    Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

    2011-10-28

    Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. A new methodology for the improvement of diagnostic immunohistochemistry in canine veterinary pathology: automated system using human monoclonal and polyclonal antibodies Uma nova metodologia para melhora do diagnóstico imunoistoquímico em patologia veterinária canina: sistema automático usando anticorpos humanos monoclonais e policlonais

    Directory of Open Access Journals (Sweden)

    G.D. Cassali

    2001-06-01

    Full Text Available The authors describe their experience with an automated immunohistochemical system applied to canine tissue samples. Twenty human cellular markers specific monoclonal and polyclonal antibodies and two different antigen retrieval methods were used in normal and neoplastic breast tissue, as well as skin samples obtained from female dogs of pure and mixed breeds. The antibodies tested were the most frequently used in human and veterinary medicine studies, employed with diagnostic purposes in breast pathology, as well as in cancer research. Most of them may be used to study other normal and abnormal tissues and included cytokeratins, progesterone receptor, c-erbB2, p53, MIB-1, PCNA, EMA, vimentin, desmin, alpha-actin, S-100, pan-cadherin, and E-cadherin. The results demonstrated that using an automated staining system it is possible to use different human markers in veterinary pathology. The advantages of automated immunohistochemistry are improved quality, reproducibility, speed, and standardisation.Os autores descrevem sua experiência com um sistema automático de imunoistoquímica aplicada à amostras de tecido canino. Foram utilizados 20 anticorpos humanos monoclonais e policlonais e dois diferentes métodos de recuperação antigênica em tecido mamário normal e neoplásico, bem como em amostras de pele obtidas de cadelas. Os anticorpos testados estão entre os mais usados em estudos de medicina humana e veterinária, com finalidade de diagnóstico em patologia mamária, bem como na pesquisa do câncer. Muitos deles podem ser usados para estudar outros tecidos normais e com alterações e incluem citoqueratinas, receptor de progesterona, c-erbB2, p53, MIB-1, PCNA, EMA, vimentina, desmina, alfa-actina, S-100, pan-caderina e E-caderina. Os resultados demonstraram que usando um sistema automático de imunoistoquímica é possível usar diferentes marcadores humanos em patologia veterinária. As vantagens da imunoistoquímica automatizada s

  16. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  17. Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

    Science.gov (United States)

    Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

    2014-11-07

    We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

  18. Large-scale in vitro expansion of polyclonal human switched-memory B lymphocytes.

    Directory of Open Access Journals (Sweden)

    Sonia Néron

    Full Text Available Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg, are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients' accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10(6-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG(1, IgG(2, IgG(3 and IgG(4 is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.

  19. Production and purification of polyclonal antibody against bovine ...

    African Journals Online (AJOL)

    SERVER

    2007-06-18

    Jun 18, 2007 ... 1Immunology Laboratory, Drug Applied Research Center, Tabriz University of medical sciences, Tabriz-Iran. 2Tropical and Infectious ... pH 7.2). Then equal volumes of diluted serum and saturated ammo- ... solution, and the container was kept on a stirrer for 20 min at room ... quality of the product. Thus ...

  20. High-level expression, purification, polyclonal antibody preparation ...

    African Journals Online (AJOL)

    OprD is a specific porin which can binds imipenem and carbapenems in Pseudomonas aeruginosa. OprD loss plays a central role in mediating carbapenem resistance. Therefore, purification of oprD protein lays a pavement for the study in vivo and in vitro. In our study, the oprD gene was cloned into pQE30 expression ...

  1. Preparation and characterization of the polyclonal antibody against ...

    African Journals Online (AJOL)

    user

    2011-03-14

    PAGE and staining the protein bands by pre-cooled 0.2 mol/l ... SDS-PAGE also showed that rGAR almost existed in the precipitate of the lysed bacteria, which suggested that the expressed. rGAR was almost the inclusion body ...

  2. High-level expression, purification, polyclonal antibody preparation ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... mechanisms of antibiotic resistance (Livermore, 2001). Carbapenem .... column volumes of sterile deionized water, five column volumes of. Charge buffer (50 mM .... antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with ... The role of specific surface loop regions in determining ...

  3. Influence of a polyclonal antibody preparation on the in situ ...

    African Journals Online (AJOL)

    User

    However, the possible health effects of the use of these additives are a cause for ... feed bunks, rubber-matted floors and automatic water fountains common to two ... 20 mg selenium; 2.700 mg zinc; 900 mg fluorine (maximum); 200.000 IU ...

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

    African Journals Online (AJOL)

    2013-09-11

    month-old male New Zealand rabbits were immunized using .... Immunofluorescence analysis of anti-rHuEPO pAb in CHO cells transfected with pTARGET/EPO. ... erythropoietin gene doping: detection strategies in the genomic era.

  5. Plant antigens cross-react with rat polyclonal antibodies against

    Czech Academy of Sciences Publication Activity Database

    Oulehlová, Denisa; Hála, Michal; Potocký, Martin; Žárský, Viktor; Cvrčková, F.

    2009-01-01

    Roč. 33, č. 1 (2009), s. 113-118 ISSN 1065-6995 R&D Projects: GA AV ČR KJB600380601; GA ČR GA204/05/0268; GA MŠk(CZ) LC06004; GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : Keyhole limpet hemocyanin * Rat antisera * Synthetic peptides Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.800, year: 2009

  6. Production of Polyclonal Antiobies to a Recombinant Potato Mop-top Virus Non-structural Triple Gene Block Protein l

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Filigarová, Marie; Pečenková, Tamara

    2006-01-01

    Roč. 154, - (2006), s. 422-427 ISSN 0931-1785 R&D Projects: GA ČR GA522/04/1329 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato mop-top virus * recombinant protein * triple gene block * polyclonal antibodies Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.817, year: 2006

  7. Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

    Science.gov (United States)

    Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

    2010-11-30

    Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001.

    Directory of Open Access Journals (Sweden)

    Nathan Holman

    Full Text Available Monoclonal antibodies that bind to human leukocyte antigen (HLA are useful tools for HLA-typing, tracking donor-recipient chimerisms after bone marrow transplants, and characterizing specific major histocompatibility complexes (MHC on cell surfaces. Unfortunately, equivalent reagents are not available for rhesus macaques, which are commonly used animal as models in organ transplant and infectious disease research. To address this deficiency, we isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a female Mamu-A1*001-negative rhesus macaque with peripheral blood mononuclear cells (PBMC from a male Mamu-A1*001-positive donor. A Fab phage display library was constructed with PBMC from the alloimmunized macaque and panned to isolate an antibody that binds to Mamu-A1*001 but not to other common rhesus macaque MHC class I molecules. The isolated antibody distinguishes PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, the Mamu-A1*001-specific antibody binds the cynomolgus macaque MHC class I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, indicating a high degree of binding specificity. The Mamu-A1*001-specific antibody will be useful for identifying Mamu-A1*001-positive rhesus macaques, for detecting Mamu-A1*001-positive cells in populations of Mamu-A1*001-negative cells, and for examining disease processes that alter expression of Mamu-A1*001 on cell surfaces. Moreover, the alloimmunization process we describe will be useful for isolating additional MHC allomorph-specific monoclonal antibodies or antibodies against other polymorphic host proteins which are difficult to isolate with traditional technologies.

  9. Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries.

    Science.gov (United States)

    Henry, Kevin A; Kim, Dae Young; Kandalaft, Hiba; Lowden, Michael J; Yang, Qingling; Schrag, Joseph D; Hussack, Greg; MacKenzie, C Roger; Tanha, Jamshid

    2017-01-01

    Human autonomous V H /V L single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged V H /V L domains. Here, we describe the design and characterization of three novel human V H /V L sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential V H /V L sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three V H /V L sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three V H /V L libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 V H s and 7 V L s in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2-3 µM), but had highly variable expression yields (range: 0.1-19 mg/L). Despite our efforts to identify the most stable V H /V L scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing V H /V L sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some V H /V L sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence

  10. Estimation of polyclonal IgG4 hybrids in normal human serum.

    Science.gov (United States)

    Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

    2014-07-01

    The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. © 2014 John Wiley & Sons Ltd.

  11. Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Marc C Levesque

    2009-07-01

    Full Text Available The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

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

    International Nuclear Information System (INIS)

    Castiglia, S.G. de

    1998-01-01

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

  13. Production, characterization and applications for Toxoplasma gondii-specific polyclonal chicken egg yolk immunoglobulins.

    Directory of Open Access Journals (Sweden)

    Álvaro Ferreira Júnior

    Full Text Available Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model.In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg and purified egg yolk antibodies (IgY by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers.Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii.

  14. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  15. Escherichia coli F4 fimbriae specific lama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhea

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Hoogendoorn, A.; Zijderveld, van F.G.; Niewold, T.A.; Meulen, van der J.

    2005-01-01

    Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs)

  16. Polyclonal immune responses to antigens associated with cancer signaling pathways and new strategies to enhance cancer vaccines.

    Science.gov (United States)

    Clay, Timothy M; Osada, Takuya; Hartman, Zachary C; Hobeika, Amy; Devi, Gayathri; Morse, Michael A; Lyerly, H Kim

    2011-04-01

    Aberrant signaling pathways are a hallmark of cancer. A variety of strategies for inhibiting signaling pathways have been developed, but monoclonal antibodies against receptor tyrosine kinases have been among the most successful. A challenge for these therapies is therapeutic unresponsiveness and acquired resistance due to mutations in the receptors, upregulation of alternate growth and survival pathways, or inadequate function of the monoclonal antibodies. Vaccines are able to induce polyclonal responses that can have a multitude of affects against the target molecule. We began to explore therapeutic vaccine development to antigens associated with these signaling pathways. We provide an illustrative example in developing therapeutic cancer vaccines inducing polyclonal adaptive immune responses targeting the ErbB family member HER2. Further, we will discuss new strategies to augment the clinical efficacy of cancer vaccines by enhancing vaccine immunogenicity and reversing the immunosuppressive tumor microenvironment.

  17. Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

    Science.gov (United States)

    Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

    2000-04-01

    VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

  18. [Antibody therapy for Alzheimer's disease].

    Science.gov (United States)

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

  19. [Progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases].

    Science.gov (United States)

    Yao, Yuan; Yu, Chuan-xin

    2013-08-01

    Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

  20. Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation

    International Nuclear Information System (INIS)

    Goldman, M.; Rose, L.M.; Hochmann, A.; Lambert, P.H.

    1982-01-01

    We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions

  1. Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

    Science.gov (United States)

    Shao, Cui-Ying; Secombes, Chris J; Porter, Andrew J

    2007-01-01

    The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

  2. Antithyroglobulin antibody

    Science.gov (United States)

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

  3. Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin

    Science.gov (United States)

    Goldman, Ellen R.; Anderson, George P.; Zabetakis, Dan; Walper, Scott; Liu, Jinny L.; Bernstein, Rachael; Calm, Alena; Carney, James P.; O’Brien, Thomas W.; Walker, Jennifer L.; Garber, Eric A. E.

    2011-01-01

    Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. PMID:22174977

  4. Human antibody technology and the development of antibodies against cytomegalovirus.

    Science.gov (United States)

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee

    2017-10-01

    Full Text Available Russell’s vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs containing 3.4 × 107 and 5.5 × 107 transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  6. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis.

    Science.gov (United States)

    Lee, Chi-Hsin; Lee, Yu-Ching; Lee, Yueh-Lun; Leu, Sy-Jye; Lin, Liang-Tzung; Chen, Chi-Ching; Chiang, Jen-Ron; Mwale, Pharaoh Fellow; Tsai, Bor-Yu; Hung, Ching-Sheng; Yang, Yi-Yuan

    2017-10-27

    Russell's vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY) antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs) containing 3.4 × 10⁷ and 5.5 × 10⁷ transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  7. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.

  8. New perspectives on recombinant human antibodies

    NARCIS (Netherlands)

    J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

    1996-01-01

    textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

  9. Detection of acute inflammation with 111In-labeled nonspecific polyclonal IgG

    International Nuclear Information System (INIS)

    Fischman, A.J.; Rubin, R.H.; Khaw, B.A.

    1988-01-01

    The detection of focal sites of inflammation is an integral part of the clinical evaluation of the febrile patient. When anatomically distinct abscesses are present, lesion detection can be accomplished by standard radiographic techniques, particularly in patients with normal anatomy. At the phlegmon stage, however, and in patients who have undergone surgery, these techniques are considerably less effective. While radionuclide methods, such as Gallium-67 (67Ga)-citrate and Indium-111 (111In)-labeled WBCs have been relatively successful for the detection of early inflammation, neither approach is ideal. In the course of studies addressing the use of specific organism-directed antibodies for imaging experimental infections in animals, we observed that nonspecific polyclonal immunoglobulin G (IgG) localized as well as specific antibodies. Preliminary experiments suggested that the Fc portion of IgG is necessary for effective inflammation localization. Since polyclonal IgG in gram quantities has been safely used for therapy in patients with immune deficiency states, we decided to test whether milligram quantities of radiolabeled IgG could image focal sites of inflammation in humans. Thus far, we have studied a series of 84 patients with suspected lesions in the abdomen, pelvis, vascular grafts, lungs, or bones/joints. In 48 of 52 patients with focal lesions detected by surgery, computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), the IgG scan correctly localized the site, while 31 patients without focal inflammation had no abnormal focal localization of the radiopharmaceutical. Four patients had false negative scans and one patient had a false positive scan. For this small series, the overall sensitivity and specificity were 92% and 95%, respectively. In this report, we review our experience with this exciting new agent

  10. Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay.

    Science.gov (United States)

    Sorsa-Leslie, Tarja; Mason, Helen D; Harris, William J; Fowler, Paul A

    2005-09-26

    We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the

  11. The research progress and medicine application of the ScFv antibody

    International Nuclear Information System (INIS)

    Qin Lili; Zhang Chunming

    2005-01-01

    Since the scholar of England and Japan had found the diphtheria antitoxin, the research of the antibody has experienced three phases: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. In recent years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-lives in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte. (authors)

  12. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  13. A freeze dried kit formulation for the preparation of 99m Tc labelled human polyclonal IgG for the detection of infection and inflammation

    International Nuclear Information System (INIS)

    Pedraza L, M.

    1996-01-01

    A freeze dried kit formulation for the preparation of 99m Tc-labelled human polyclonal IgG for the detection of infection and inflammation employing direct methods for protein labeling was developed. The method comprises reduction of intrinsic disulphide bridges within the antibody molecule by the use of the reductant 2-mercaptoethanol. Following a subsequent purification, the resulting reduced antibody is labeled via Sn 2+ reduction of pertechnetate in the presence of a weak competing ligand ethane-1-hydroxy-1,1-diphosphonate (EHDP). High labeling efficiencies (>90%) were obtained with high in vitro stability and without effect upon antibody immunoreactivity. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in radiopharmaceutical solution. 99m Tc-polyclonal IgG prepared by the kit method was evaluated for scintigraphic localization of inflammatory lesions and abscesses in rabbits. Data demonstrated that the 99m Tc-polyclonal IgG after kit-reconstitution shows excellent stability and is an effective imaging agent of infection and/or inflammation. (Author)

  14. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

    Science.gov (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  15. Current diagnostic efficacy of Tc-99m-labeled antitumor antibodies

    International Nuclear Information System (INIS)

    Morrison, R.T.; Lyster, D.M.; Szasz, I.; Alcorn, L.N.; Rhodes, B.A.; Breslow, K.; Burchiel, S.W.

    1983-01-01

    The authors have recently evaluated technetium 99 labeled antibodies specific to human chorionic gonadotropin (hCG) for the in vivo detection of a variety of human tumors. Both mouse monoclonal and sheep polyclonal antibodies were evaluated in this study. Another antibody specific to hCG studied, is an antigen-agglutinating monoclonal F(ab') 2 fragment. Some preliminary results are reported

  16. HETEROGENEITY OF POLYCLONAL IMMUNOGLOBULINS NUCLEASE ACTIVITY IN RHEUMATOID AND REACTIVE ARTHRITIS

    Directory of Open Access Journals (Sweden)

    M. V. Volkova

    2017-01-01

    Full Text Available Catalytic properties of immunoglobulins are widely studied within recent years. It was found that nuclease activity of immunoglobulins is increased in systemic autoimmune diseases. Given some pathogenetic features of rheumatoid arthritis and reactive arthritis, it is appropriate to clarify the nature of nuclease activity in these diseases. Determination of DNAse activity of immunoglobulins with different DNA substrates, and search for specific substrates for distinct clinical entities could serve these purposes. The aim of present work is to determine DNase activity of the polyclonal class G immunoglobulins in rheumatoid and reactive arthritis using various methods.Different methods are used to evaluate nuclease activity. In this paper we present newly developed and modified techniques for determination of DNAse activity of polyclonal IgGs. Particular attention was paid to the electrophoretic method of DNase activity assessment. Polyclonal IgG isolated from blood serum of patients with rheumatoid arthritis and reactive arthritis were used for assays. In this study, we demonstrated the presence of an inhomogeneous DNase activity of immunoglobulins in relation to different substrates.Along with calf thymus DNA, we used bacterial plasmid DNA and PCR products based on bacterial gene sequences. Levels of DNase activity by rivanol clot method with calf thymus DNA as substrate proved to be higher in patients with rheumatoid arthritis than the control values (p < 0.01. DNase abzyme activity in patients with rheumatoid arthritis was elevated, as compared to the patients with reactive arthritis (p < 0.01.When examining ability of the IgG to hydrolyze procaryotic DNA (bacterial plasmid DNA and PCR products, based on bacterial genes, we obtained heterogeneous results. Different Ig samples showed varying degrees of DNA hydrolysis. Abzyme hydrolysis of DNA substrates longer than 700 bp was more pronounced, as compared to short DNA substrates (100 base pairs

  17. Murine Visceral Leishmaniasis: IgM and Polyclonal B-Cell Activation Lead to Disease Exacerbation

    Science.gov (United States)

    Deak, Eszter; Jayakumar, Asha; Wing Cho, Ka; Goldsmith-Pestana, Karen; Dondji, Blaise; Lambris, John D.; McMahon-Pratt, Diane

    2010-01-01

    In visceral leishmaniasis, the draining lymph node (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with L. infantum. Although not unexpected, at early times post-infection there is a marked B cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JHD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JHD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JHD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to wild type BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5aR (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-γ). Overall these studies indicate that polyclonal B cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention. PMID:20213734

  18. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  19. Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

    Directory of Open Access Journals (Sweden)

    Anton M Sholukh

    Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

  20. Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.

