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Sample records for monospecific polyclonal antibodies

  1. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies.

    Science.gov (United States)

    Hjelm, Barbara; Forsström, Björn; Igel, Ulrika; Johannesson, Henrik; Stadler, Charlotte; Lundberg, Emma; Ponten, Fredrik; Sjöberg, Anna; Rockberg, Johan; Schwenk, Jochen M; Nilsson, Peter; Johansson, Christine; Uhlén, Mathias

    2011-11-01

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

  2. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

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

    2015-01-01

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

  3. Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

    Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

  4. Production and Purification of Polyclonal Antibodies.

    Science.gov (United States)

    Nakazawa, Masami; Mukumoto, Mari; Miyatake, Kazutaka

    2016-01-01

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

  5. 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.

  6. Polyclonal Antibody Therapies for Clostridium difficile Infection

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    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. Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

    DEFF Research Database (Denmark)

    Mirza, Nadia Muhammad Akram; Schulz, Alexander; Halkier, Barbara Ann

    2016-01-01

    Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal...... rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated...... that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross...

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

    African Journals Online (AJOL)

    SERVER

    2007-06-18

    Jun 18, 2007 ... that ion-exchange chromatography could be an appropriate method for purification of IgG ... researches, polyclonal antibodies are routinely used as .... production and characterization of anti- human IgG monoclonal antibody ...

  9. Development of Anti-Isoproturon Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    LI Fang-shi; SUN Feng; LIU Xian-jin; CUI Heng-hua

    2007-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and

  10. Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

    Institute of Scientific and Technical Information of China (English)

    SHEN Xing-gui; LI Wan-nan; WANG Jing; JIANG Yi-qun; LI Qing-shan; FU Xue-qi

    2007-01-01

    Phosphatase of regenerating liver 3(PRL3), which belongs to the superfamily of protein tyrosine phosphatases (PTPs), represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

  11. Preparation and Characterization of Olaquindox Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    CHE,Huilian; SHI Baoxia; CHEN,Jinlan; ZHU,Xuejia; HE,Jiguo

    2009-01-01

    2-[N-(2-Hydroxyethyl)-carbamoyl]-3-methyl-oxoquinoxaline-l,4-oxides (OLA) was first mono-esterificated by succinic acid anhydride, and was then turned into a derivative with carboxyl. The synthetic product was purified by recrystallization with a yield of 52.47%. Its chemical structure was determined by NMR, IR and MS spectra. The determined structure was used to synthese OLA hapten, which had the molecular structural characteristics of OLA. The OLA hapten and bovine serum albumin (BSA) were conjugated using the activated ester method, while the hapten and ovalbumin (OVA) were coupled by the mixed anhydride method. The UV scanning and infrared spectrum results showed that the hapten and the carrier protein were successfully coupled to BSA and OVA, with the combined ratios of 3.8 : 1 and 5 : 1, respectively. OLA-BSA was used as the immunogen to immunize four New Zealand white rabbits, and the high titer anti-serum produced relatively. The titers were 1 " 6400, 1 : 1600, 1 : 12800 and 1 : 6400, respectively, determined by indirect ELISA OLA-OVA as the coating antigen. The intermediate inhibition concentration (IC50) of OLA in the indirect ELISA test was 743.3 ng/mL. The lowest detection limit (IC20) was 5.71 ng/mL. High-purity OLA polyclonal antibody has been obtained by saturated ammonium sulfate and ion-exchange chromatography.

  12. Neoglycoproteins as carbohydrate antigens: synthesis, analysis, and polyclonal antibody response.

    Science.gov (United States)

    Kerékgyártó, Márta; Fekete, Anikó; Szurmai, Zoltán; Kerékgyártó, János; Takács, László; Kurucz, István; Guttman, András

    2013-08-01

    The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by CGE and the number of conjugated maltose residues was determined by MALDI-TOF MS. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by ELISA as evidence for the presence of sugar-containing epitope-specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degrees suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies.

  13. Preparation and characterization of polyclonal antibodies against ARL-1 protein

    Institute of Scientific and Technical Information of China (English)

    Jun-Fei Jin; Liu-Di Yuan; Li Liu; Zhu-Jiang Zhao; Wei Xie

    2003-01-01

    AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein.METHODS: ARL-L gene was inserted into the E.coli expression vector pGEX-4T-1(His)6C and vector pQE-30. Recombinant ARL1 proteins named ARL-(His)6 and ARL-GST were expressed.They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His)6 were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting.RESULTS: Recombinant proteins of ARL-(His)6 with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)6 were 15.1% and 27.7 %among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His)6 C-ARL and pQE-30-ARL individually.CONCLUSION: Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His)6 are highly expressed and purified.