    OpenAIRE

    Sastry, L; Alting-Mees, M; Huse, W D; Short, J M; Sorge, J A; Hay, B N; Janda, K D; Benkovic, S J; Lerner, R A

    1989-01-01

    Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establi...

  1. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Haisma, H.; Hilgers, J.

    1987-01-01

    Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

  2. Immune reconstitution with two different rabbit polyclonal anti-thymocytes globulins.

    Science.gov (United States)

    Bamoulid, Jamal; Crepin, Thomas; Gaiffe, Emilie; Laheurte, Caroline; Moulin, Bruno; Frimat, Luc; Rieu, Philippe; Mousson, Christiane; Durrbach, Antoine; Heng, Anne-Elisabeth; Rebibou, Jean-Michel; Saas, Philippe; Courivaud, Cécile; Ducloux, Didier

    2017-12-01

    Broad T cell depletion by polyclonal anti-thymocyte globulins (ATG) has been used for many years as a part of immunosuppressive treatment in transplantation. Currently, two different ATG are used in clinical practice, Thymoglobulin and Grafalon. Due to differences in the immunization source, these products contain different specificities and quantity of antibodies. These differences may have clinical consequences. We conducted a nested study in a large prospective multicentric cohort of kidney transplant to determine whether Grafalon-treated and Thymoglobulin-treated patients experience different lymphocyte reconstitution and clinical outcomes. 182 patients matched for age, gender, CMV status, CMV prophylaxis, number of previous transplantation, and maintenance immunosuppressive treatment were included (Thymoglobulin, [n=91]; Grafalon®, [n=91]). One-year post-transplant, recent thymic emigrants were significantly decreased (12±10% vs 21±12%; p<0.001) in Grafalon-treated patients. By contrast, T cell activation (CD38+DR+Ki67+) and senescence (CD8+CD57+CD28-) was increased in Thymoglobulin-treated patients. Compared to Grafalon, Thymoglobulin was not associated with a significantly different rate of acute rejection. CMV disease (p=0.013) was more frequent in Thymoglobulin-treated patients. Grafalon and Thymoglobulin seem to be equivalent to prevent acute rejection. CMV disease is more frequent in Thymoglobulin-treated patients. One year post-transplant immune profile profoundly differs according to the type of ATG. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Specificity and polyreactivity of the antibody response during natural HIV-1 infection

    OpenAIRE

    Wang, Xin

    2006-01-01

    The specificity and polyreactivity of the antibody response in natural HIV-1 infection were studied. First, to investigate the overall antibody response, overlapping linear peptides were used to screen sera taken from HIV-1-infected individuals. The polyclonal antibody response was relatively stable during long-term infection, compared with acute infection, and mostly directed against immunodominant regions. Low level, transient antibody responses were detected against membrane proximal exter...

  4. Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus

    DEFF Research Database (Denmark)

    Fernandez-Alonso, M.; Lorenzo, G.; Perez, L.

    1998-01-01

    antibodies (MAbs), only 2 non-neutralizing MAbs, I10 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None......Antibody Linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout...... antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Coll 1996; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995; Virology 212:348-355). Among 17 monoclonal...

  5. Immunoparesis and polyclonal immunoglobulin recovery after auto-SCT for patients with multiple myeloma treated at a single institution.

    Science.gov (United States)

    Jimenez-Zepeda, Victor H; Duggan, Peter; Neri, Paola; Chaudhry, Ahsan; Tay, Jason; Bahlis, Nizar

    2017-11-21

    Immunoparesis and polyclonal immunoglobulin recovery have been recently described as common indicators of immune dysfunction in patients with multiple myeloma. In the present study, we aimed to assess the impact of immunoparesis and polyclonal immunoglobulin recovery at day-100 post autologous stem cell transplant (auto-SCT) on clinical outcomes. A total of 302 patients were included for the analysis of immunoparesis, and 197 were evaluable for polyclonal immunoglobulin recovery evaluation. Immunoparesis was observed in 93.5% of cases, with 47% of cases having polyclonal immunoglobulin recovery at 12 months post auto-SCT. Median overall and progression-free survival were longer in the group of patients with complete or partial normalization of polyclonal immunoglobulins. Patients receiving consolidation had a lower level of polyclonal reconstitution. In conclusion, polyclonal immunoglobulin recovery by 12 months post-auto-SCT is associated with superior overall and progression free survival in patients with MM. Efforts to better enhance polyclonal recovery deserve further investigation.

  6. Featured Library: Parrish Library

    OpenAIRE

    Kirkwood, Hal P, Jr

    2015-01-01

    The Roland G. Parrish Library of Management & Economics is located within the Krannert School of Management at Purdue University. Between 2005 - 2007 work was completed on a white paper that focused on a student-centered vision for the Management & Economics Library. The next step was a massive collection reduction and a re-envisioning of both the services and space of the library. Thus began a 3 phase renovation from a 2 floor standard, collection-focused library into a single floor, 18,000s...

  7. Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

    International Nuclear Information System (INIS)

    Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi

    1991-01-01

    To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

  8. Production of polyclonal antibodies to a recombinant coat protein of Potato virus Y

    Czech Academy of Sciences Publication Activity Database

    Kmoníčková, Jitka; Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Dědič, P.; Čeřovská, Noemi

    2008-01-01

    Roč. 53, č. 5 (2008), s. 438-442 ISSN 0015-5632 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : ESCHERICHIA-COLI * ANTISERUM * ELISA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.172, year: 2008

  9. Reliable tool for detection of novel Coxiella burnetii antigens, using immobilized human polyclonal antibodies

    Czech Academy of Sciences Publication Activity Database

    Flores-Ramírez, G.; Danchenko, M.; Quevedo-Diaz, M.; Škultéty, L'udovít

    2017-01-01

    Roč. 1047, MAR 15 SI (2017), s. 84-91 ISSN 1570-0232 Institutional support: RVO:61388971 Keywords : Qfever * Biofunctionalized magnetic beads * LC-MS/MS Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 2.603, year: 2016

  10. Development of polyclonal and monoclonal antibodies to Rose rosette virus nucleoprotein

    Science.gov (United States)

    Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale US domestic production valued at ca. $400 million. Over the past few decades Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus), has become a major threat to t...

  11. Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Rosecká, Pavla; Dědič, P.; Filigarová, Marie

    2003-01-01

    Roč. 151, č. 4 (2003), s. 195-200 ISSN 0931-1785 R&D Projects: GA ČR GA522/01/1121 Institutional research plan: CEZ:AV0Z5038910 Keywords : potato mop-top virus * recombinant coat protein * Escherichia Coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.557, year: 2003

  12. Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus A

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Velemínský, Jiří

    2002-01-01

    Roč. 46, - (2002), s. 147-151 ISSN 0001-723X R&D Projects: GA ČR GA310/00/0381 Institutional research plan: CEZ:AV0Z5038910 Keywords : Potato virus A * recombinant coat protein * Escherichia coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.660, year: 2002

  13. Production of Potent Fully Human Polyclonal Antibodies Against Zaire Ebola Virus in Transchromosomal Cattle

    Science.gov (United States)

    2016-07-01

    both humans and non-human primates (NHP) causing severe hemorrhagic fevers with symptoms of disease including sudden onset of fever , chills, headache...and anorexia followed by sore throat, vomiting, diarrhea, hemorrhaging, and the appearance of a petechial rash1-3. Filoviruses are categorized as...hemorrhagic fever . Clinics in laboratory medicine 30, 161-177 (2010). 6. Parren, P.W., et al., Pre- and postexposure prophylaxis of Ebola virus

  14. Monoclonal antibodies against plant viruses

    International Nuclear Information System (INIS)

    Sandler, E.; Dietzgen, R.G.

    1984-01-01

    Ever since antigenic properties of plant viruses were discovered antisera have been raised and used for plant virus diagnosis and for the analysis of virus structure as well. From the early qualitative diagnosis method of precipitating the virus in clarified sap of an infected plant and the first quantitative application of the precipitin test vast progress has been made with regard to the development of highly sensitive and highly quantitative methods for virus detection. Of equal importance was the improvement of methods for separating virus from host cell components since the specificity of antisera raised against a virus could be increased by using an antigen for immunization highly concentrated and largely freed from contaminating host substances. The introduction of the enzyme-linked immunosorbent assay (ELISA) into plant virology allows detection of virus in nanogram quantities. Still, the conventionally raised antisera, no matter how pure an antigen was used for immunization, are polyclonal. They contain products of thousands of different antibody-secreting plasma cell clones which can be directed against all antigenic determinants (epitopes) of the virus, but also against antigens of the host plant that may not have been entirely separated from the immunizing virus during the purification procedure. Even after cross adsorption of polyclonal antisera some residual heterogeneity can be expected to remain. Within these boundaries the information gained with polyclonal antisera on virus structure and on virus diagnosis has to be interpreted

  15. Development of antibody against sulfamethazine

    International Nuclear Information System (INIS)

    Li Ziying; Xi Wenge; Liu Yibing; Zhang Liling; Guo Weizheng; Han Shiquan

    2004-01-01

    Sulfamethazine (SMT) is widely used to treat bacterial and protozoan infections in food animals. So its residue has been detected in various food products, and in Europe, the tolerance level for sulfonamides in meat and milk is 100 ng/g. To ensure that residues in animal food products do not exceed this limit, a simple, sensitive, and rapid method to determinate their residues in animal tissues is needed. In this paper the development of polyclonal or monoclonal antibodies against sulfamethazine (SMT) and a simplified method to identify residual sulfamethazine by radio immunoassay (RIA) is presented. Polyclonal antibodies (PcAbs) against sulfamethazine (SMT) were obtained by immunizing rabbits with SMT-conjugated bovine serum albumin (BSA). The association constants (Ka) of the PcAbs were higher than 108 and the cross-reactivities with Sulfadiazine(SD), Sulfaquinoxaline(SQX) which were structurally related compounds were lower than 0.05%(RIA). Simultaneous, six strains of hybridoma cell were prepared which can secrete monoclonal antibodies (McAbs) against SMT . The Ka of the McAbs against SMT were higher than 107 and the cross-reactivities with SD, SQX were lower than 0.1%(RIA). (authors)

  16. Production and purification of polyclonal anti-hamster ...

    African Journals Online (AJOL)

    . ... IgG showed high titer and high specificity in the designed ELISA. Purified antibody and its conjugation with HRP are used in research and diagnosis of hamster disease. Key words: Production, purification, hamster immunoglobulins.

  17. Taking aim at cancer with monoclonal antibodies

    International Nuclear Information System (INIS)

    Klausner, A.

    1986-01-01

    Conjugating radioisotopes to monoclonal antibodies could have certain advantages in cancer therapy. Radioactive compounds have the double-edged ability to kill cells that are up to centimeter or more away. This is a plausible way to overcome tumor heterogeneity, but it also means that normal cells near the tumor could be affected. Hybritech (San Diego, CA) has been supplying antibody linked to the radioisotope yttrium-90 for a number of clinical trials. Work at Johns Hopkins University (Baltimore, MD) has focused on polyclonal antibodies to hepatoma. Monoclonal antibodies will be used there soon, and trials could be expanded eventually to include breast, lung, and prostate cancer as well. Hybritech also expects that the yttrium-antibody conjugates developed with NCI will enter the clinic later this year for treating leukemia and lymphoma systems; treatments for melanomas should follow

  18. In vivo localization of radiolabeled monoclonal antibody to carcinoembryonic antigen (CEA) in a CEA-producing tumor

    International Nuclear Information System (INIS)

    Kamei, Tetsuya; Seto, Hikaru; Taki, Kuniyasu; Soya, Toshio; Kakishita, Masao; Maeda, Masatoshi; Honda, Takashi; Koshimura, Saburou.

    1987-01-01

    To compare accumulation of the 125 I-labeled antibodies(anti-carcinoembryonic antigen(CEA) monoclonal antibody and polyclonal antibody) to a CEA-producing tumor (SC-2-JCK), an in vivo localization study was performed in nude mice. The tumor-to-blood ratio at 120 hours after injection rose to 4.6 for the monoclonal antibody, but remained at 1.3 for the polyclonal antibody. However, no differences were noted between the antibodies up to 72 hours after injection. In autoradiograms, selective accumulation of the tracer was noted in the tumor for both antibodies. However, no superiority or inferiority of imaging for either of the antibodies could be definitely determined. (author)

  19. Monoclonal antibodies: potential role in radiation therapy and oncology

    International Nuclear Information System (INIS)

    Order, S.E.

    1982-01-01

    Specificity, which is a hallmark of the immune system, will be used in radiation oncology in both diagnosis and therapy through the application of radiolabelled monoclonal and polyclonal antibodies. Antigenic specificities, antibody preparations, and the tumor as a target for radiolabelled antibody is reviewed. Several clinical situations, i.e. single tumor cell suspensions, intraperitoneal single cells and masses, and solid tumors are reviewed in regard to both immune antibody targeting and specific differences between tumors in these regions. The concentration of tumor associated antigens is introductory to radiolabelled antibodies in diagnosis. In the radiation therapy of solid tumors, data regarding tumor dose, tumor effective half-life, varied antibody preparations, and the use of radiolabelled antibody as a method of tumor implantation is discussed using antiferritin 131 I-IgG as a model in hepatoma. The theoretical applications of monoclonal antibody integrated in cancer therapy are then presented as a new goal for future development

  20. Growth and yield of mixed polyclonal stands of Populus in short-rotation coppice

    Energy Technology Data Exchange (ETDEWEB)

    Benbrahim, Mohammed; Gavaland, Andre [INRA centre de Toulouse (France). Unite Agroforesterie et foret Paysanne; Gauvin, Jean [INRA centre d' Orleans (France). Unite d' Amelioration des arbres forestiers

    2000-07-01

    Eight clones of poplar were used to compare the growth and productivity of monoclonal and polyclonal mixed plantations in short-rotation coppice. At the end of the eight growing season, the diameter at breast height (DBH) and height of trees were measured and dry weight and yield were estimated. Polyclonal mixtures did not affect mortality. Few differences in growth were observed between clones in monoclonal plots. Polyclonal mixture slightly affected the growth and tree size of the clones compared with monoclonal plots. No increase in stand heterogeneity in relation to clone deployment was observed. A neighbourhood index was calculated for each tree and was significantly affected by polyclonal mixture. However, the relationship between the neighbourhood index and the DBH indicated that this effect did not cause a great change in DBH. Consequently, dry weight and yield productivity were not affected by clone deployment.

  1. Antibody production in rabbits administered Freund's complete adjuvant and carprofen concurrently.

    Science.gov (United States)

    Fishback, Joanna E; Stronsky, Sabrina M; Green, Catherine A; Bean, Krystal D; Froude, Jeffrey W

    2016-02-01

    Freund's complete adjuvant (FCA) is a commonly used immunopotentiator that can boost polyclonal antibody production in animal models such as rabbits, but FCA is also known to cause inflammation and pain. It is important to balance the welfare of animals with the goal of efficiently producing antibodies, but little is known about how common treatments for pain and inflammation, such as non-steroidal anti-inflammatory drugs (NSAIDs), affect the production of polyclonal antibodies. The purpose of this study was to measure polyclonal antibody production in rabbits that were administered FCA either with or without a concurrent treatment of a NSAID, carprofen. Rabbits were divided into two groups and were administered identical treatments of an antigen with adjuvant, and the treatment group also received carprofen injections at different stages of the study. Carprofen treatment did not significantly affect polyclonal antibody production, which suggests that carprofen and other NSAIDs can be used alongside FCA in rabbits to achieve desired levels of antibody production while minimizing pain and distress associated with the use of FCA.

  2. Monoclonal antibodies against pregnancy-specific β1-glycoprotein (SP1) in immunohistochemistry and radioimmunoassay

    International Nuclear Information System (INIS)

    Wahlstroem, T.; Heikinheimo, M.

    1983-01-01

    Monoclonal mouse antibodies against pregnancy-specific beta-1-glycoprotein (SP 1 ) have been studied for their suitability in immunoperoxidase staining and radioimmunoassay methodologies. These antibodies were useful in staining normal placentas, hydatidiform moles, invasive moles and choriocarcinomas. They showed good specificity, with minimal background staining, and will thus be superior to conventional polyclonal antisera in immunohistochemistry. However, the presently tested monoclonal anti-SP 1 antibodies were found not to be suitable for radioimmunoassay. (Auth.)

  3. Research Library

    Science.gov (United States)

    Los Alamos National Laboratory Research Library Search Site submit Contact Us | Remote Access Standards Theses/Dissertations Research Help Subject Guides Library Training Video Tutorials Alerts Research Library: delivering essential knowledge services for national security sciences since 1947 Los

  4. Homeostatic 'bystander' proliferation of human peripheral blood B cells in response to polyclonal T-cell stimulation in vitro.