  14. Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage.

    Science.gov (United States)

    Gay, Carolina; Maruñak, Silvana; Teibler, Pamela; Leiva, Laura; Acosta, Ofelia

    2013-03-01

    Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Preparation and Characterization of Polyclonal Antibodies against VLDL Receptor

    Institute of Scientific and Technical Information of China (English)

    屈伸; 陈涛; 吴凡; 尹燕华; 毕昊

    2004-01-01

    Summary: The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared poly clonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.

  16. 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.

  17. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology.

    Science.gov (United States)

    Berry, Jody D; Gaudet, Ryan G

    2011-09-01

    Antibody preparations have a long history of providing protection from infectious diseases. Although antibodies remain the only natural host-derived defense mechanism capable of completely preventing infection, as products, they compete against inexpensive therapeutics such as antibiotics, small molecule inhibitors and active vaccines. The continued discovery in the monoclonal antibody (mAb) field of leads with broadened cross neutralization of viruses and demonstrable synergy of antibody with antibiotics for bacterial diseases, clearly show that innovation remains. The commercial success of mAbs in chronic disease has not been paralleled in infectious diseases for several reasons. Infectious disease immunotherapeutics are limited in scope as endemic diseases necessitate active vaccine development. Also, the complexity of these small markets draws the interest of niche companies rather than big pharmaceutical corporations. Lastly, the cost of goods for mAb therapeutics is inherently high for infectious agents due to the need for antibody cocktails, which better mimic polyclonal immunoglobulin preparations and prevent antigenic escape. In cases where vaccine or convalescent populations are available, current polyclonal hyperimmune immunoglobulin preparations (pIgG), with modern and highly efficient purification technology and standardized assays for potency, can make economic sense. Recent innovations to broaden the potency of mAb therapies, while reducing cost of production, are discussed herein. On the basis of centuries of effective use of Ab treatments, and with growing immunocompromised populations, the question is not whether antibodies have a bright future for infectious agents, but rather what formats are cost effective and generate safe and efficacious treatments to satisfy regulatory approval. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Characterization of a novel polyclonal anti-hypusine antibody.

    Science.gov (United States)

    Nishiki, Yurika; Farb, Thomas B; Friedrich, Jessica; Bokvist, Krister; Mirmira, Raghavendra G; Maier, Bernhard

    2013-01-01

    The translation factor eIF5A is the only protein known to contain the amino acid hypusine, which is formed posttranslationally. Hypusinated eIF5A is necessary for cellular proliferation and responses to extracellular stressors, and has been proposed as a target for pharmacologic therapy. Here, we provide the first comprehensive characterization of a novel polyclonal antibody (IU-88) that specifically recognizes the hypusinated eIF5A. IU-88 will be useful for the investigation of eIF5A biology and for the development of assays recognizing hypusinated eIF5A.

  19. [Preparation and identification of polyclonal antibody of Streptococcus salivarius 57.I urease].

    Science.gov (United States)

    Wang, Yan; Tao, Dan-Ying; Feng, Xi-Ping

    2016-12-01

    To prepare and identify polyclonal antibody of Streptococcus salivarius 57.I urease. The biggest structural subunit of Streptococcus salivarius 57.I urease, UreC, was obtained by gene clone, IPTG-induced expression, and purification through affinity chromatography. Anti-sera and polyclonal antibody were raised by immunizing rabbits with purified UreC. Western blot was utilized to detect the specific combination of polyclonal antibody with UreC. Purified UreC protein was prepared and used as antigen to immunize rabbits. Polyclonal antibody was obtained, and Western blot displayed a specific band of the polyclonal antibody with UreC about 62 kD as anticipated. Polyclonal antibody against Streptococcus salivarius 57.I urease is obtained, which provides an important tool to explore the function of urease and its relationship with dental caries.

  20. Detection of Salmonella typhimurium using polyclonal antibody immobilized magnetostrictive biosensors

    Science.gov (United States)

    Guntupalli, R.; Hu, Jing; Lakshmanan, Ramji S.; Wan, Jiehui; Huang, Shichu; Yang, Hong; Barbaree, James M.; Huang, T. S.; Chin, Bryan A.