    Science.gov (United States)

    Jasiulewicz, Aleksandra; Lisowska, Katarzyna A; Pietruczuk, Krzysztof; Frąckowiak, Joanna; Fulop, Tamas; Witkowski, Jacek M

    2015-11-01

    The mechanisms of maintenance of adequate numbers of B lymphocytes and of protective levels of immunoglobulins in the absence of antigenic (re)stimulation remain not fully understood. Meanwhile, our results presented here show that both peripheral blood naive and memory B cells can be activated strongly and non-specifically (in a mitogen-like fashion) in 5-day in vitro cultures of anti-CD3- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells of healthy people. This polyclonal, bystander activation of the B cells includes multiple divisions of most of them (assessed here by the flow cytometric technique of dividing cell tracking) and significant antibody [immunoglobulin M (IgM) and IgG] secretion. Observed proliferation of the CD19(+) B cells depends on contact with stimulated T helper (Th) cells (via CD40-CD40L interaction) and on the response of B cells to secreted interleukins IL-5, IL-10 and IL-4, and is correlated with the levels of these Th-derived molecules, while it does not involve the ligation of the BCR/CD19 complex. We suggest that the effect might reflect the situation occurring in vivo as the homeostatic proliferation of otherwise non-stimulated, peripheral B lymphocytes, providing an always ready pool for efficient antibody production to any new (or cognate) antigen challenge. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody

    NARCIS (Netherlands)

    Zapata, Sonia; Trueba, Gabriel; Bulach, Dieter M.; Boucher, David; Adler, Ben; Hartskeerl, Rudy

    2010-01-01

    A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    ) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also......), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i...

  7. Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column.

    Science.gov (United States)

    Wu, Zhihua; Li, Kun; Zhan, Shaode; Tong, Ping; Li, Xin; Yang, Anshu; Chen, Hongbing

    2017-09-14

    Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits' antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.

  8. [Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

    Science.gov (United States)

    Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

    2012-07-01

    To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

  9. Antimitochondrial antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  10. Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

    Science.gov (United States)

    Olovnikova, N I; Belkina, E V; Nikolaeva, T L; Miterev, G Yu; Chertkov, I L

    2006-01-01

    Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

  11. Use of polyclonal IgG in HIV infection and AIDS

    International Nuclear Information System (INIS)

    Buscombe, J.R.; Oyen, W.J.G.; Corstens, F.H.M.

    1995-01-01

    Nuclear Medicine should have a pivitol role to play in the investigation of patients infected with the Human Immunodeficiency Virus (HIV). Unfortunately the use of scintigraphic techniques to localize infection have not become widely used in Europe. Neither 67 Ga citrate or labelled leukocytes are ideal. In a search for new agents which can be used to identify the presence of infection both 99m Tc and 111 In labelled polyclonal immunoglobulin-C have been investigated. It was found that 99m TC labelled polyclonal immunoglobulin-G was not able to localize infection in either the chest or the abdomen. In contrast 111 In labelled polyclonal immunoglobulin-G had both high sensitivity and specificity for imaging infection in HIV infected patients. If these preliminary results are confirmed immunoglobulin-G could find an important clinical application in this specialized patient group

  12. Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

    Science.gov (United States)

    Zhou, Jun; Hou, Fujing; Li, Ye; Su, Xiurong; Li, Taiwu; Jin, Chunhua

    2016-07-01

    Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

  13. Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

    Science.gov (United States)

    Collazo, M. Thelma; Garza, John A.; Hayhurst, Andrew

    2010-01-01

    Background There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype. Methods and Findings A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism. Conclusions Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively. PMID:20098614

  14. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...... laboratory conditions. A particular emphasis was put on using molecular techniques in conjunction with microenvironmental measurements (O2, pH, irradiance), a combination that is rarely found but provides a much more detailed understanding of “cause and effect” in complex natural systems...

  15. Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

    Science.gov (United States)

    Zhang, Qian; Noble, Kyle A.; Mao, Yuan; Young, Nicolas L.; Sathe, Shridhar K.; Roux, Kenneth H.; Marshall, Alan G.

    2013-07-01

    The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

  16. Effect of low doses of ionizing radiation on the thymus-dependent humoral immune response and the polyclonal activation of B-lymphocytes

    International Nuclear Information System (INIS)

    Sharetskij, A.N.; Surinov, B.P.; Abramova, M.R.

    2000-01-01

    The results of studies on the effect of the low-dose (10 cGy with the dose rate of 1cGy/min) γ-radiation on the indices of the mice system and local immune response are presented. The sheep erythrocytes were used as a thymus-dependent antigen. It is shown that the total irradiation with the above dose rate induced the increase in the primary thymus-dependent humoral immune response on the sheep erythrocytes and polyclonal activation of the B-lymphocytes. The sharp oppression of the antibody formation was observed in the immune response dynamics after the phase of the radiation-induced elevation. The injection of hydroquinone right after the irradiation resulted in elimination of the radiation stimulation of the polyclonal response of the B-cells. The essential decrease in the immunoantilogarithmic radiation effect took place in the animals treated with thymogen. The possible negative consequences of the low-dose ionizing radiation impact on the body immune system are discussed [ru

  17. Radioimmunodetection of tumor with Ga-67 labeled antibodies

    International Nuclear Information System (INIS)

    Furukawa, Takako; Endo, Keigo; Ohmomo, Yoshiro

    1986-01-01

    Antibodies against tumor associated antigen; anti-AFP polyclonal antibody, anti-thyroglobulin monoclonal antibody and anti-hCG monoclonal antibody, were labeled with Ga-67, using deferoxamine (DF) as a bifunctional chelating agent. The immunoreactivity and in vivo stability of the Ga-67 labeled antibodies were examined. The effect of DF conjugation to antibodies on the antigen-binding activity was evaluated by RIA and Scatchard analysis or tanned sheep red blood cell hemagglutination technique. When DF was conjugated to antibody at the molar ratio of 1 : 1, the antibody activity of the DF-conjugated antibodies was fully retained. Whereas, in heavily conjugated antibodies, the maximum antigen binding capacity was reduced. Biodistribution study in normal mice demonstrated the high in vivo stability of Ga-67 labeled antibodies. The labeling of DF-antibody conjugated with Ga-67 was performed easily and quickly, with a high labeling efficiency, requiring no further purification. Thus, this labeling method, providing in vivo stability of Ga-67 labeled antibody and full retention of immunoreactivity, would be useful for the radioimmunodetection of various cancers. (author)

  18. Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

    OpenAIRE

    Baltus, E; Hanocq-Quertier, J; Hanocq, F; Brachet, J

    1988-01-01

    The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

  19. NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

    Directory of Open Access Journals (Sweden)

    Saeko Yanaka

    2017-09-01

    Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

  20. [Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

    Science.gov (United States)

    Chen, Miao; Yang, Jin-Ju; Li, Shu-Zhen; Liu, Xiao-Lan; Liu, Ying; Zhang, Lin-Jie; Gao, Jian-En; Sun, Qi-Hong

    2008-07-01

    To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2). A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. The GST-PON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Western blot analysis proved the rabbit polyclonal antibodies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SY5Y cells. The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.

  1. Library Computing

    Science.gov (United States)

    Library Computing, 1985

    1985-01-01

    Special supplement to "Library Journal" and "School Library Journal" covers topics of interest to school, public, academic, and special libraries planning for automation: microcomputer use, readings in automation, online searching, databases of microcomputer software, public access to microcomputers, circulation, creating a…

  2. Quantitative imaging with radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Hammond, N.D.

    1988-01-01

    The ability to image tumor by using radiolabeled monoclonal antibody products has been widely demonstrated. The questions of safety and efficacy remain open and require further experience, but at least in some clinical situations radioimmunoimaging has provided clinically useful information. Imaging tumor with radiolabeled monoclonal and polyclonal antibodies has been widely reported, and several summaries have recently appeared. For extensive review of recent clinical imaging the reader is referred to these excellent sources. Having demonstrated the possibility of imaging tumor with radiolabeled antibody, the question now apparent is: will the imaging modality provide information new and different from the already available with established techniques in computed tomography, magnetic resonance imaging, and standard nuclear medicine?

  3. Antigenic profile of human recombinant PrP: generation and chracterization of a versatile polyclonal antiserum

    NARCIS (Netherlands)

    Sachsamanoglou, M.; Paspaltzis, I.; Petrakis, S.; Verghese-Nikolakaki, S.; Panagiotidis, C.H.; Voitlander, T.; Budka, H.; Langeveld, J.P.M.; Sklaviadis, T.

    2004-01-01

    We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the

  4. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain

    DEFF Research Database (Denmark)

    Vanwolleghem, Thomas; Bukh, Jens; Meuleman, Philip

    2008-01-01

    The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these Ig...... were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV...... infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve...

  5. Recursive deconvolution of combinatorial chemical libraries.

    OpenAIRE

    Erb, E; Janda, K D; Brenner, S

    1994-01-01

    A recursive strategy that solves for the active members of a chemical library is presented. A pentapeptide library with an alphabet of Gly, Leu, Phe, and Tyr (1024 members) was constructed on a solid support by the method of split synthesis. One member of this library (NH2-Tyr-Gly-Gly-Phe-Leu) is a native binder to a beta-endorphin antibody. A variation of the split synthesis approach is used to build the combinatorial library. In four vials, a member of the library's alphabet is coupled to a...

  6. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  7. Antithyroid microsomal antibody

    Science.gov (United States)

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  8. Clinical experience in humans with radiolabeled antibody for tumor detection

    International Nuclear Information System (INIS)

    Morrison, R.T.; Lyster, D.M.; Szasz, I.; Alcorn, L.N.; Huckell, V.F.; Rhodes, B.; Breslow, K.; Burchiel, S.

    1982-01-01

    I-131 and Tc-99m labeled polyclonal or monoclonal antibody and fragments of antibody, specific to human chorionic gonadotropin (hCG) or to a melanoma cell surface antigen (MCSA) were injected into proven cancer patients. Using standard homeostasis parameters, and scanning techniques, the safety and efficacy of each antibody was evaluated. Antibody fragments were expected to clear faster from the circulation allowing for earlier imaging and a better target-to-non-target ratio. The technetium label may perturb the antiboby's kinetics so that clearance is more rapid for both whole antibody and fragments. After a statistical evaluation of all parameters measured pre and post injection it was concluded that no acute toxicity reactions were present in any patient studied. Scan results were not acceptable for a tumor detecting procedure used in routine practice. Tumor upake was seen in less than 10% of scans

  9. Newly formed skeletal muscle fibers are prone to false positive immunostaining by rabbit antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Kliem, Anette; Schrøder, Henrik Daa

    2011-01-01

    rely on controls that reveal non-specific binding by the secondary antibody and neglect that the primary rabbit antibody itself may cause false positive staining of the muscle. We suggest that reliable immuno-based protein detection in newly formed muscle fibers at least requires a nonsense rabbit......Reports on muscle biology and regeneration often implicate immuno(cyto/histo)chemical protein characterization using rabbit polyclonal antibodies. In this study we demonstrate that newly formed myofibers are especially prone to false positive staining by rabbit antibodies and this unwanted staining...

  10. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    Science.gov (United States)

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  11. Optimization of antibody immobilization for on-line or off-line immunoaffinity chromatography

    DEFF Research Database (Denmark)

    Beyer, Natascha Helena; Schou, Christian; Højrup, Peter

    2009-01-01

    -POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow....... A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We...

  12. Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries.

    Science.gov (United States)

    Mattioli, S; Imberti, L; Stellini, R; Primi, D

    1995-09-01

    Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure.

  13. Thyroid Antibodies

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  14. Regulation of Polysaccharide- and Protein- Specific Antibody Responses to Intact Extracellular Bacteria

    Science.gov (United States)

    2016-03-11

    OVA peptide (amino acids 323-339), presented by MHC-II I-Ad, were purchased from Taconic Farms (Hudson, NY). They were thereafter bred in our...overnight at 4°C and plates were then washed 3x with PBS + 0.1% Tween 20. Alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG Abs 41 | P a g e...phosphatase-conjugated polyclonal goat anti-mouse IgG antibodies (200 ng/ml) in PBS + 1.0% BSA were then added, and plates were incubated at 37°C for

  15. Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA.

    Science.gov (United States)

    Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew R M; Lemere, Cynthia A; Messer, Anne; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Schuurman, Janine; Adams, Gregory P; Reichert, Janice M

    Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.

  16. About the Library - Betty Petersen Memorial Library

    Science.gov (United States)

    branch library of the NOAA Central Library. The library serves the NOAA Science Center in Camp Springs , Maryland. History and Mission: Betty Petersen Memorial Library began as a reading room in the NOAA Science Science Center staff and advises the library on all aspects of the library program. Library Newsletters

  17. Selection of matched pair of monoclonal antibodies for development of immunoradiometric assay (IRMA) : our experience with IRMA of TSH

    International Nuclear Information System (INIS)

    Kadwad, V.B.; Jyotsna, N.; Sivaprasad, N.

    1998-01-01

    Full text: In immunoradiometricassay (IRMA) two antibodies raised against two different epitopes of the same antigen are used, one bound to a solid phase (capture antibody) and the other labelled with 125 I (detector antibody). The development of any IRMA thus involves proper selection of the capture and detector antibody, preparation of solid phase, labelling of the antibody and assay optimization. Extensive studies have been carried out on these aspects in our laboratory with greater emphasis on the behavior of different pairs of antibodies as sandwich partners : monoclonal-monoclonal and monoclonal-polyclonal antibodies. The parameters studied include the ease of radio-iodination of different monoclonal antibodies, the effect of interchange of capture and detector antibody etc. Keeping TSH antibody as a model, two different monoclonal antibodies, a polyclonal antibody and a tracer from a commercial TSH IRMA kit were used in this study. Based on our studies an assay procedure for in-house IRMA of TSH has been developed with a sensitivity of 0.1 μIU/ml and validated

  18. Library Use

    DEFF Research Database (Denmark)

    Konzack, Lars

    2012-01-01

    A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model.......A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model....

  19. A freeze dried kit formulation for the preparation of {sup 99m} Tc labelled human polyclonal IgG for the detection of infection and inflammation; Desarrollo del nucleo equipo {sup 99m} Tc-IgG humana para la deteccion `In vivo` de procesos inflamatorios

    Energy Technology Data Exchange (ETDEWEB)

    Pedraza L, M

    1996-11-01

    A freeze dried kit formulation for the preparation of {sup 99m}Tc-labelled human polyclonal IgG for the detection of infection and inflammation employing direct methods for protein labeling was developed. The method comprises reduction of intrinsic disulphide bridges within the antibody molecule by the use of the reductant 2-mercaptoethanol. Following a subsequent purification, the resulting reduced antibody is labeled via Sn{sup 2+} reduction of pertechnetate in the presence of a weak competing ligand ethane-1-hydroxy-1,1-diphosphonate (EHDP). High labeling efficiencies (>90%) were obtained with high in vitro stability and without effect upon antibody immunoreactivity. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in radiopharmaceutical solution. {sup 99m} Tc-polyclonal IgG prepared by the kit method was evaluated for scintigraphic localization of inflammatory lesions and abscesses in rabbits. Data demonstrated that the {sup 99m} Tc-polyclonal IgG after kit-reconstitution shows excellent stability and is an effective imaging agent of infection and/or inflammation. (Author).

  20. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  1. Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

    Directory of Open Access Journals (Sweden)

    Sotiropoulou Georgia

    2012-12-01

    Full Text Available Abstract Background Human kallikrein-related peptidase 6 (KLK6 has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.

  2. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  3. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  4. Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

    DEFF Research Database (Denmark)

    Litman, Thomas; Jensen, Ulla; Hansen, Alastair

    2002-01-01

    Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corres...

  5. Comparison of monoclonal and polyclonal ELISAs for fecal elastase in patients with cystic fibrosis and pancreatic insufficiency.

    Science.gov (United States)

    Borowitz, Drucy; Lin, Rong; Baker, Susan S

    2007-02-01

    Two enzyme-linked immunosorbent assay methodologies are used to detect pancreatic insufficiency: monoclonal and polyclonal. We sought to compare these assays in patients with cystic fibrosis and to correlate these with the coefficient of fat absorption (CFA). As part of a larger study, subjects had stool elastase measured by both methods while taking exogenous enzymes. Subjects subsequently stopped enzymes and had a fecal fat balance study performed; the CFA was then calculated. One hundred twenty-four subjects participated in this substudy. The median values for the monoclonal and polyclonal assays were 0.3 and 22.75 microg/g, respectively. The correlation coefficient between the 2 tests was 0.86 (P definition of pancreatic insufficiency was set at a CFA definition of pancreatic insufficiency was set at <100 microg/g, then the monoclonal and polyclonal assay positive predictive values were 97.6% (120 of 123) and 97.4% (111 of 114), respectively. The positive predictive value of both monoclonal and polyclonal fecal elastase in patients with cystic fibrosis is extremely good; however, correlation of either test with CFA was poor. The median value for the polyclonal elastase assay is higher than for the monoclonal assay, which could potentially lead to lower sensitivity of the polyclonal assay at lower cutpoints for the monoclonal assay is used.

  6. Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

    Science.gov (United States)

    Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

    2016-11-01

    Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

  7. Technetium-99m pertechnetate - a tracer for radiolabelling antibody for inflammation detection

    International Nuclear Information System (INIS)

    Shaharuddin Mohd; Wan Hamirul Bahrin Wan Kamal; Shahrin A Hamid; Ang Woan Tze; Rosnani Hashim

    1999-01-01

    The polyclonal antibody, Human Immunoglobulin G (HlgG) was reduced by using 2-mercaptoethanol with molar ratio of 1000:1 (i.e. mercaptoethanol:antibody). The reduction of the antibody, was carried out for 30 minutes at room temperature. The reduced antibody was purified by using Sephadex G-25 fine column. The antibody kit for the detection of inflammation was prepared aseptically in Class 1 Laminar Flow cabinet. The kit passed the sterility test. Upon reconstitution of the antibody kit with sodium pertechnetate-99m ( 99m Tc) solution, the labelling efficiency obtained was more than 95%. This preparation was stable up to 24-hour stored at room temperature. Gamma camera scans showed the accumulation of technetium-99m labelled antibody ( 99m Tc-HIgG) at the turpentine-induced inflammation of female Sprague-Dawley rats. This indicated the possibility of using 99m Tc-HIgG for inflammation detection. (author)

  8. Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

    Science.gov (United States)

    Nagata, Keiko; Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

    2017-04-01

    Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

  9. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

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

  10. Nigerian Libraries

    African Journals Online (AJOL)

    Bridging the digital divide: the potential role of the National Library of Nigeria · EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. Juliana Obiageri Akidi, Joy Chituru Onyenachi, 11-19 ...

  11. Library Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Carnegie Library of Pittsburgh locations including address, coordinates, phone number, square footage, and standard operating hours. The map below does not display...

  12. academic libraries

    African Journals Online (AJOL)

    Information Impact: Journal of Information and Knowledge Management

    Information Impact: Journal of Information and Knowledge Management ... Key words: academic libraries, open access, research, researchers, technology ... European commission (2012) reports that affordable and easy access to the results ...

  13. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  14. Modification of Antibody Function by Mutagenesis.

    Science.gov (United States)

    Dasch, James R; Dasch, Amy L

    2017-09-01

    The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

  15. Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies.

    NARCIS (Netherlands)

    Grebenchtchikov, N.J.; Ven-Jongekrijg, J. van der; Pesman, G.J.; Geurts-Moespot, A.; Meer, J.W.M. van der; Sweep, C.G.J.

    2005-01-01

    Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is

  16. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga

    2014-08-01

    Full Text Available Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.

  17. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  18. Probing cocaine-antibody interactions in buffer and human serum.

    Directory of Open Access Journals (Sweden)

    Muthu Ramakrishnan

    Full Text Available Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST, isothermal titration calorimetry (ITC, and surface plasmon resonance (SPR we have evaluated the affinity properties of a representative mouse monoclonal (mAb08 as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum.MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC. This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies.High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide

  19. KIT of human polyclonal IGG for the I diagnostic of infectious processes

    International Nuclear Information System (INIS)

    Perera, Alejandro

    1997-01-01

    Early diagnosis of infections can be performed by using non-invasive techniques of Nuclear Medicine, even before appearing anatomical damage of the tissues. The main aim of the present work was to obtain a freeze-dried kit for direct labelling of human polyclonal IgG, for the detection of septic processes by scintigraphy. 99mT c-IgG labelling procedure was carried out by Schwarz method Its was obtained the best yields of 99m Tc using using sodium pyrophosphate decahydrate as a week chelating agent

  20. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies

    International Nuclear Information System (INIS)

    Williams, J.A.; Wessels, B.W.; Wharam, M.D.; Order, S.E.; Wanek, P.M.; Poggenburg, J.K.; Klein, J.L.

    1990-01-01

    Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumor-associated antigens in nude mice bearing human glioma xenografts. Monoclonal P96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. 111In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (microCi) of tumor activity per gram per 100 microCi injected activity compared to 4.5 microCi following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than 10%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumor-associated antigen, demonstrate positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the 111In-radiolabeled antibody, 90Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of 90Y-radiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 microCi 90Y-radiolabeled P96.5, average absorbed doses of 3770, 980, 353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively

  1. The library

    International Nuclear Information System (INIS)

    1980-01-01

    A specialized library is essential for conducting the research work of the Uranium Institute. The need was recognized at the foundation of the Institute and a full-time librarian was employed in 1976 to establish the necessary systems and begin the task of building up the collection. A brief description is given of the services offered by the library which now contains books, periodicals, pamphlets and press cuttings, focussed on uranium and nuclear energy, but embracing economics, politics, trade, legislation, geology, mining and mineral processing, environmental protection and nuclear technology. (author)

  2. Libraries on the MOVE.

    Science.gov (United States)

    Edgar, Jim; And Others

    1986-01-01

    Presents papers from Illinois State Library and Shawnee Library System's "Libraries on the MOVE" conference focusing on how libraries can impact economic/cultural climate of an area. Topics addressed included information services of rural libraries; marketing; rural library development; library law; information access; interagency…

  3. Personal Virtual Libraries

    Science.gov (United States)

    Pappas, Marjorie L.

    2004-01-01

    Virtual libraries are becoming more and more common. Most states have a virtual library. A growing number of public libraries have a virtual presence on the Web. Virtual libraries are a growing addition to school library media collections. The next logical step would be personal virtual libraries. A personal virtual library (PVL) is a collection…

  4. America's Star Libraries

    Science.gov (United States)

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  5. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

    DEFF Research Database (Denmark)

    Buus, Søren; Rockberg, Johan; Forsström, Björn

    2012-01-01

    Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning...... against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high......-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against...

  6. Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

    Science.gov (United States)

    Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

    1988-01-01

    Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

  7. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Huy, Tran Quang, E-mail: huytq@nihe.org.vn [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam); Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam)

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  8. Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

    Directory of Open Access Journals (Sweden)

    Leena Mattsson

    2017-12-01

    Full Text Available This study describes the development and the challenges in the development of an on-chip immunoassay for histamine using commercially available antibodies. Histamine can be used as an indicator of food freshness and quality, but it is also a relevant marker in clinical diagnostics. Due to its low molecular weight, simple structure and thus low immunogenicity production of high specificity and affinity antibodies is difficult. From six commercial anti-histamine antibodies tested, only two bound the histamine free in the solution. A fluorescent on-chip immunoassay for histamine was established with a dynamic range of 8–111 µg/mL using polyclonal anti-histamine antibody H7403 from Sigma (Mendota Heights, MN, USA. The anti-histamine antibodies described and used in published literature are thoroughly reviewed and the quality of commercial antibodies and their traceability and quality issues are highlighted and extensively discussed.

  9. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  10. Induction of polyclonal B cell activation and differentiation by the AIDS retrovirus (HTLV-III/LAV)

    International Nuclear Information System (INIS)

    Higgins, S.E.; Schnittman, S.M.; Lane, H.C.; Folks, T.; Koenig, S.; Fauci, A.S.

    1986-01-01

    The immune systems of individuals infected with HTLV-III/LAV are characterized by a profound defect in cellular immunity together with paradoxical polyclonal B cell activation. The present study examined the direct effects of HTLV-III/LAV on B lymphocytes. Peripheral blood B cells from healthy donors were incubated with a variety of HTLV-III/LAV isolates for 1 h and 3 H-thymidine incorporation was measured at multiple time points. Responses ranged from 9000-28,000 cpm and peaked on day 4. This B cell activation was not enhanced by the addition of interleukin-2 to culture, was not synergistic with Staphylococcus aureus Cowan I, was not modulated by the addition of T lymphocytes to culture, and was not associated with B cell transformation. Supernatant Ig could first be detected in virus-activated cultures at day 4, plateaued by day 8, and yielded a mean of 12,500 ng IgG+IgM/ml/50,000 B cells. Thus, HTLV-III/LAV is a potent T cell independent B cell mitogen capable of inducing B cell activation, proliferation, and differentiation comparable in magnitude to that of the most potent B cell activators. This biological property of HTLV-III/LAV may help explain the profound polyclonal B cell activation observed in patients with AIDS and may provide investigators with another probe for investigating the mechanisms of B cell activation

  11. Platelet antibodies of the IgM class in immune thrombocytopenic purpura

    International Nuclear Information System (INIS)

    Cines, D.B.; Wilson, S.B.; Tomaski, A.; Schreiber, A.D.

    1985-01-01

    The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, the authors studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. They observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP. They obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3. The authors demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP

  12. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

    Science.gov (United States)

    Sorice, M; Pittoni, V; Griggi, T; Losardo, A; Leri, O; Magno, M S; Misasi, R; Valesini, G

    2000-01-01

    The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-β2-glycoprotein I (β2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific ‘pure’ anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti-β2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins. PMID:10792380

  13. Library Automation.

    Science.gov (United States)

    Husby, Ole

    1990-01-01

    The challenges and potential benefits of automating university libraries are reviewed, with special attention given to cooperative systems. Aspects discussed include database size, the role of the university computer center, storage modes, multi-institutional systems, resource sharing, cooperative system management, networking, and intelligent…

  14. MARKETING LIBRARY SERVICES IN ACADEMIC LIBRARIES: A ...

    African Journals Online (AJOL)

    MARKETING LIBRARY SERVICES IN ACADEMIC LIBRARIES: A TOOL FOR SURVIVAL IN THE ... This article discusses the concept of marketing library and information services as an ... EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT

  15. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  16. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  17. Antibodies recognizing both IgM isotypes in Atlantic salmon

    DEFF Research Database (Denmark)

    Hedfors, Ida Aagård; Bakke, Hege; Skjødt, Karsten

    2012-01-01

    these molecules. The present study aimed at identifying tools to separate IgM positive (IgM(+)) B cells from IgM negative (IgM(-)) non-B cell populations using flow cytometry. Several monoclonal antibodies (mAbs), and one polyclonal antibody (pAb) to both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon...... (Salmo salar) IgM, either commercially available or locally produced were tested for their recognition of Atlantic salmon IgM(+) cells. Leukocytes were isolated from peripheral blood (PB), spleen (S) and head kidney (HK) and stained with all mAbs and the pAb, to possibly verify the approximate number...... of IgM(+) cells in the respective tissues in salmon. To our surprise, this seemingly simple task did not reveal similar staining patterns for all antibodies as expected, but rather large differences in the number of positively stained cells were discovered. In short, positively stained cells by each...

  18. IDENTIFICATION OF THE CORE RESIDUES OF THE EPITOPE OF A MONOCLONAL-ANTIBODY RAISED AGAINST GLYCOPROTEIN-D OF HERPES-SIMPLEX VIRUS TYPE-1 BY SCREENING OF A RANDOM PEPTIDE LIBRARY

    NARCIS (Netherlands)

    SCHELLEKENS, GA; LASONDER, E; FEIJLBRIEF, M; KOEDIJK, DGAM; DRIJFHOUT, JW; SCHEFFER, AJ; WELLINGWESTER, S; WELLING, GW

    1994-01-01

    Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal

  19. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  20. Antibody repertoire profiling with mimotope arrays

    OpenAIRE

    Pashova, Shina; Schneider, Christoph; von Gunten, Stephan; Pashov, Anastas

    2016-01-01

    Large-scale profiling and monitoring of antibody repertoires is possible through next generation sequencing (NGS), phage display libraries and microarrays. These methods can be combined in a pipeline, which ultimately maps the antibody reactivities onto defined arrays of structures - peptides or carbohydrates. The arrays can help analyze the individual specificities or can be used as complex patterns. In any case, the targets recognized should formally be considered mimotopes unless they are ...

  1. Imaging of colorectal carcinoma with radiolabeled antibodies.

    Science.gov (United States)

    Goldenberg, D M; Goldenberg, H; Sharkey, R M; Lee, R E; Higgenbotham-Ford, E; Horowitz, J A; Hall, T C; Pinsky, C M; Hansen, H J

    1989-10-01

    Colorectal cancer has been the tumor type most frequently studied with radiolabeled antibodies. Among the various antibodies, a majority of patients with colorectal cancer have received xenogeneic polyclonal or monoclonal antibodies against carcino-embryonic antigen. This review summarizes the current status of colorectal cancer imaging with radiolabeled antibodies, ie, radioimmunodetection (RAID), and examines the published studies involving carcinoembryonic antigen (CEA) antibodies and 17-1A, 19-9, and B72.3, and other monoclonal antibodies. In order to better address the issue of the current and future clinical usefulness of this emerging technology, particular attention is given to the protocols, methods, and results of the published studies. Despite differences in study parameters, antibodies and forms, labels, administration routes and doses, and scanning instruments and methods, it has been found that (1) almost no adverse reactions have been evident; (2) antibody fragments are preferred over whole immunoglobulin G reagents because they achieve higher tumor-to-background ratios earlier, thus reducing or precluding the need for dual-isotope subtraction methods or long delays before imaging; (3) use of antibody fragments, including the monovalent Fab' form, permits imaging with short-lived radionuclides of excellent photon properties, such as 123I and 99mTc; (4) circulating antigens against which the imaging antibody is directed can complex with the injected antibody, but such complexes have not prevented successful RAID; (5) patients with high serum titers of the appropriate antigen target usually have higher rates of positive RAID; (6) patients who are seronegative for the tumor antigen being studied can have positive RAID findings, which can represent the detection of occult lesions; (7) single photon emission computed tomography appears to provide better image resolution than planar scanning; (8) regardless of the sensitivity reported in any particular

  2. Library news

    CERN Multimedia

    CERN Library

    2010-01-01

    The CERN Library has been providing electronic access to the "Techniques de l'Ingénieur" database for the past 8 months. As a reminder, this is a multidisciplinary database of over 4000 technical and scientific articles in French, covering a broad range of topics such as mechanical engineering, safety, electronics and the environment. In a few simple steps, you can create your own account, select the types of documents you are interested in and configure your settings so as to receive alerts when articles in your field of activity are published. You can now access this resource from outside CERN using the "remote access to electronic resources" service. Further information is available here. Direct access to the database. Remote access to electronic resources. If you have any questions or comments, don't hesitate to contact us at: library.desk@cern.ch.

  3. Library Benchmarking

    Directory of Open Access Journals (Sweden)

    Wiji Suwarno

    2017-02-01

    Full Text Available The term benchmarking has been encountered in the implementation of total quality (TQM or in Indonesian termed holistic quality management because benchmarking is a tool to look for ideas or learn from the library. Benchmarking is a processof measuring and comparing for continuous business process of systematic and continuous measurement, the process of measuring and comparing for continuous business process of an organization to get information that can help these organization improve their performance efforts.

  4. Antibody bond to the microcrystalline cellulose in progesterone radioimmunoassay

    International Nuclear Information System (INIS)

    Krnavek, B.

    1992-01-01

    A suspension of microcrystalline cellulose with bonded globulin fraction of the polyclonal antibody against progesterone was prepared and applied to the radioimmunoanalysis of progesterone in full milk and in blood serum. The results were compared with those obtained using RETRO-test kits; the comparison gave evidence that the novel technique can fully replace the RETRO-test, the elimination of the separating medium (activated carbon, polyethylene glycol) being an asset. The obtained correlation coefficient and regression equation for a simultaneous determination of 120 samples by the two methods were r = 0.964 and y = 1.113x - 0.939, respectively

  5. Next-Generation Sequencing of Antibody Display Repertoires

    Directory of Open Access Journals (Sweden)

    Romain Rouet

    2018-02-01

    Full Text Available In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

  6. Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G; Choudhary, Neelima; Routh, Patty A; Cholon, Deborah M; Hobbs, Marcia M; Almond, Glen W; Orndorff, Paul E; Elkins, Christopher

    2011-08-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

  7. Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid▿

    Science.gov (United States)

    Leduc, Isabelle; Fusco, William G.; Choudhary, Neelima; Routh, Patty A.; Cholon, Deborah M.; Hobbs, Marcia M.; Almond, Glen W.; Orndorff, Paul E.; Elkins, Christopher

    2011-01-01

    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme. PMID:21646451

  8. Application of murine monoclonal antibodies to the serodiagnosis of tuberculosis

    International Nuclear Information System (INIS)

    Ivanyl, J.; Coates, A.R.M.; Krambovitis, E.

    1982-01-01

    The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of 125 I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

  9. Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2010-01-01

    Full Text Available The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs that can reduce the tumor mass. Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

  10. Catalytic Antibodies

    Indian Academy of Sciences (India)

    biological processes and is intended to catalyze a reaction for which no real enzyme is ... the reaction. In order to enhance the rates of chemical reactions, enzymes, ..... of such antibodies has already been exploited in the production of a biosensor. ..... tant to the pharmaceutical and fine chemical industries for the synthesis ...

  11. 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

    2013-08-01

    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.

  12. Advances in recombinant antibody manufacturing.

    Science.gov (United States)

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

  13. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.

    2008-01-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  14. Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus monodon

    Digital Repository Service at National Institute of Oceanography (India)

    Vrinda, S.; Jasmin, C.; Sivakumar, K.C.; Jose, B.; Philip, R; BrightSingh, I

    thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231 bp, that codes for 77 amino acids, was cloned into the Escherichia coli...

  15. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

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

    OpenAIRE

    Narat, Mojca; Biček, Ajda; Vadnjal, Robert; Benčina, Dušan

    2004-01-01

    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...

  17. Libraries for users services in academic libraries

    CERN Document Server

    Alvite, Luisa

    2010-01-01

    This book reviews the quality and evolution of academic library services. It revises service trends offered by academic libraries and the challenge of enhancing traditional ones such as: catalogues, repositories and digital collections, learning resources centres, virtual reference services, information literacy and 2.0 tools.studies the role of the university library in the new educational environment of higher educationrethinks libraries in academic contextredefines roles for academic libraries

  18. Antiparietal cell antibody test

    Science.gov (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; ...

  19. Monoclonal antibodies to carcino-embryonic antigen

    International Nuclear Information System (INIS)

    Teh, Jinghee; McKenzie, I.F.C.