    2006-05-01

    Novel mass-sensitive, magnetostrictive sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless or remote. These biosensors can thus be used in-situ for detecting pathogens and biological threat agents. In this work, we have used a magnetostrictive platform immobilized with a polyclonal antibody (the bio-molecular recognition element) to form a biosensor for the detection of Salmonella typhimurium. Upon exposure to solutions containing Salmonella typhimurium bacteria, the bacteria were bound to the sensor and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Responses of the sensors to different concentrations of S. typhimurium were recorded and the results correlated with those obtained from scanning electron microscopy (SEM) images of samples. Good agreement between the measured number of bound bacterial cells (attached mass) and frequency shifts were obtained. The longevity and specificity of the selected polyclonal antibody were also investigated and are reported.

  1. Synthesis of Polyclonal Antibodies against Aflatoxin B1

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    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.

  2. Preparation and Application of Polyclonal Antibody against a Recombinant Laccase

    Institute of Scientific and Technical Information of China (English)

    Yinghai Xu; Yuzhi Hong; Yazhong Xiao; Wei Fang

    2007-01-01

    A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD.Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1:32 in double immunodiffusion test and of 1:128,000 in enzyme-linked immunosorbent assay (ELISA). The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities.

  3. 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)

  4. 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.

  5. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, T.P.

    2003-12-31

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

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

    Science.gov (United States)

    2016-07-01

    1 Production of potent fully human polyclonal antibodies against Zaire Ebola virus in transchromosomal cattle John M. Dye1, Hua Wu2, Jay...mail: jjiao@sabbiotherapeutics.com Keywords: Ebola virus, virus neutralization assay, human polyclonal antibodies, transchromosomal bovine...recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV)-Makona isolate. Serum collected from these hyperimmunized Tc

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  8. Artificial antigen synthesis and the development of polyclonal antibody-based immunoassay for citreoviridin determination.

    Science.gov (United States)

    Zhuang, Zhen Hong; Que, Shan Jin; Gao, Yue Ming; Yuan, Jun; Ye, Zhou; Du, Min; Lin, Guang Mei; Liu, Li Cai; Wang, Shi Hua

    2014-01-01

    Citreoviridin, a mycotoxin produced by Penicillium citreonigrum is a common contaminant of wide range of agri-products and detrimental to human and animal health. Therefore it is important to develop a rapid, sensitive, and specific immunoassay for citreoviridin detection. In this study, polyclonal antibody against citreoviridin was developed. For the preparation of citreoviridin-bovine serum albumin conjugate (CIT-BSA), hydroxyl groups on adjacent carbon atoms were oxidized by sodium periodate, so the product with reactive aldehyde residues was suitable for coupling with amine. Anti-citreoviridin polyclonal antibody was prepared by immunizing mice with CIT-BSA conjugate. The specificity and sensitivity of the polyclonal antibody was determined by indirect competitive ELISA. Results showed that the IC50 value of the polyclonal antibody was 0.56 μg/mL and no cross-reactivity was found between antiserum and other mycotoxins used in the experiment. The citreoviridin recovery rates by this polyclonal antibody were calculated through rice powder spiked by artificial citreoviridin. The recovery rates ranged were found from 70.5 ± 0.08 % to 94.7 ± 0.09% for inter-assay, and from 77.5 ± 0.04% to 95.4 ± 0.18% for intra-assay, which indicated that this polyclonal antibody could detect trace amount of CIT from the tested samples. Consequently, this study provided a specific and sensitive anti-citreoviridin polyclonal antibody, which made the determination of citreoviridin easier, quicker, and more accurate.

  9. Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Goli, Leila Balaei; Babaei, Mahdokht; Ghods, Roya

    2010-04-01

    R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity.

  10. Renalase-specific polyclonal antibody and its application in the detection of renalase's expression.

    Science.gov (United States)

    Wang, Feng; Zhao, Qing; Xing, Tao; Li, Junhui; Wang, Niansong

    2012-10-01

    Renalase is generated mainly by the kidneys, and renalase's expression in chronic kidney disease patients is reduced due to renal dysfunction. In this study, human renalase recombinant protein with prokaryotic expression was used for immunization of male New Zealand rabbits, and polyclonal antibodies against human renalase were obtained. To prepare and verify renalase polyclonal antibody, renalase recombinant protein was used as antigen and male New Zealand rabbits were immunized to obtain anti-serum for identification. On the basis of renalase antibody, the expression of renalase in renal tubular epithelial cells and renal tissue was detected. Two anti-renalase polyclonal antibodies were obtained. Renalase was constitutively expressed in human renal tubular epithelial cells, with the maximum expression in proximal convoluted tubules in renal tissue, and a small amount of expression in the glomeruli. Anti-renalase polyclonal antibodies were successfully prepared, which lays a foundation for further studies.