    1990-01-01

    With the aim of producing new MoAb to colorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonic tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb I-1 and O-1 were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens. The six antibodies gave a range of reactions with normal and malignant tissues, indicating that they most likely reacted with different epitopes. Thus, apart from the reactions of 17C4, LK-4 and XPX-13 with fresh and formalin-fixed granulocytes, none of the antibodies reacted with formalin-fixed normal tissues. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to carcino-embryonic antigen polyclonal antibodies, that is the MoAb gave no obvious advantage. 9 refs., 1 tab., 3 figs

  20. Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H

    2014-01-01

    . All 19 trials were with high risk of bias. Of the 19 trials, 16 trials were two-arm trials, and three trials were three-arm trials. Hence, we found 25 trial comparisons with antibody induction agents: interleukin-2 receptor antagonist (IL-2 RA) versus no induction (10 trials with 1454 participants....... Furthermore, serum creatinine was statistically significantly higher when T-cell specific antibody induction was compared with no induction (MD 3.77 μmol/L, 95% CI 0.33 to 7.21; low-quality evidence), as well as when polyclonal T-cell specific antibody induction was compared with no induction, but this small...... T-cell specific antibody induction, drug-related adverse events were less common among participants treated with interleukin-2 receptor antagonists (RR 0.23, 95% CI 0.09 to 0.63; low-quality evidence), but this was caused by the results from one trial, and trial sequential analysis could not exclude...

  1. Antibody phage display applications for nuclear medicine imaging and therapy

    International Nuclear Information System (INIS)

    Winthrop, M.D.; Denardo, G.L.; Denardo, S.J.

    2000-01-01

    Antibody-based constructs genetically engineered from genes of diverse origin provide a remarkable opportunity to develop functional molecular imaging techniques and specific molecular targeted radionuclide therapies. Phage display libraries of antibody fragment genes can be used to select antibody-based constructs that bind any chosen epitope. A large naive human antibody-based library was used to illustrate binding of antibody constructs to a variety of common and unique antigens. Antibody-based libraries from hybridoma cells, lymphocytes from immunized humans or from mice and human antibody repertoires produced in transgenic mice have also been described. Several orders of magnitude of affinity enhancement can be achieved by random or site specific mutations of the selected binding peptide domains of the scFv. Affinities (K d ) as high as 10 - 11 M (10 pM) for affinity-matured scFv have been documented. Such gene libraries thus offer an almost limitless variety of antibody-based molecular binding peptide modules that can be used in creative ways for the construction of new targeting agents for functional or molecular imaging and therapy

  2. Direct labelling of monomeric antibody fragments Fab' with 99mTc

    International Nuclear Information System (INIS)

    Li Jun; Wang Shizhen; Yang Ziyi

    1994-01-01

    Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG

  3. Dissection of antibody specificities induced by yellow fever vaccination.

    Directory of Open Access Journals (Sweden)

    Oksana Vratskikh

    Full Text Available The live attenuated yellow fever (YF vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual

  4. Marketing the Virtual Library

    Science.gov (United States)

    Fagan, Jody Condit

    2009-01-01

    Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

  5. State Virtual Libraries

    Science.gov (United States)

    Pappas, Marjorie L.

    2003-01-01

    Virtual library? Electronic library? Digital library? Online information network? These all apply to the growing number of Web-based resource collections managed by consortiums of state library entities. Some, like "INFOhio" and "KYVL" ("Kentucky Virtual Library"), have been available for a few years, but others are just starting. Searching for…

  6. Public Libraries in Bangladesh.

    Science.gov (United States)

    Khan, M. H.

    1984-01-01

    Overview of library movement in Bangladesh highlights British (1851-1947) and Pakistan periods (1947-1971), separation of Bangladesh from Pakistan, libraries in development plans (1951-1970), three important public libraries, development of national library, book resources, a library network plan, legislation, finance, leadership, library…

  7. Study of rat kidney transamidinase structure and regulation with monoclonal antibodies and the purification and characterization of human kidney transamidinase

    International Nuclear Information System (INIS)

    Gross, M.D.

    1985-01-01

    The isolation of monoclonal antibodies to transamidinase made possible the development of an immunosorbent inhibition assay for transamidinase protein using a 125 I-labeled monoclonal antibody. This assay is a more direct measurement of transamidinase protein than the determination of the amount of polyclonal antibody required to precipitate the transamidinase activities. Rats were fed diets supplemented with creatine and/or glycine, and the amounts of transamidinase protein were determined with the assay using the monoclonal antibody. The transamidinase activities of kidneys from the rats fed the various supplemented diets ranged from 10 to 40% of the control values, whereas, the amounts of transamidinase protein were, in all instances no lower than 66% of the control values. Purified homogeneous rat kidney transamidinase and rat kidney supernatants were subjected to isoelectric focussing and four to five fractions of the enzyme were obtained. Polyclonal antibodies, but not the monoclonal antibodies were found by Western blotting experiments to recognize all the forms of the enzyme obtained by the isoelectric focussing. The author concluded that the monoclonal antibodies recognized forms of the enzyme that changed very little in amount, relative to the alterations in enzyme activities, when rats were fed a diet containing creatine

  8. Targeting Antibodies to Carbon Nanotube Field Effect Transistors by Pyrene Hydrazide Modification of Heavy Chain Carbohydrates

    Directory of Open Access Journals (Sweden)

    Steingrimur Stefansson

    2012-01-01

    Full Text Available Many carbon nanotube field-effect transistor (CNT-FET studies have used immobilized antibodies as the ligand binding moiety. However, antibodies are not optimal for CNT-FET detection due to their large size and charge. Their size can prevent ligands from reaching within the Debye length of the CNTs and a layer of charged antibodies on the circuits can drown out any ligand signal. In an attempt to minimize the antibody footprint on CNT-FETs, we examined whether pyrene hydrazide modification of antibody carbohydrates could reduce the concentration required to functionalize CNT circuits. The carbohydrates are almost exclusively on the antibody Fc region and this site-specific modification could mediate uniform antibody orientation on the CNTs. We compared the hydrazide modification of anti-E. coli O157:H7 polyclonal antibodies to pyrenebutanoic acid succinimidyl ester-coated CNTs and carbodiimide-mediated antibody CNT attachment. Our results show that the pyrene hydrazide modification was superior to those methods with respect to bacteria detection and less than 1 nM labeled antibody was required to functionalize the circuits.

  9. Estimation of sensitivity, specificity and predictive values of two serologic tests for the detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in the absence of a reference test (gold standard)

    DEFF Research Database (Denmark)

    Enøe, Claes; Andersen, Søren; Sørensen, Vibeke

    2001-01-01

    Latent-class models were used to determine the sensitivity, specificity and predictive values of a polyclonal blocking enzyme-linked immunosorbent assay (ELISA) and a modified complement-fixation test (CFT) when there was no reference test. The tests were used for detection of antibodies against ...

  10. Multigroup cross section library; WIMS library

    International Nuclear Information System (INIS)

    Kannan, Umasankari

    2000-01-01

    The WIMS library has been extensively used in thermal reactor calculations. This multigroup constants library was originally developed from the UKNDL in the late 60's and has been updated in 1986. This library has been distributed with the WIMS-D code by NEA data bank. The references to WIMS library in literature are the 'old' which is the original as developed by the AEA Winfrith and the 'new' which is the current 1986 WIMS library. IAEA has organised a CRP where a new and fully updated WIMS library will soon be available. This paper gives an overview of the definitions of the group constants that go into any basic nuclear data library used for reactor calculations. This paper also outlines the contents of the WIMS library and some of its shortcomings

  11. Scintigraphy with 99mTc labelled polyclonal human IgG in rheumatoid arthritis patients

    International Nuclear Information System (INIS)

    Stanchev, V.; Batalov, A.; Atanasov, A.

    1999-01-01

    The study design to assess the diagnostic relevance of scintigraphy with 99m Tc labelled polyclonal human IgG (HIG) for detecting active synovitis in rheumatoid arthritis patients. Fifteen patients presenting rheumatoid arthritis and 3 healthy volunteers are studied on digital camera (Diacam, Siemens). Following iv injection of 500 MBq 99m Tc - HIG, a 3- phase scintigraphy of the knee joints is performed and 4 hours later multiple planar views of the peripheral joint are recorded. Scintigraphic data are comparatively studied with the clinical indicators pointing to active synovitis - joint swellings and pain. Markedly expressed 99m Tc - HIG uptake is noted in joints apparently the most actively involved in the arthritis process clinically, whereas most of the joints without evidence of active synovitis revealed background activity only. The obtained scintigraphic results correlate strongly with the clinical indicator joint swelling (93.2%), and somewhat less with the presence of pain (81.5%). 13.5 per cent of the joints without clinically detectable swelling and 25.6% those free of pain are HIG-positive. 99m Tc - HIG scintigraphy is a highly sensitive noninvasive method of detecting active synovitis, promoting objective assessment of the joint inflammatory process in the course of treatment and follow-up study of rheumatoid arthritis patients

  12. Glycation of polyclonal IgGs: Effect of sugar excipients during stability studies.

    Science.gov (United States)

    Leblanc, Y; Bihoreau, N; Jube, M; Andre, M-H; Tellier, Z; Chevreux, G

    2016-05-01

    A number of intravenous immunoglobulin preparations are stabilized with sugar additives that may lead over time to undesirable glycation reactions especially in liquid formulation. This study aimed to evaluate the reactivity of sugar excipients on such preparations in condition of temperature, formulation and concentration commonly used for pharmaceutical products. Through an innovative LC-MS method reported to characterize post-translational modifications of IgGs Fc/2 fragments, a stability study of IVIg formulated with reducing and non-reducing sugars has been undertaken. The rate of polyclonal IgGs glycation was investigated during 6months at 5, 25, 30 and 40°C. High levels of glycation were observed with reducing sugars such as glucose and maltose in the first months of the stability study from 25°C. Non-reducing sugars presented a low reactivity even at the highest tested temperature (40°C). Furthermore, a site by site analysis was performed by MS/MS to determine the glycation sites which were mainly identified at Lys246, Lys248 and Lys324. This work points out the high probability of glycation reactions in some commercialized products and describes a useful method to characterize IVIg glycated products issued from reducing sugar excipients. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    Directory of Open Access Journals (Sweden)

    Damián ePérez-Mazliah

    2012-09-01

    Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

  14. The role of Tc-99m polyclonal human immunoglobulin G scintigraphy in Graves' ophthalmopathy

    International Nuclear Information System (INIS)

    Ortapamuk, H.; Hosal, B.; Naldoken, S.

    2002-01-01

    The aim of this study was to clarify whether Tc-99m HIG (Polyclonal Human Immunoglobulin G) can image and determine the severity of orbital involvement in patients with Graves' ophthalmopathy. Twenty-six patients between 19 and 56 years old with Graves' ophthalmopathy were examined. All patients received approximately 370 MBq Tc-99m HIG by intravenous (i.v.) injection. Planar and SPECT examination were performed 4 hours after the injection. Visual and semiquantitative evaluations were performed for both orbits by two independent observers. Clinically active ophthalmopathy patients had noticeably increased orbital accumulation of Tc-99m HIG. In patients with inactive disease, and 14 of 19 had no uptake, whereas 5 patients had orbital radioactivity accumulation. The duration of Graves' ophthalmopathy did not correlate with the presence of active ophthalmopathy and Tc-99m HIG grade. There was no correlation between clinical classification and clinical activity (r=278). There was a good correlation between clinical activity and the radioactivity grade with r=0.666 (p=0.01). The clinical classification closely correlated with Tc-99m HIG grade (r=0.423, p=0.05) Tc-99m HIG scan can clearly identified clinically active patients, and subclinical inflammation can be shown by this scintigraphic evaluation. The current preliminary results suggested that Tc-99m HIG SPECT might be useful for the assessment of disease activity in Graves' ophthalmopathy. (author)

  15. LIBRARY SKILL INSTRUCTION IN NIGERIAN ACADEMIC LIBRARIES

    African Journals Online (AJOL)

    DJFLEX

    www.globaljournalseries.com; Info@globaljournalseries.com. LIBRARY SKILL INSTRUCTION IN NIGERIAN ACADEMIC. LIBRARIES. P. C. AZIAGBA AND E. H. UZOEZI. (Received 10, September 2009; Revision Accepted 8, February 2010). ABSTRACT. This survey was undertaken to portray the level of library involvement ...

  16. Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

    Science.gov (United States)

    Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

    2017-09-15

    Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Biting back: BiTE antibodies as a promising therapy for acute myeloid leukemia.

    Science.gov (United States)

    Walter, Roland B

    2014-06-01

    The experience with gemtuzumab ozogamicin has highlighted both the potential value and limitations of antibodies in acute myeloid leukemia (AML). Recently, bispecific T-cell engager (BiTE) antibodies have emerged as a means to harness polyclonal cytotoxic T-cells and cause highly efficient lysis of targeted tumor cells. Promising early results have been obtained with the CD19-directed BiTE antibody, blinatumomab, in patients with acute lymphoblastic leukemia. A first candidate for AML is the CD33/CD3 molecule, AMG 330, for which several recent preclinical studies demonstrated high potency and efficacy in destroying CD33(+) human AML cells. Many questions remain to be addressed, but BiTE antibodies may offer an exciting new tool in a disease for which the outcomes in many patients remain unsatisfactory.

  18. The development of methods for obtaining monoclonal antibody-producing cells

    Directory of Open Access Journals (Sweden)

    Michał Skowicki

    2016-04-01

    Full Text Available Monoclonal antibodies (mAbs are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.

  19. Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

    Directory of Open Access Journals (Sweden)

    Franck R Petry

    Full Text Available Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3, type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5, and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46. For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409 while others did not (pS199, pT205, pS396, pS404, pS422, A0024. With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i using secondary antibodies designed to bind only non-denatured Igs, ii preparation of a heat-stable fraction, iii clearing Igs from the homogenates, and iv using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes

  20. Basics of Antibody Phage Display Technology.

    Science.gov (United States)

    Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas H

    2018-06-09

    Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

  1. Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

    Science.gov (United States)

    Sierralta, W D; Thole, H H

    1996-05-01

    The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

  2. Libraries and Learning

    Science.gov (United States)

    Rainie, Lee

    2016-01-01

    The majority of Americans think local libraries serve the educational needs of their communities and families pretty well and library users often outpace others in learning activities. But many do not know about key education services libraries provide. This report provides statistics on library usage and presents key education services provided…

  3. Growing Competition for Libraries.

    Science.gov (United States)

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  4. The library marketing toolkit

    CERN Document Server

    Potter, Ned

    2012-01-01

    A guide that offers coverage of various elements of library marketing and branding for different sectors including archives and academic, public and special libraries. It is suitable for those who are involved in promoting their library or information service, whether at an academic, public or special library or in archives or records management.

  5. Automating the Small Library.

    Science.gov (United States)

    Skapura, Robert

    1987-01-01

    Discusses the use of microcomputers for automating school libraries, both for entire systems and for specific library tasks. Highlights include available library management software, newsletters that evaluate software, constructing an evaluation matrix, steps to consider in library automation, and a brief discussion of computerized card catalogs.…

  6. Less is More: A Comparison of Antibody-Gold Nanoparticle Conjugates of Different Ratios.

    Science.gov (United States)

    Byzova, Nadezhda A; Safenkova, Irina V; Slutskaya, Elvira S; Zherdev, Anatoly V; Dzantiev, Boris B

    2017-11-15

    This comprehensive study is related to gold nanoparticles (GNPs) conjugated with antibodies. The goal of the study is to determine the minimal concentration of antibodies for conjugate synthesis when the conjugates have high antigen-capturing activity. Two systems were studied: gold nanoparticles conjugated with monoclonal antibodies (mAb-GNP) specific to Helicobacter pylori and gold nanoparticles conjugated with polyclonal antibodies (pAb-GNP) specific to mouse immunoglobulins. Several conjugates were synthesized with different GNP-to-antibody molar ratios (from 1:1 to 1:245) through nondirectional and noncovalent immobilization on a surface of GNPs with a diameter of 25.3 ± 4.6 nm. The maximal antigen-capturing activities and equilibrium constants of the conjugates correlate with the formation of a constant hydrodynamic radius of the conjugates for mAb-GNP (GNP to antibody molar ratio 1:58) and with the stabilizing concentration by flocculation curves for pAb-GNP (GNP to antibody molar ratio 1:116). The application of the conjugates to the lateral flow immunoassay shows that the antibody concentrations used for the conjugation can be reduced (below the stabilizing concentration) without losing activity for the mAb-GNP conjugates. The findings highlight that the optimal concentration of antibodies immobilized on the surface of GNPs is not always equal to the stabilizing concentration determined by the flocculation curve.

  7. Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

    Science.gov (United States)

    Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

    2014-04-01

    A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  8. Improved detection of a staphylococcal infection by monomeric and protein A-purified polyclonal human immunoglobulin

    International Nuclear Information System (INIS)

    Calame, W.

    1993-01-01

    The present study was undertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99m Tc-labelled monomeric human immunoglobulin (m-Ig), 99m Tc-labelled, protein A-purified, human immunoglobulin (A-IG) and 99m Tc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99m Tc-labelled Igs bound to bacteria in vitro: The percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-to-non-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purified Ig yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99m Tc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99m Tc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein A-purified monomeric Ig was the highest of all tracer agents tested. (orig.)

  9. Antibodies against glycolipids enhance antifungal activity of macrophages and reduce fungal burden after infection with Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Renata Amelia eBueno

    2016-02-01

    Full Text Available Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

  10. Basic studies on the tumor imaging using antibodies to human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Ohta, Hitoya; Torizuka, Kanji

    1984-01-01

    Using polyclonal antibodies to human α-fetoprotein (AFP), the effect of iodination on the antibody activity and tumor accumulation of radioiodinated antibodies in tumor bearing nude mice were examined. Antibodies, obtained from horse antiserum and purified by affinity chromatography, were iodinated by the chloramine-T method and their antibody activity was evaluated using RIA and Scatchard plot analysis. When high concentrations of chloramine-T were used or more than 2.6 iodine atoms were incorporated per antibody molecule, the antigen binding capacity rather than the affinity constant was affected by the iodination. The antibody activity was completely destroyed at an iodine to antibody molar ratio of 15.4. Antibodies, however, which were iodinated under low concentrations of chloramine-T and contained less than 0.8 iodines per antibody molecule, showed almost full retention of their antibody activity. Nude mice transplanted with AFP producing human testicular tumor or AFP non-producing human urinary bladder tumor were administered intravenously with 131 I-labeled antibodies to human AFP. Scintigrams were taken at 1, 2, 4 and 7 days after the injection of labeled antibodies. At day 7, nude mice were sacrificed and organs and tumor were removed, weighed and counted. In nude mice bearing testicular tumor, tumor image became gradually clear with decreasing background activity and tumor to blood ratio, obtained, was 0.82 for testicular tumor compared to 0.42 for bladder tumor. These results indicated a specific in vivo localization of 131 I-labeled antihuman AFP antibodies in AFP producing tumor. (author)

  11. PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTO

    Directory of Open Access Journals (Sweden)

    Edgar Antonio Reyes Montaño

    2011-12-01

    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

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

    Science.gov (United States)

    Tsai, Wen-Yang; Lin, Hong-En; Wang, Wei-Kung

    2017-01-01

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

  13. 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

    2004-01-01

    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.

  14. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Heidi A. Neubauer

    2017-03-01

    Full Text Available Sphingosine kinase 2 (SK2 is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs. Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout (Sphk2-/- MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

  15. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

    Directory of Open Access Journals (Sweden)

    Andrew R Williams

    Full Text Available No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

  16. America's Star Libraries, 2010: Top-Rated Libraries

    Science.gov (United States)

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  17. IBC's 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences and the 2012 Annual Meeting of The Antibody Society: December 3-6, 2012, San Diego, CA.

    Science.gov (United States)

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A; Proetzel, Gabriele; Yong, May; Begent, Richard H J; Reichert, Janice M

    2013-01-01

    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3-6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3-5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4-5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society's special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5-6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.

  18. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

  19. A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

    Science.gov (United States)

    Torres, Oscar B; Duval, Alexander J; Sulima, Agnieszka; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2018-06-01

    We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

  20. Progranulin antibodies in autoimmune diseases.

    Science.gov (United States)

    Thurner, Lorenz; Preuss, Klaus-Dieter; Fadle, Natalie; Regitz, Evi; Klemm, Philipp; Zaks, Marina; Kemele, Maria; Hasenfus, Andrea; Csernok, Elena; Gross, Wolfgang L; Pasquali, Jean-Louis; Martin, Thierry; Bohle, Rainer Maria; Pfreundschuh, Michael

    2013-05-01

    Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Expression of human protein S100A7 (psoriasin, preparation of antibody and application to human larynx squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Barbieri Manuela R

    2011-11-01

    Full Text Available Abstract Background Up-regulation of S100A7 (Psoriasin, a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag rabbit serum (polyclonal antibody anti-rS100A7. The molecular weight of rS100A7 (His-tag protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da. Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

  2. The synthesis of a small library of prospective growth hormone secretagogues

    Directory of Open Access Journals (Sweden)

    JELENA JOKSIMOVIC

    1999-10-01

    Full Text Available Employing tools of combinatorial chemistry, an original methodological approach has been developed and applied for the design and synthesis of a small library of peptide-like compounds, prospective growth hormone (GH secretagogues. For this purpose seven building blocks of tBoc- and Fmoc-protected amino acids was used. In this way, a small, tripeptoid library on polyethylene glycol monomethyl ether 5000 (PEG 5000 as a soluble support was obtained. The library was screened by a new, simple system, based on polyclonal rabbit antiserum raised against "GH secretagogue pharmacophore" of a known growth hormone secretagogue GHRP-6 (Hexarelin® and the most promising GH secretagogue candidate was selected.

  3. Measurement of lymphatic function with technetium-99m-labelled polyclonal immunoglobulin

    International Nuclear Information System (INIS)

    Svensson, W.; Glass, D.M.; Bradley, D.; Peters, A.M.

    1999-01-01

    A reliable method for measuring lymph flow in physiological units would be valuable, especially in conditions in which it is uncertain whether lymph flow is increased or decreased. The requirements of a radiopharmaceutical for such measurement include stable radionuclide labelling and rapid access to lymphatic vessels following tissue injection but no access to blood vessels. A soluble macromolecule is likely to come closest to meeting these requirements. Technetium-99m- labelled human polyclonal immunoglobulin (HIG) was therefore investigated firstly in comparison with 99m Tc-labelled human serum albumin (HSA) in patients undergoing routine lymphoscintigraphy and secondly with respect to injection site in a group of volunteers with post-mastectomy oedema (PMO). Subcutaneous injection of 99m Tc-HIG into the web space of a distal extremity gave images in which lymphatic vessels were more clearly defined compared with images obtained after injection of 99m Tc-HSA. Lymph nodes were also more clearly defined, suggesting specific retention of HIG, possibly through Fc-mediated binding. Peripheral blood sampling showed a delayed arrival in blood of radioactivity after 99m Tc-HIG compared with 99m Tc-HSA, although ultimately, the blood recovery of 99m Tc-HIG was significantly higher (P 99m Tc-HSA. Clearance rates of radioactivity from the injection site were not sinificantly different, however, between the two agents. In patients with PMO, web space injection of 99m Tc-HIG gave excellent images of normal lymphatic vessels, of lymph nodes and of abnormal lymph drainage such as dermal backflow in swollen arms. In contrast, neither lymphatic vessels nor lymph nodes were visualised after injection into the skin of the dorsum of the distal forearm. Although there was no difference in clearance rates from the injection sites between normal and swollen arms with either agent in PMO, clearance was significantly faster following injection into the web space (0.11% per minute for

  4. Libraries and Accessibility: Istanbul Public Libraries Case

    Directory of Open Access Journals (Sweden)

    Gül Yücel

    2016-12-01

    Full Text Available In the study; the assessment of accessibility has been conducted in Istanbul public libraries within the scope of public area. Public libraries commonly serve with its user of more than 20 million in total, spread to the general of Turkey, having more than one thousand branches in the centrums and having more than one million registered members. The building principles and standards covering the subjects such as the selection of place, historical and architectural specification of the region, distance to the centre of population and design in a way that the disabled people could benefit from the library services fully have been determined with regulations in the construction of new libraries. There are works for the existent libraries such as access for the disabled, fire safety precautions etc. within the scope of the related standards. Easy access by everyone is prioritized in the public libraries having a significant role in life-long learning. The purpose of the study is to develop solution suggestions for the accessibility problems in the public libraries. The study based on the eye inspection and assessments carried out within the scope of accessibility in the public libraries subsidiary to Istanbul Culture and Tourism Provincial Directorate Library and Publications Department within the provincial borders of Istanbul. The arrangements such as reading halls, study areas, book shelves etc. have been examined within the frame of accessible building standards. Building entrances, ramps and staircases, horizontal and vertical circulation of building etc. have been taken into consideration within the scope of accessible building standards. The subjects such as the reading and studying areas and book shelf arrangements for the library have been assessed within the scope of specific buildings. There are a total of 34 public libraries subsidiary to Istanbul Culture and Tourism Provincial Directorate on condition that 20 ea. of them are in the

  5. Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

    International Nuclear Information System (INIS)

    Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

    1986-01-01

    Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

  6. Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus monodon.

    Science.gov (United States)

    Vrinda, S; Jasmin, C; Sivakumar, K C; Jose, Blessy; Philip, Rosamma; Bright Singh, I S

    2016-07-01

    Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Screening individual hybridomas by microengraving to discover monoclonal antibodies

    Science.gov (United States)

    Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

    2014-01-01

    The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

  8. Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera

    Digital Repository Service at National Institute of Oceanography (India)

    Vrinda, S.; Jasmin, C.; Sivakumar, K.C.; Jose, S.; Jose, B.; Philip, R; BrightSingh, I

    September 2016 Available online 15 September 2016 Keywords: Gonad-inhibiting hormone (GIH) Fusion/chimeric proteinaa r t i c l e i n f o b s t r a c tRegulating gonad inhibition and vitellogen Penaeus monodon using mature GIH fusion polyclonal antisera... restriction site overhangs to match with that of the expression vector; pET32a+. The forward primer (GIH-F- 5′-GAAT TCAACATCCTGGACAGCAAATGCAGGGGTGC-3′) contained EcoR I site (italics) and 29 nucleotide residues encoding the N terminal amino acid residues...

  9. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    Science.gov (United States)

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  10. Libraries serving dialogue

    CERN Document Server

    Dupont, Odile

    2014-01-01

    This book based on experiences of libraries serving interreligious dialogue, presents themes like library tools serving dialogue between cultures, collections dialoguing, children and young adults dialoguing beyond borders, story telling as dialog, librarians serving interreligious dialogue.

  11. Israeli Special Libraries

    Science.gov (United States)

    Foster, Barbara

    1974-01-01

    Israel is sprinkled with a noteworthy representation of special libraries which run the gamut from modest kibbutz efforts to highly technical scientific and humanities libraries. A few examples are discussed here. (Author/CH)

  12. Marketing library and information services in academic libraries in ...

    African Journals Online (AJOL)

    Marketing library and information services in academic libraries in Niger State, Nigeria. ... This study was designed to investigate the marketing of library services in academic libraries in Niger state, ... EMAIL FULL TEXT EMAIL FULL TEXT

  13. Population dynamics and in vitro antibody pressure of porcine parvovirus indicate a decrease in variability.

    Science.gov (United States)

    Streck, André Felipe; Homeier, Timo; Foerster, Tessa; Truyen, Uwe

    2013-09-01

    To estimate the impact of porcine parvovirus (PPV) vaccines on the emergence of new phenotypes, the population dynamic history of the virus was calculated using the Bayesian Markov chain Monte Carlo method with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed with consecutive passages of the 'Challenge' strain (a virulent field strain) and NADL2 strain (a vaccine strain) in a PK-15 cell line supplemented with polyclonal antibodies raised against the vaccine strain. A decrease in genetic diversity was observed in the presence of antibodies in vitro or after vaccination (as estimated by the in silico model). We hypothesized that the antibodies induced a selective pressure that may reduce the incidence of neutral selection, which should play a major role in the emergence of new mutations. In this scenario, vaccine failures and non-vaccinated populations (e.g. wild boars) may have an important impact in the emergence of new phenotypes.

  14. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))

    1990-05-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

  15. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    International Nuclear Information System (INIS)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C.

    1990-01-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with 131 I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author)

  16. The alternative library

    OpenAIRE

    Collinson, Timothy; Williams, A.

    2004-01-01

    Much time and effort has been devoted to designing and developing library Web sites that are easy to navigate by both new students and experienced researchers. In a review of the Southampton Institute Library it was decided that in addition to updating the existing homepage an alternative would be offered. Drawing on theory relating to user interface design, learning styles and creative thinking, an Alternative Library navigation system was added to the more traditional library homepage. The ...

  17. FENDL multigroup libraries

    International Nuclear Information System (INIS)

    Ganesan, S.; Muir, D.W.

    1992-01-01

    Selected neutron reaction nuclear data libraries and photon-atomic interaction cross section libraries for elements of interest to the IAEA's program on Fusion Evaluated Nuclear Data Library (FENDL) have been processed into MATXSR format using the NJOY system on the VAX4000 computer of the IAEA. This document lists the resulting multigroup data libraries. All the multigroup data generated are available cost-free upon request from the IAEA Nuclear Data Section. (author). 9 refs

  18. Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

    International Nuclear Information System (INIS)

    Wahl, R.L.; Fisher, S.

    1987-01-01

    Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility. (author)

  19. Changing State Digital Libraries

    Science.gov (United States)

    Pappas, Marjorie L.

    2006-01-01

    Research has shown that state virtual or digital libraries are evolving into websites that are loaded with free resources, subscription databases, and instructional tools. In this article, the author explores these evolving libraries based on the following questions: (1) How user-friendly are the state digital libraries?; (2) How do state digital…

  20. School Libraries and Innovation

    Science.gov (United States)

    McGrath, Kevin G.

    2015-01-01

    School library programs have measured success by improved test scores. But how do next-generation school libraries demonstrate success as they strive to be centers of innovation and creativity? These libraries offer solutions for school leaders who struggle to restructure existing systems built around traditional silos of learning (subjects and…

  1. Reforming Prison Libraries.

    Science.gov (United States)

    Coyle, William J.

    1989-01-01

    Discusses the current widespread acceptance of the public library model for prison libraries, in which preferences of the inmates are the chief consideration in programing and collection development. It is argued that this model results in recreational programs and collections that fail to fulfill the prison library's role in education and…

  2. LANL Research Library

    Science.gov (United States)

    Los Alamos National Laboratory The LANL Research Library website has been moved to http ://www.lanl.gov/library/. Please update your bookmarks. If you are not redirected to the new location within 10 http:// | Last Modified: Send email to the Library

  3. Joint-Use Libraries

    Science.gov (United States)

    Casstevens, Susan

    2017-01-01

    The joint-use library is a place where people of all ages, interests, and income levels can find items of interest at no personal cost. The mission of A. H. Meadows Public and High School Library in Midlothian, Texas, is to offer what other public libraries provide: educational and entertainment resources to a community. Yet, the staff also wants…

  4. Learning Boost C++ libraries

    CERN Document Server

    Mukherjee, Arindam

    2015-01-01

    If you are a C++ programmer who has never used Boost libraries before, this book will get you up-to-speed with using them. Whether you are developing new C++ software or maintaining existing code written using Boost libraries, this hands-on introduction will help you decide on the right library and techniques to solve your practical programming problems.

  5. Technostress and Library Values.

    Science.gov (United States)

    Gorman, Michael

    2001-01-01

    Discusses information overload and society's and libraries' responses to technology. Considers eight values that libraries should focus on and how they relate to technology in libraries: democracy, stewardship, service, intellectual freedom, privacy, rationalism, equity of access, and building harmony and balance. (LRW)

  6. Marketing and Library Management.

    Science.gov (United States)

    Murphy, Kurt R.

    1991-01-01

    Examines the role of marketing in the management of libraries. The role of public relations (PR) in the total marketing concept is discussed, surveys that have explored PR efforts in academic and public libraries are described, and changes affecting libraries that marketing efforts could help to manage are discussed. (seven references) (LRW)

  7. Virtual Libraries: Service Realities.

    Science.gov (United States)

    Novak, Jan

    2002-01-01

    Discussion of changes in society that have resulted from information and communication technologies focuses on changes in libraries and a new market for library services with new styles of clients. Highlights client service issues to be considered when transitioning to a virtual library situation. (Author/LRW)

  8. Merchandising Your Library.

    Science.gov (United States)

    Sivulich, Kenneth G.

    1989-01-01

    Discusses library circulation figures as a reflection of the success of library services and describes merchandising techniques that have produced a 137 percent circulation increase at Queens Borough Public Library over the past seven years. Merchandising techniques such as minibranches, displays, signage, dumps, and modified shelving are…

  9. Editorial Library: User Survey.

    Science.gov (United States)

    Surace, Cecily J.

    This report presents the findings of a survey conducted by the editorial library of the Los Angeles Times to measure usage and satisfaction with library service, provide background information on library user characteristics, collect information on patterns of use of the Times' clipping files, relate data on usage and satisfaction parameters to…

  10. Marketing Academic Libraries

    Science.gov (United States)

    Mallon, Melissa, Ed.

    2013-01-01

    Ask any academic librarian if marketing their library and its services is an important task, and the answer will most likely be a resounding "yes!" Particularly in economically troubled times, librarians are increasingly called upon to promote their services and defend their library's worth. Since few academic libraries have in-house marketing…

  11. Antibodies against CKI1(RD), a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: From antigen preparation to in planta immunolocalization

    Czech Academy of Sciences Publication Activity Database

    Borkovcová, P.; Pekárová, B.; Válková, M.; Dopitová, R.; Brzobohatý, Břetislav; Janda, L.; Hejatko, J.

    2014-01-01

    Roč. 100, APR 2014 (2014), s. 6-15 ISSN 0031-9422 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068 Grant - others:GA ČR(CZ) GAP501/11/1150; GA ČR(CZ) GA13-25280S Program:GA; GA Institutional support: RVO:68081707 Keywords : Receiver domain * Polyclonal antibodies * Immunoprecipitation Subject RIV: BO - Biophysics Impact factor: 2.547, year: 2014

  12. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

  13. Local Preparation and Evaluation of Double - antibody Liquid Phase Radioimmunoassay System for Detection of Human Testosterone

    International Nuclear Information System (INIS)

    Shafik, H.M.; Sallam, Kh.M.; Ebeid, N.H.; Elshaer, M.R.; Elshae, M.R.