  11. [Preparation and application of rabbit anti-mouse Setd8 polyclonal antibody].

    Science.gov (United States)

    Ma, Haitao; Guo, Jiaqian; Xia, Jing; Niu, Changmin; Shen, Xueyi; Sun, Hongya; Zheng, Ying

    2017-02-01

    Objective To purify the recombinant Setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody. Methods The recombinant plasmid pET-30a-Setd8 was constructed by double enzyme digestion and linkage, and then transformed into E.coli BL21. The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph. The purified protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The titer and specificity of the antibody were identified by ELISA, Western blotting and immunohistochemistry. Results The prokaryotic expression vector pET-30a-Setd8 was constructed successfully. After induced by IPTG, the recombinant Setd8 protein was expressed effectively in E.coli BL21. Polyclonal antibody against Setd8 was generated by immunizing rabbits with the routine method. ELISA showed that the titer of rabbit anti-Setd8 antiserum was 1:1 000 000. Western blotting demonstrated that the polyclonal antibody could recognize the native mouse Setd8 protein. Immunohistochemistry revealed that Setd8 protein recognized by the polyclonal antibody was mainly distributed in the nucleus of spermatogonia in adult mouse testis. Conclusion Using the prokaryotic expression vector pET-30a-Setd8, we have prepared successfully the polyclonal antibody with high affinity and specificity.

  12. Prokaryotic expression of f protein from PPRV and characterization of its polyclonal antibody.

    Science.gov (United States)

    Wang, Qiuxia; Dou, Yongxi; Yang, Xiangfang; Meng, Xuelian; Zhai, Junjun; Zhu, Xueliang; Luo, Xuenong; Chen, Lei; Cai, Xuepeng

    2013-02-01

    The goal of this study was to evaluate the specificity of a polyclonal antibody against the F protein from Peste des petits ruminants virus (PPRV). A pET30a/F prokaryotic expression vector was successfully constructed and its recombinant protein was expressed. The result of Western blot analysis showed that the fusion protein pET30a/F possessed good immunoreactivity and the purified recombinant protein was then used as the antigen to raise anti-pET30a/F polyclonal antibody in rabbits. The polyclonal antibody titer against the recombinant F protein was confirmed by indirect ELISA, and the protein's specificity against pET30/F polyclonal antibody was confirmed by both Western blot and indirect immunofluorescence assay in transfected cells. In short, we obtained the high-level expression of recombinant F protein as well as high titers of rabbit polyclonal antibody specificity against F protein in pCAGGS/F transfected cells. This special polyclonal antibody offers a valuable and useful tool for further study of the pathogenesis of PPRV early infection and the structural and functional characterization of PPRV F protein.

  13. EFFECT OF POLYCLONAL AND MONOCLONAL-ANTIBODIES ON SURFACE-PROPERTIES OF STREPTOCOCCUS-SOBRINUS

    NARCIS (Netherlands)

    VANRAAMSDONK, M; VANDERMEI, HC; DESOET, JJ; BUSSCHER, HJ; DEGRAAFF, J

    1995-01-01

    In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined

  14. Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

    Science.gov (United States)

    Fan, Weifei; Tang, Qiao; Shen, Chenyou; Qin, Di; Lu, Chun; Yan, Qin

    2015-11-01

    Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

  15. [Preparation and application of melanoma-associated antigen D4 polyclonal antibody].

    Science.gov (United States)

    He, Shujia; Gu, Yongyao; Xiao, Shaowen; Zhang, Qingmei; Chen, Fang; Luo, Bin; Fu, Jun; Xie, Xiaoxun

    2017-01-01

    Objective To prepare the rabbit polyclonal antibody recognizing human melanoma-associated antigen D4 (MAGE-D4), and identify its immune characteristics for preliminary application. Methods MBP/MAGE-D4 fusion protein was expressed from pMAL-C2/MAGE-D4 recombinant plasmid constructed in the previous work. Then the purified MBP/MAGE-D4 protein was used to immunize the New Zealand white rabbits for generating polyclonal antibody. Subsequently, the anti-MAGE-D4 antibody was purified with protein A affinity chromatograph and identified by SDS-PAGE. The titer and specificity of the antibody were further detected by indirect ELISA and Western blotting, respectively. MAGE-D4 expression and localization of lung cancer tissues were analyzed by immunohistochemical staining. Results MAGE-D4 polyclonal antibody with high purity was obtained. Its titer was about 1:256 000. Western blot analysis demonstrated that the antibody could specifically react to the recombinant MAGE-D4 protein. Immunohistochemical staining showed that the antibody could recognize the endogenous MAGE-D4 protein in lung cancer tissues, and its positive rate was 69.6% (17/23). Conclusion The MAGE-D4 polyclonal antibody with high specificity and sensitivity has been successfully prepared.

  16. Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Testis-specific protease 50 (TSP50) is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSP50 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector pcDNA3.1. Rabbit anti-TSP50 polyclonal antibodies were prepared by means of intramuscular injection of pcDNA3.1-TSP50 into the rabbits. Titers of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSP50 fusion protein as an antigen. In addition, we examined the expression of TSP50 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.

  17. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection.

    Science.gov (United States)

    Stevens, Natalie E; Hatjopolous, Antoinette; Fraser, Cara K; Alsharifi, Mohammed; Diener, Kerrilyn R; Hayball, John D

    2016-07-06

    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus.

  18. Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: Establishment of an efficient ELISA for antigen detection in feces

    OpenAIRE

    Czerny, C. P.; Eichhorn, Werner

    1989-01-01

    Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40 000. Only one out of 908 hybridoma colonies tested secreted antibodies ...

  19. Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody.

    Science.gov (United States)

    Kang, Changkeun; Han, Dae-Yong; Park, Kwang-Il; Pyo, Min-Jung; Heo, Yunwi; Lee, Hyunkyoung; Kim, Gon Sup; Kim, Euikyung

    2014-08-01

    Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging.

  20. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.

    Science.gov (United States)

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

    2016-02-24

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  2. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  3. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.

    1996-01-01

    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  4. Assessment of Neutralising Activity of Colostrum-Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1 Anthrax Lethal Toxin Cytototoxity Assay

    Science.gov (United States)

    2005-12-01

    Assessment of Neutralising Activity of Colostrum - Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1 Anthrax Lethal Toxin...activity of colostrum -derived, polyclonal, bovine antibodies. Antibodies against lethal factor and protective antigen were found to protect...APPROVED FOR PUBLIC RELEASE Assessment of Neutralising Activity of Colostrum - Derived, Polyclonal, Bovine Antibodies: Use of the J774A.1

  5. Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

    Science.gov (United States)

    Saxena, Shikha; Kumar, Gandham Ravi; Singh, Prafull; Chaturvedi, Uttara; Saxena, Lovleen; Kumar, Rajiv; Sahoo, Aditya Prasad; Doley, Juwar; Rajmani; Kumar, Amit; Kumar, Sudesh; Tiwari, Ashok K

    2012-05-01

    In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

  6. Polyclonal antibody localizes glia maturation factor beta-like immunoreactivity in neurons and glia.

    Science.gov (United States)

    Wang, B R; Zaheer, A; Lim, R

    1992-09-18

    A rabbit polyclonal antibody (91-01) was raised against recombinant human glia maturation factor beta (r-hGMF-beta). The antibody did not cross-react with a number of other growth factors on ELISA test. When compared with the monoclonal antibody G2-09 previously obtained, 91-01 immunoblotted the same protein band in rat brain extract. However, unlike G2-09 which immunostained only astrocytes and Bergmann glia, 91-01 stained neurons as well. Many but not all neurons in the central and peripheral nervous system were positive for GMF-beta. The larger cell population stained by the polyclonal antibody was most likely due to its increased sensitivity, although other explanations are possible. The presence of GMF-beta-like immunoreactivity in both neurons and glia raises the possibility of a wider range of cell-cell interaction than was previously considered.