    2016-01-01

    Preparation, evaluation and optimization of testosterone radioimmunoassay (RIA) system using liquid phase double antibody is considered to be the main objective. Three primary components were prepared and characterized to obtain valid and accurate system. These components were polyclonal testosterone antibody, the "1"2"5I-testosterone tracer and set of testosterone standards. The production of polyclonal testosterone antibody was undertaken by immunizing two groups of females white New-Zealand rabbits with testosterone-3-(O-carboxy methyloxime): BSA as immunogen through primary immunization and five boosters. Both R 1 and R 4 gave anti-serum has a high immuno reactivity. The preparation of "1"2"5I-testosterone tracer was carried out using three different conjugates (testosterone-3-TME, testosterone-3-histamine and testosterone-3-BSA) by electrophilic substitution mechanism using chloramine-T as oxidizing agent. Tracers were characterized in terms of radiochemical yield %, radiochemical purity %, specific activity and immuno reactivity. A set of testosterone standards were prepared using highly purified testosterone antigen. Optimization and validation tests of the local liquid phase RIA system were carried out. In conclusion, the results showed that, the local testosterone RIA system is sensitive, specific and accurate with significant cost reduction in comparison with commertial kit and extended use of the method for routine investigation of variety of diseases especially hypogonadism and associated male infertility

  14. FY 2009 Public Libraries Survey

    Data.gov (United States)

    Institute of Museum and Library Services — Dig into FY 2009 data on public library systems (referred to as administrative entities in the Public Libraries Survey) and main libraries, branches, and bookmobiles...

  15. FY 2010 Public Libraries Survey

    Data.gov (United States)

    Institute of Museum and Library Services — Dig into FY 2010 data on public library systems (referred to as administrative entities in the Public Libraries Survey) and main libraries, branches, and bookmobiles...

  16. FY 2011 Public Libraries Survey

    Data.gov (United States)

    Institute of Museum and Library Services — Dig into FY 2011 data on public library systems (referred to as administrative entities in the Public Libraries Survey) and main libraries, branches, and bookmobiles...

  17. FY 2008 Public Libraries Survey

    Data.gov (United States)

    Institute of Museum and Library Services — Dig into FY 2008 data on public library systems (referred to as administrative entities in the Public Libraries Survey) and main libraries, branches, and bookmobiles...

  18. Sperm, nuclear, phospholipid, and red blood cell antibodies and isotype RF in infertile couples and patients with autoimmune rheumatic diseases.

    Science.gov (United States)

    Fichorova, R; Nakov, L; Baleva, M; Nikolov, K; Gegova, I

    1996-12-01

    To determine if measuring of nonorgan-specific autoantibodies is useful for better understanding and management of unexplained infertility. Sera were obtained from 70 infertile couples, 57 rheumatic patients, and 76 fertile donors. Sperm antibodies (SA) were detected by the tests of Kibrick and Friberg, anti-histones, anti-cardiolipin antibodies, and RF isotypes by ELISA, antinuclear antibodies by indirect immunofluorescence, and anti-red blood cell antibodies by Capture-R. Multiple autoimmune reactivity (both partners positive and/or more than one type of autoantibody involved), higher than naturally occurring in fertile individuals, was found in 55% of the idiopathically infertile couples. IgA-RF was the dominant autoimmune marker. SA revealed similar rates in patients with rheumatic diseases and in infertiles with or without other autoantibodies. Although no single autoimmunity marker could predict occurrence of SA, the coincidence of enhanced polyclonal autoimmunity in both partners of infertile couples might potentiate their negative effect on reproduction.

  19. Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini.

    Science.gov (United States)

    Ryu, Je-Young; Siswanto, Antoni; Harimoto, Kenichi; Tagawa, Yoh-ichi

    2013-06-01

    The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.

  20. German Librarianship and Munich Libraries

    Directory of Open Access Journals (Sweden)

    Osman Ümit Özen

    1994-06-01

    Full Text Available There are 27 municipal libraries including the Central Public Library in Munich. The other important libraries in the city are Bayern State National Library, Maximillian University Library, a technical highschool library and the "Deutsches Musuem" Library. All these libraries are financed locally. The author introduces these libraries briefly and compares German libraries with Turkish libraries. He concludes that although theoretically there are not distinctive differences, in practice, buildings and their layout are better in Germany where more variety of services are offered. In Turkey standardization has not been realized yet. Turkey needs to computerize and network to improve the services offered in an efficient way.

  1. Library/vendor relationships

    CERN Document Server

    Brooks, Sam

    2014-01-01

    A view of the mutual dependence between libraries and vendorsAs technology advances, libraries are forced to reach beyond their own resources to find effective ways to maintain accuracy and superior service levels. Vendors provide databases and integrated library systems that perform those functions for profit. Library/Vendor Relationships examines the increasing cooperation in which libraries find they must participate in, and vice versa, with the vendors that provide system infrastructure and software. Expert contributors provide insights from all sides of this unique collaboration, offering

  2. Teleporting the library?

    DEFF Research Database (Denmark)

    Heilesen, Simon

    2009-01-01

    In 2007, six Danish public libraries established a virtual library, Info Island DK, in Second Life. This article discusses the library project in terms of design. The design processes include the planning and implementation of the virtual library structure and its equipment, as well...... as the organizing and carrying out of activities in the virtual setting. It will be argued that, to a large extent, conventions have determined design and use of the virtual library, and also that design has had an impact on the attitudes and understanding of the participants....

  3. A universal phage display system for the seamless construction of Fab libraries.

    Science.gov (United States)

    Nelson, Renae S; Valadon, Philippe

    2017-11-01

    The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Library system of Italy

    Directory of Open Access Journals (Sweden)

    Nataša Gerbec

    2003-01-01

    Full Text Available In the European extent, Italy is the cradle of libraries and library sciences. In the past, Italian national public libraries played an important role through their vast book treasury. But only during the last thirty years have public libraries been developed following the Anglo-American public library model. Italy does not have any uniform or general legislation concerning libraries. On the state level, this area is regulated by some separate acts, while on the regional level there is a collection of various acts and regulations. Libraries are not strictly divided into general categories. It is required that the professionals engaged in Italian libraries should have secondary or university education. The level of their professional tasks depends on the type of library and its capacity. The competency for the development in the field of librarianship is assigned to The Ministry of Cultural and Environment Heritage as well as to its subordinate institutions (Central Institute for the Union catalogue of Italian Libraries and for Bibliographic Information, Central Institute for Book Pathology, Observatory for International Libraries Programmes.

  5. Planning & Urban Affairs Library Manual.

    Science.gov (United States)

    Knobbe, Mary L., Ed.; Lessel, Janice W., Ed.

    Written especially for persons without a library degree who are operating a small urban study or planning agency library on a part-time basis. Subjects covered are: (1) library function and staff function, duties and training; (2) physical layout and equipment of library; (3) establishing and maintaining the library; (4) library administration;…

  6. Special Libraries and Multitype Networks.

    Science.gov (United States)

    Segal, JoAn S.

    1989-01-01

    Describes the history of multitype library networks; examines the reasons why special libraries and other network participants have resisted the inclusion of special libraries in these networks; and discusses the benefits to both special libraries and to other libraries in the network that would result from special library participation. (17…

  7. NOAA Miami Regional Library > Home

    Science.gov (United States)

    Library Collections Open Access Resources Research Tools E-resources NOAA S. and NOAA N.E. Library Institutional Repository DIVE INTO About the Library | Collections | Research Tools | Library Services & NOAA Miami Regional Library @ AOML & NHC NOAA Miami Regional Library at National Hurricane

  8. Libraries in society

    DEFF Research Database (Denmark)

    Kristiansson, Michael; Skouvig, Laura Henriette Christine

    2008-01-01

    The purpose of the paper is to investigate the phenomenon of openness in relation to library development. The term openness is presented and related to library development from historical and theoretical perspectives. The paper elaborates on the differences over time on to how openness has been...... understood in a library setting. Historically, openness in form of the open shelves played a crucial role in developing the modern public library. The paper examines this openness-centred library policy as adopted by Danish public libraries in the beginning of the 20th century by applying the theories...... by Michel Foucault on discourse and power to the introduction of open shelves. Furthermore, the paper discusses current challenges facing the modern public library in coping with openness issues that follow from changes in society and advances in technology. These influences and developments are not least...

  9. The participatory public library

    DEFF Research Database (Denmark)

    Rasmussen, Casper Hvenegaard

    2016-01-01

    of theoretical approaches and practical examples to obtain a varied understanding of user participation in public libraries. Research fields outside library and information science have developed a wide range of theoretical approaches on user participation. Examples from cultural policy, museum studies......Purpose From collection to connection has been a buzzword in the library world for more than a decade. This catchy phrase indicates that users are seen not only as borrowers, but as active participants. The aim of this paper is to investigate and analyse three questions in relation to user...... participation in public libraries in a Nordic perspective. How can participation in public libraries be characterised? Why should libraries deal with user participation? What kinds of different user participation can be identified in public libraries? Design/methodology/approach The paper uses a selection...

  10. Flight Software Math Library

    Science.gov (United States)

    McComas, David

    2013-01-01

    The flight software (FSW) math library is a collection of reusable math components that provides typical math utilities required by spacecraft flight software. These utilities are intended to increase flight software quality reusability and maintainability by providing a set of consistent, well-documented, and tested math utilities. This library only has dependencies on ANSI C, so it is easily ported. Prior to this library, each mission typically created its own math utilities using ideas/code from previous missions. Part of the reason for this is that math libraries can be written with different strategies in areas like error handling, parameters orders, naming conventions, etc. Changing the utilities for each mission introduces risks and costs. The obvious risks and costs are that the utilities must be coded and revalidated. The hidden risks and costs arise in miscommunication between engineers. These utilities must be understood by both the flight software engineers and other subsystem engineers (primarily guidance navigation and control). The FSW math library is part of a larger goal to produce a library of reusable Guidance Navigation and Control (GN&C) FSW components. A GN&C FSW library cannot be created unless a standardized math basis is created. This library solves the standardization problem by defining a common feature set and establishing policies for the library s design. This allows the libraries to be maintained with the same strategy used in its initial development, which supports a library of reusable GN&C FSW components. The FSW math library is written for an embedded software environment in C. This places restrictions on the language features that can be used by the library. Another advantage of the FSW math library is that it can be used in the FSW as well as other environments like the GN&C analyst s simulators. This helps communication between the teams because they can use the same utilities with the same feature set and syntax.

  11. Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

    Science.gov (United States)

    Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

    2017-06-12

    The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

  12. Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus

    Directory of Open Access Journals (Sweden)

    Karlsson Johnny

    2009-05-01

    Full Text Available Abstract Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the

  13. Lyme disease antibody

    Science.gov (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 . ...

  14. Antinuclear antibody panel

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003535.htm 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 ...

  15. Acetylcholine receptor antibody

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood of ...

  16. Nuclear medicine: Monoclonal antibodies

    International Nuclear Information System (INIS)

    Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

    1986-01-01

    Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

  17. Platelet antibodies blood test

    Science.gov (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  18. Assessing Library Automation and Virtual Library Development in Four Academic Libraries in Oyo, Oyo State, Nigeria

    Science.gov (United States)

    Gbadamosi, Belau Olatunde

    2011-01-01

    The paper examines the level of library automation and virtual library development in four academic libraries. A validated questionnaire was used to capture the responses from academic librarians of the libraries under study. The paper discovers that none of the four academic libraries is fully automated. The libraries make use of librarians with…

  19. /sup 99m/Tc radiolabelling and quality control tests of anti-melanoma monoclonal antibodies and F(ab')/sub 2/ fragments for immunoscintigraphy

    International Nuclear Information System (INIS)

    Callegaro, L.; Deleide, G.; Dovis, M.; Cecconato, E.; Scassellati, G.A.

    1986-01-01

    Tumour radioimmunodetection was first developed by using radiolabelled polyclonal antibodies, raised in goats against tumour associated antigens (TAA). The availability of monoclonal antibodies to TAA has definitely contributed to more extensive in vivo use of radiolabelled antibodies. However, many factors are involved in tumour radioimmunolocalization, related either to the antibody and radioisotope features or to the natural history of the tumour itself. The experimental protocol developed by the authors allows a full evaluation of the properties of a particular MoAb.This paper illustrates the work done with on a particular set of monoclonal antibodies, raised against human melanoma associated antigens, with the aim of visualizing primary and metastatic lesions in melanoma patients

  20. Wikis in Libraries

    Directory of Open Access Journals (Sweden)

    Matthew Bejune

    2007-09-01

    Full Text Available Wikis have recently been adopted to support a variety of collaborative activities within libraries. This article and its companion wiki, LibraryWikis (http://librarywikis.pbwiki.com/, seek to document the phenomenon of wikis in libraries. This subject is considered within the framework of computer-supported cooperative work (CSCW. The author identified thirty-three library wikis and developed a classification schema with four categories: (1 collaboration among libraries (45.7 percent; (2 collaboration among library staff (31.4 percent; (3 collaboration among library staff and patrons (14.3 percent; and (4 collaboration among patrons (8.6 percent. Examples of library wikis are presented within the article, as is a discussion for why wikis are primarily utilized within categories I and II and not within categories III and IV. It is clear that wikis have great utility within libraries, and the author urges further application of wikis in libraries.

  1. News from the Library

    CERN Multimedia

    CERN Library

    2010-01-01

    The LHC Library to be merged with the Central Library. Not everyone knows that CERN Scientific Information Service currently counts three physical libraries on site. The Central Library is located in Building 52 and there are two satellite libraries located respectively in building 30 (the LHC Library) and in building 864 on Prévessin site (the SPS Library). Moreover, the Legal Service Library is located in Building 60. In the past, there have been at CERN up to 6 satellite libraries; they were essential at a time when information was only in paper form and having multiple copies of documents located in several places at CERN was useful to facilitate scientific research. Today, this need is less critical as most of our resources are online. That is why, following a SIPB (Scientific Information Policy Board) decision, the collections of the LHC Library will be merged this summer with the Central collection. This reorganization and centralization of resources will improve loan services. The SP...

  2. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  3. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    Novak, J.; Kselikova, M.; Urbankova, J.

    1980-01-01

    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125 I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  4. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    International Nuclear Information System (INIS)

    Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Lurilli, A.P.; Ponzo, F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A.C.

    1995-01-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99m Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  5. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Liberatore, M. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Neri, D. [Cambridge Centre for Protein Engineering - MRC Centre (United Kingdom); Neri, G. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Pini, A. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Lurilli, A.P. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Ponzo, F. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Spampinato, G. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Padula, F. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Pala, A. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Colella, A.C. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy)

    1995-11-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for {sup 99m}Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  6. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

  7. E.coli and investigation of antibody titer in rats

    Directory of Open Access Journals (Sweden)

    masoud abdollahi

    2017-03-01

    Full Text Available Introduction: Plant ribosome inactivating proteins act as N-glycosidase enzyme and produce by several family of Caryophyllaceae such as Saponaria Officinalis. Different Isoforms of RIPs expressed by Saponaria Officinalis. SO6 isoform depurinate Adenine 4324 in the conserved GAGA loop of 28SrRNA and disrupts protein synthesis. The aim of this study was expression of SO6 isoform in E.coli and investigation of antibody titer in rats. Methods: In this experimental study, SO6 synthetic gene was excised from recombinant pUC57- SO6 plasmid with BamHI and SalI restriction enzymes and subcloned into pET28a (+ expression vector. The expression of recombinant protein was induced by IPTG. Recombinant SO6 was purified by nickel affinity chromatography. Western blotting was performed to confirm the recombinant protein. Rats were immunized intraperitoneal with purified protein and IgG serum titer was assayed by ELISA. Results: PCR reaction and enzyme digestion confirmed subcloning of SO6 gene into pET28a (+ expression vector. A 29.5kDa protein band on SDS-PAGE showed a high level of recombinant protein expression. Polyclonal antibodies recognized SO6. ELISA confirmed significant antibody titer after injection of protein in test group compared with the control group. Conclusion: The recombinant purified SO6 antigen can be used for anti-cancer and vaccine candidate research.

  8. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

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

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

    NARCIS (Netherlands)

    Griep, R.

    1999-01-01

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

  10. The Library International Partnerweek 2011

    DEFF Research Database (Denmark)

    Presentation at the Library International Partnerweek, held at Copenhagen Technical Library at the Copenhagen University College of Engineering. Participant: Ms. Carmen Priesto Estravid from Madrid Technical University, E.U.I.T. Obras Públicas, Library. Spain Ms.Tuulikki Hattunen from TUAS Library....... Finland Ms. Anitta Ôrm from Kemi-Tornio UAS Library. Finland Mr. Manfred Walter from HTW-Berlin. Germany Mr. Peter Hald from Copenhagen Technical Library. Denmark Mr. Ole Micahelsen from Copenhagen Technical Library. Denmark...

  11. KAERI photonuclear library

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Jong Hwa; Lee, Young Ouk; Han, Yin Iu

    2000-03-01

    This report contains summary information and figures depicting the KAERI photonuclear data library that extends up to 140 MeV of incident photon. The library consists of 143 isotopes from C-12 to Bi-209, providing the photoabsorption cross section and the emission spectra for neutron, proton, deuteron, triton, alpha particles, and all residual nuclides in ENDF6 format. The contents of this report and ENDF-6 format data library are available at http://atom.kaeri.re.kr/.

  12. The CERN Library

    CERN Multimedia

    Hester, Alec G

    1968-01-01

    Any advanced research centre needs a good Library. It can be regarded as a piece of equipment as vital as any machine. At the present time, the CERN Library is undergoing a number of modifications to adjust it to the changing scale of CERN's activities and to the ever increasing flood of information. This article, by A.G. Hester, former Editor of CERN COURIER who now works in the Scientific Information Service, describes the purposes, methods and future of the CERN Library.

  13. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mitani, M.; Cianciolo, G.J.; Snyderman, R.; Yasuda, M.; Good, R.A.; Day, N.K.

    1987-01-01

    Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effect on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These finding suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.

  14. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses

    International Nuclear Information System (INIS)

    Mitani, M.; Cianciolo, G.J.; Snyderman, R.; Yasuda, M.; Good, R.A.; Day, N.K.

    1987-01-01

    Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effect on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These finding suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17

  15. Matrix effects in applying mono- and polyclonal ELISA systems to the analysis of weathered oils in contaminated soil.