  7. Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

    Directory of Open Access Journals (Sweden)

    Ballantyne John

    2011-09-01

    Full Text Available Abstract Background Previously we demonstrated that DNA vaccination of nonhuman primates (NHP with a small subset of vaccinia virus (VACV immunogens (L1, A27, A33, B5 protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV whereas A33 and B5 target the enveloped virion (EV. Results Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal Orthopoxvirus infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality. Conclusions A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.

  8. Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins*

    Science.gov (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-01-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. PMID:24722731

  9. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    Science.gov (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  10. [Preparation and identification of rabbit anti-AKR1B10 polyclonal antibody].

    Science.gov (United States)

    Hu, Jian; Wang, Qingmei; Huang, Li; Zeng, Yuanqing; Hu, Zheng; Luo, Dixian

    2016-11-01

    Objective To prepare rabbit anti-aldo-keto reductase family 1 member B10 (AKR1B10) polyclonal antibody and identify its specificity. Methods AKR1B10 cDNA was amplified by reverse transcription PCR (RT-PCR) and inserted into prokaryotic expression vector pET-15b to form recombinant plasmid pET-15b-AKR1B10. The recombinant plasmid pET-15b-AKR1B10 was transformed into E.coli DH5α. Isopropylthio-β-D-galactoside (IPTG) was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α. The expression products from different clones of E.coli DH5α were identified by SDS-PAGE. The positive bacteria were picked out and amplified. His-Tag-AKR1B10 protein was purified from the expression product of the positive clones by His-tagged purification column. The purified recombinant protein His-Tag-AKR1B10 was used to immunize New Zealand white rabbits. Antisera were acquired after two months. Anti-AKR1B10 polyclonal antibodies were purified by antigen purification column with AKR1B10 recombinant protein. Lastly, the purified polyclonal antibodies were identified by SDS-PAGE, ELISA, Western blotting. Results The recombinant plasmid pET-15b-AKR1B10 was constructed successfully, and the recombinant protein His-Tag-AKR1B10 with high purity was acquired. The purified polyclonal antibodies were able to specifically recognize AKR1B10 protein. Conclusion The rabbit anti-AKR1B10 polyclonal antibodies is prepared successfully with high specificity.

  11. ELISPOT Assay for Measurement of Antigen-Specific and Polyclonal Antibody Responses.

    Science.gov (United States)

    Lycke, Nils; Coico, Richard

    2015-02-02

    The enzyme-linked immunospot (ELISPOT) assay for detection of antigen-specific and polyclonal antibody responses by single antibody-secreting cells has become the method of choice due to its cell-based quantitative value. Antigen stability and specificity and the diversity of antigens that can be used in the assay have contributed to the translational application of ELISPOT as demonstrated by many FDA-approved clinical tests that employ this technique. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two-color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell.

  12. Immunisation - Choice of host, adjuvants and boosting schedules with emphasis on polyclonal antibody production.

    Science.gov (United States)

    Delahaut, Philippe

    2017-03-01

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

  13. 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; Xing, Mingwei; Wang, Junwei

    2014-08-01

    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.

  14. SERUM IMMUNOGLOBULIN OF THE MANDARIN FISH, SINIPERCA CHUATSI WITH DEVELOPMENT OF POLYCLONAL ANTIBODY

    Institute of Scientific and Technical Information of China (English)

    张永安; 聂品

    2002-01-01

    Serum immunoglobulin from the mandarin fish, or the so-called Chinese perch, S iniperca chuatsi (Basilewsky), was successfully purified using affinity chroma togr aphy. Heavy and light chains were detected on electrophoresis gel, with molecula r weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuat si immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.

  15. Serum immunoglobulin of the mandarin fish, Siniperca chuatsi with development of polyclonal antibody

    Science.gov (United States)

    Zhang, Yong-An; Nie, Pin

    2002-12-01

    Serum immunoglobulin from the mandarin fish, or the so-called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.

  16. 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

  17. VH and VL Domains of Polyspecific IgM and Monospecific IgG Antibodies Contribute Differentially to Antigen Recognition and Virus Neutralization Functions.