    Science.gov (United States)

    Pollard, S J T; Farmer, J G; Knight, D M; Young, P J

    2002-01-01

    Commercial mono- and polyclonal enzyme-linked immunosorbent assay (ELISA) systems were applied to the on-site analysis of weathered hydrocarbon-contaminated soils at a former integrated steelworks. Comparisons were made between concentrations of solvent extractable matter (SEM) determined gravimetrically by Soxhlet (dichloromethane) extraction and those estimated immunologically by ELISA determination over a concentration range of 2000-330,000 mg SEM/kg soil dry weight. Both ELISA systems tinder-reported for the more weathered soil samples. Results suggest this is due to matrix effects in the sample rather than any inherent bias in the ELISA systems and it is concluded that, for weathered hydrocarbons typical of steelworks and coke production sites, the use of ELISA requires careful consideration as a field technique. Consideration of the target analyte relative to the composition of the hydrocarbon waste encountered appears critical.

  16. The Personal Virtual Library

    CERN Document Server

    Le Meur, Jean-Yves

    1998-01-01

    Looking for "library" in the usual search engines of the World Wide Web gives: "Infoseek found 3,593,126 pages containing the word library" and it nicely proposes: "Search only within these 3,59 3,126 pages ?" "Yahoo! Found 1299 categories and 8669 sites for library" "LycOs: 1-10 von 512354 relevanten Ergebnissen" "AltaVista: About 14830527 documents match your query" and at the botto m: "Word count: library: 15466897" ! Excite: Top 10 matches and it does not say how many can be browsed... "Library" on the World Wide Web is really popular. At least fiveteen million pages ar e supposed to contain this word. Half of them may have disappeared by now but one more hit will be added once the search robots will have indexed this document ! The notion of Personal Library i s a modest attempt, in a small environment like a library, to give poor users lost in cyber-libraries the opportunity to keep their own private little shelves - virtually. In this paper, we will l ook at the usual functionalities of library systems...

  17. Integrated circuit cell library

    Science.gov (United States)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  18. NEIC Library Services

    Science.gov (United States)

    The National Enforcement Investigation Center (NEIC) Environmental Forensic Library partners with NEIC's forensic scientists to retrieve, validate and deliver information to develop methods, defensible regulations, and environmental measurements.

  19. Library and Education

    Directory of Open Access Journals (Sweden)

    Gheorghe Buluţă

    2011-01-01

    Full Text Available The psycho-social phenomena generated by mass-media and the new information and communication technologies at the level of the young generations have led to new communication practices that bypass libraries and revolutionized the intellectual labor practices, with texts being rather used than read. In this context, our article examines the need to increase the library's role in developing the quality of education and research and brings to attention a few possible solutions which include a partnership between various types of libraries and between librarians' associations and NGOs to facilitate education through library and safeguard reading.

  20. Libraries and licensing

    Directory of Open Access Journals (Sweden)

    Maja Žumer

    2001-01-01

    Full Text Available In the mid 90s, the abundance of various electronic publications exposed libraries to the problems of licensing electronic content. Various licensing principles have been prepared recently to help libraries in the process; it can be said that in general, the knowledge of licensing issues has improved in libraries of all types. Libraries form consortia in order to gain stronger negotiating positions and obtain better conditions.In the article, new licensing principles are presented in more detail, as well as some domestic and foreign experiences with consortia forming.

  1. International Youth Library

    Directory of Open Access Journals (Sweden)

    Osman Ümit Özen

    1994-03-01

    Full Text Available International Youth Library, the biggest youth library in the world, was founded in 1948 in Munich, Germany, by Jella Lepman. She aimed to unite all the children of the world through books by establishing this library. IYL is still trying to achieve this end supporting scholarship programmes in children’s literature research, participating in or organizing meetings on children’s literature, and working with other national and international organizations deeding with children’s literature. Unfortunately the library is facing some problems recently which have risen from economic difficulties which also inhibits promotional activities.

  2. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  3. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter; Frøkiær, Hanne; Hearty, Stephen

    2007-01-01

    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-produci...

  4. Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

    Directory of Open Access Journals (Sweden)

    Laurence Rolland

    1992-06-01

    Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

  5. Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

    Science.gov (United States)

    Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

    2012-02-01

    Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

  6. Generation of genic diversity among Streptococcus pneumoniae strains via horizontal gene transfer during a chronic polyclonal pediatric infection.

    Directory of Open Access Journals (Sweden)

    N Luisa Hiller

    2010-09-01

    Full Text Available Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

  7. Obtaining a citric tristeza virus p65 protein antibody and preliminary results of p65 in vivo expression

    Directory of Open Access Journals (Sweden)

    Yanneth Torres

    2003-07-01

    Full Text Available The citric tristeza virus (CTV belongs to the Closteroviridae family which indudes the only vegetal viruses possessing genes homologous to HSP70 thermal cellular shock proteins in their genome. Such is the case of the gene encoding for the CTV p65 protein which presents high homology with the HSP70 protein family. It has been shown recently that HSP70h viral proteins (such as CTV p65 are involved both in viral assembly, as a microtubule binding protein, and in cell-cell movement. Since CTV is the most deleterious citrus pathogen, understanding this protein's role in the pathogenesis process is important. Rabbits were immunised with four synthetic peptides (corresponding to CTV p65 thermal shock protein's carboxyl-terminal region to obtain polyclonal antibodies. All the peptides used were immunogenic, even though two of them showed greater response. Whilst none of the antibodies obtained reacted to non-infected plant extract, the p65 proteins was detected in extracts taken from citric plants infected with CTV Based on the antibody's reaction to two Colombian isolates having different serological characteristics, the p65 antibody's immunological behaviour appeared to be independent of the symptomatic severity of the CTV isolates. It was shown that the ORF encoded for the HSP70 homologue in CTV was expressed in vivo, even though the p65 antibody was only detected in concentrated protein extracts taken from infected plants, supporting reports from other studies that the concentration of this protein in plants infected with CTV is low. This is the first time that a polyclonal CTV antibody has been obtained in Colombia against p65 (a protein intervening in viral assembly and movement. Adapting a technique for obtaining p65 antibodies by using synthetic peptides as immunogens could be useful in the future for detecting or diagnosing p65 proteins present in different Colombian CTV isolates, especially in developing studies contributing towards greater

  8. Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non-structural Triple Gene Block Protein 1 and Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Dědič, P.

    2012-01-01

    Roč. 160, č. 5 (2012), s. 251-254 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus M * recombinant protein * coat protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.000, year: 2012

  9. In vivo pharmacological study on the effectiveness of available polyclonal antivenom against Hemiscorpius lepturus venom

    Directory of Open Access Journals (Sweden)

    A Jalali

    2011-01-01

    Full Text Available The available Razi Institute antivenom is still, empirically, used by intramuscular (IM administration for the treatment of scorpion stings in humans by six medically dangerous species including Hemiscorpius lepturus (H. lepturus. The aim of this study was to assess the neutralizing ability and effectiveness of the antivenom in inhibiting hemoglobinuria, biochemical changes, increased microalbuminuria and urinary lactate dehydrogenase (LDH following H. lepturus sting. Simultaneous intramuscular administration of 10 μL and 100 μL of antivenom, after 24 hours, had no significant preventive effect on the extent and degree of hemoglobinuria or proteinuria produced in venom-treated rats. After IM administration of antivenom, no significant changes in decreased red blood cell (RBC count and hemoglobin were observed. Immediate intramuscular administration of 10 μL of antivenom had no significant effects on both LDH and microalbuminuria. The present findings did not present correlation with clinical signs. Therefore, to fully assess the efficacy of the available antivenom and make appropriate recommendations, more in vivo or in vitro investigations including antigen-antibody interaction, enzymatic analysis and route-dependent administration are required.

  10. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    Science.gov (United States)

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  11. Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications.

    Science.gov (United States)

    D'Angelo, Sara; Staquicini, Fernanda I; Ferrara, Fortunato; Staquicini, Daniela I; Sharma, Geetanjali; Tarleton, Christy A; Nguyen, Huynh; Naranjo, Leslie A; Sidman, Richard L; Arap, Wadih; Bradbury, Andrew Rm; Pasqualini, Renata

    2018-05-03

    We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.

  12. Development of antibodies against the rat brain somatostatin receptor.

    Science.gov (United States)

    Theveniau, M; Rens-Domiano, S; Law, S F; Rougon, G; Reisine, T

    1992-05-15

    Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).

  13. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  14. Production and purification of IgY antibodies from chicken egg yolk

    Directory of Open Access Journals (Sweden)

    Wala Ahmad Amro

    2018-06-01

    Full Text Available The isolation of polyclonal antibodies from the serum of immunized mammals has significantly contributed to scientific research and diagnosis. The fact that recent technologies allow the production of antibodies in the yolk of eggs laid by hens, has led to the development of an alternative method for antibody generation that is less stressful to animals. As hens are kept under almost all their natural conditions, antibodies are isolated from the collected eggs; this technology is expected to become an interesting alternative to the conventionally serum-based techniques that eventually require to sacrifice the animal.Here we present a modified protocol for the isolation of IgY antibodies from immunized chickens and provide comparison between two chicken breeds in relative to IgY yield per egg. Our results show the possibility of generating large quantities of highly pure IgY from chicken eggs and also show large differences in the yield of IgY production between the two studied breeds. The results of this work indicate that IgY technology can be used for the production of primary antibodies for immunological work and disease diagnosis. Keywords: IgY, Chicken egg yolk, Gel filtration chromatography, Salmonella typhimurium

  15. Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

    Science.gov (United States)

    van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

    2018-02-20

    A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

  16. An antibody toolkit for the study of membrane traffic in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Falko Riedel

    2016-07-01

    Full Text Available The use of Drosophila melanogaster as a model organism has been pivotal to understanding the developmental processes of metazoans. However, the use of flies for studying subcellular organization is hampered by a paucity of reliable reagents to label specific organelles. Here, we describe the generation of mouse monoclonal antibodies against a set of markers of the secretory and endocytic pathways, along with goat polyclonal antibodies against two Golgi proteins. We show that the monoclonal antibodies are highly specific and sufficiently sensitive to detect endogenous proteins in crude extracts by immunoblotting with little background staining. By immunofluorescence the major compartments of the membrane traffic system (including the endoplasmic reticulum, the Golgi, and early and late endosomes are labeled by at least one antibody. Moreover, the antibodies can be used to label organelles in fly tissues including salivary glands and wing imaginal discs. We anticipate that these antibodies will provide a useful tool kit to facilitate the investigation of how the endomembrane system functions and varies in the diverse tissue types of metazoans.

  17. Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

    Directory of Open Access Journals (Sweden)

    Swarmistha Devi Aribam

    Full Text Available Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

  18. Antibodies to the extracellular pore loop of TRPM8 act as antagonists of channel activation.

    Directory of Open Access Journals (Sweden)

    Silke Miller

    Full Text Available The mammalian transient receptor potential melastatin channel 8 (TRPM8 is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced 45Ca2+ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies.

  19. Chemoradiotherapy of cancer using boronated monoclonal antibodies. Progress report, December 1, 1982-November 30, 1983

    International Nuclear Information System (INIS)

    Soloway, A.H.

    1984-01-01

    The feasibility was established of using antibodies for the delivery of 10 B. Problems faced included 1) preservation of antibody activity following boronation, 2) antigenic receptor site density of the target cells, and 3) delivery of a critical number of 10 B atoms per cell. The linkage of a heavily boronated polymeric species to antibody by means of a single functional group allow for the delivery of a large number 10 B atoms per antibody molecule without a significant reduction in affinity. Both the polyclonally derived anti-thymocyte globulin (ATG) and the monoclonal anti-colorectal carcinoma antibody (17-1A) recognize antigens that are expressed with a density of approximately 10 6 epitopes per cell. The major concept that we advance is that just as effective cancer chemotherapy is based on the use of a combination of drugs, similarly a combination of compounds could be employed to deliver the requisite amount of 10 B to tumor target cells. This could include compounds such as Na 2 B 12 H 11 Sh together with boronated antibodies directed against tumor associated antigens. (DT)

  20. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Directory of Open Access Journals (Sweden)

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  1. High-density fecal Enterococcus faecium colonization in hospitalized patients is associated with the presence of the polyclonal subcluster CC17

    NARCIS (Netherlands)

    Ruiz-Garbajosa, P.; de Regt, M.; Bonten, M.; Baquero, F.; Coque, T. M.; Canton, R.; Harmsen, H. J. M.; Willems, Rob J. L.

    Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the

  2. Public Relations in Special Libraries.

    Science.gov (United States)

    Rutkowski, Hollace Ann; And Others

    1991-01-01

    This theme issue includes 11 articles on public relations (PR) in special libraries. Highlights include PR at the Special Libraries Association (SLA); sources for marketing research for libraries; developing a library image; sample PR releases; brand strategies for libraries; case studies; publicizing a consortium; and a bibliography of pertinent…

  3. Music Libraries: Centralization versus Decentralization.

    Science.gov (United States)

    Kuyper-Rushing, Lois

    2002-01-01

    Considers the decision that branch libraries, music libraries in particular, have struggled with concerning a centralized location in the main library versus a decentralized collection. Reports on a study of the Association of Research Libraries that investigated the location of music libraries, motivation for the location, degrees offered,…

  4. Anti-dog IgG secondary antibody successfully detects IgG in a variety of aquatic mammals

    Science.gov (United States)

    Roehl, Katherine; Jankowski, Mark D.; Hofmeister, Erik K.

    2016-01-01

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

  5. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

    Science.gov (United States)

    Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

    2014-05-01

    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

  6. Library Systems: FY 2013 Public Libraries Survey (Administrative Entity)

    Data.gov (United States)

    Institute of Museum and Library Services — Find key information on library systems around the United States.These data include imputed values for libraries that did not submit information in the FY 2013 data...

  7. Library Systems: FY 2012 Public Libraries Survey (Administrative Entity)

    Data.gov (United States)

    Institute of Museum and Library Services — Find key information on library systems around the United States.These data include imputed values for libraries that did not submit information in the FY 2012 data...

  8. Library Systems: FY 2014 Public Libraries Survey (Administrative Entity Data)

    Data.gov (United States)

    Institute of Museum and Library Services — Find key information on library systems around the United States.These data include imputed values for libraries that did not submit information in the FY 2014 data...

  9. Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

    Science.gov (United States)

    Ruan, Guang-Ping; Ma, Li; Meng, Xiao-Jing; Meng, Min-Jie; Wang, Xiao-Ning; Lin, Ying; Wu, Zheng-Qiang; He, Xiao-Wei; Wang, Ju-Fang; Zhu, Yong

    2007-01-01

    HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

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

    Directory of Open Access Journals (Sweden)

    Christine Rasetti-Escargueil

    2017-10-01

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

  11. Determination of a Screening Metric for High Diversity DNA Libraries.

    Science.gov (United States)

    Guido, Nicholas J; Handerson, Steven; Joseph, Elaine M; Leake, Devin; Kung, Li A

    2016-01-01

    The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

  12. Determination of a Screening Metric for High Diversity DNA Libraries.

    Directory of Open Access Journals (Sweden)

    Nicholas J Guido

    Full Text Available The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

  13. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Special Libraries in Singapore.

    Science.gov (United States)

    Leong, Alice

    1979-01-01

    Distinguishes five main categories of special libraries in Singapore: those of private organizations, foreign governments, government departments, statutory boards, and regional organizations. Statistical data are provided for library holdings, professional staff employment, and subject profiles, and suggestions for improving various aspects of…

  15. Iranian Library Update.

    Science.gov (United States)

    Harvey, John F.

    1979-01-01

    Discusses the state of Iranian libraries since the revolution: the printing industry flourishes because of obsolete copyright laws, and the government is attempting to dewesternize media and education. Also considered are budget cuts, the revolution's cost to libraries, and its effect on individual librarians. (SW)

  16. The Memory Library

    DEFF Research Database (Denmark)

    Olesen-Bagneux, Ole

    2014-01-01

    of classification and retrieval processes is presented. The key element is to understand the library both as a physical structure and as a structure in the memory of the Alexandrian scholars. In this article, these structures are put together so to propose a new interpretation of the library....

  17. Privacy and Library Records

    Science.gov (United States)

    Bowers, Stacey L.

    2006-01-01

    This paper summarizes the history of privacy as it relates to library records. It commences with a discussion of how the concept of privacy first originated through case law and follows the concept of privacy as it has affected library records through current day and the "USA PATRIOT Act."

  18. Hospital Library Administration.

    Science.gov (United States)

    Cramer, Anne

    The objectives of a hospital are to improve patient care, while the objectives of a hospital library are to improve services to the staff which will support their efforts. This handbook dealing with hospital administration is designed to aid the librarian in either implementing a hospital library, or improving services in an existing medical…

  19. Munitions Classification Library

    Science.gov (United States)

    2016-04-04

    members of the community to make their own additions to any, or all, of the classification libraries . The next phase entailed data collection over less......Include area code) 04/04/2016 Final Report August 2014 - August 2015 MUNITIONS CLASSIFICATION LIBRARY Mr. Craig Murray, Parsons Dr. Thomas H. Bell, Leidos

  20. TRAC Searchable Research Library

    Science.gov (United States)

    2016-05-01

    Relational Data Modeling (VRDM) computational paradigm. VRDM has the key attributes of being cloud available, using domain semantics for configured...Figure 1. Methodology for TRAC Searchable Research Library Development. ........................... 5 Figure 2. The conceptual model for the cloud ...TRAC Searchable Research Library project was initiated by TRAC- HQ to address a current capability gap in the TRAC organization. Currently TRAC does not