    Science.gov (United States)

    Pasman, Y; Kaushik, A K

    2016-07-01

    We analysed contributions of variable heavy (FdVH ) and variable light (FdVL ) domains in comparison to scFv (FdVH +FdVL ) of naturally occurring polyspecific bovine IgM with an exceptionally long CDR3H and an induced monospecific bovine herpes virus-1 (BoHV-1) neutralizing IgG1 antibody in the context of to antigen-binding site and antibody function. Various recombinant FdVH , FdVL and scFv were constructed and expressed in Pichia pastoris from the bovine IgM and IgG1 antibody encoding cDNA. The scFv1H12 showed polyspecific antigen binding similar to parent IgM antibody, though subtle differences, for example, higher thyroglobulin recognition. Such differences reflect influence of the constant region on the antigen-binding site configuration. Unlike, variable light domain FdVL 1H12, the variable heavy domain FdVH 1H12 alone recognized multiple antigens that differed from the recognition pattern of scFv1H12 (FdVH +FdVL ) and the parent IgM antibody. Nonetheless, role of FdVL 1H12 in providing structural support to FdVH in antigen recognition is noted, apart from its intrinsic antigen recognition ability. Surface plasmon resonance analysis revealed low to moderate affinity of scFv1H12 to IgG antigen. By contrast, the individual FdVH 073 and FdVL 074, originating from induced BoHV-1 neutralizing IgG1 antibody, recognized target epitope on BoHV-1 weakly when compared to FdVH +FdVL (scFv3-18L). Interestingly, both the FdVH and FdVL domains of induced IgG antibody are required to achieve BoHV-1 neutralization. To conclude, there exist subtle functional differences in the contribution of FdVH and FdVL to antigen-binding site generation of polyspecific IgM and monospecific IgG antibodies relevant to antigen recognition and virus neutralization functions.

  18. Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody.

    Science.gov (United States)

    Sun, Mingjuan; Zou, Rongjiang; Dong, Xiaoyi; Zong, Ying; Gao, Yun; Wang, Lianghua; Jiao, Binghua

    2013-01-01

    Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

  19. Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

    Institute of Scientific and Technical Information of China (English)

    YANG Yan; HUANG Huang; ZHANG Zhenghua; WANG Baoju; TIAN Yongjun; LU Mengji; YANG Dongliang

    2007-01-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  20. 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.

  1. Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

    Institute of Scientific and Technical Information of China (English)

    Han Yu Chen; Hui Sheng Zhuang; Chun Zhou

    2009-01-01

    Three polychlorinated biphenyls(PCBs)congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized.The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens.Three of the resultant polyclonal antibodies(Pabs)were obtained.The antiserum exhibited relatively high antibody titres(1:32-64)in double agar diffusion.◎2008 Hui Sheng Zhuang.Published by Elsevier B.V.on behalf of Chinese Chemical Society.All rights reserved.

  2. Preparation of polyclonal antibodies against pig trachea apomucin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, H.H.; Carlson, D.M.

    1987-05-01

    Purified procine tracheal mucin (PTM) was deglycosylated by treatment with trifluoromethanesulfonic acid (TFMS). A preferrential release of fucose, galactose > GlcNAc > GalNAc was observed during the deglycosylation. After five hours of TFMS treatment at 5C, over 90% of the carbohydrate was released leaving mainly Ga1NAc attached to the core protein. The residual Ga1NAc was further removed by three series of the Smith Degradation. The resulting apomucin consisted mainly of (mol %) Thr, 14; Ser, 12; Gly, 12; Glx, 10; Ala, 9; Pro, 8; Val, 7; Asx, 7. Sugars were not detected by gas chromatography. Antibodies obtained by immunizing rabbits with the apomucin recognized only the apomucin and not the holomucin in a double-sandwich ELISA assay. The ELISA is highly specific and has detection limits in the ng range. This antibody preparation was used in precipitation assays of cell-free translation products of pig trachea epithelium RNAs. Translocation products labeled with (TH)Ser and (TH)Thr showed significantly higher counts than with control serum.

  3. Monoclonal and polyclonal antibodies for ante- and post-mortem detection of PrPSc in sheep

    Directory of Open Access Journals (Sweden)

    Michele Dietrich Moura Costa

    2015-04-01

    Full Text Available Scrapie is a disease that affects sheep and goats and is characterized by the accumulation of an abnormal isoform (PrPSc of the cellular prion protein, PrPC, in the central nervous system (CNS and in lymphoid tissues. Detection of PrPSc in these tissues can be attempted by a variety of techniques, including immunohistochemistry (IHC and western blotting (WB, for which a wide range of monoclonal and polyclonal antibodies are commercially available. The objective of this study was to test and compare the efficacy of monoclonal antibodiesF89/160.1.5, F99/97.6.1, and P4 and polyclonal antibodies M52 and R486 in the detection of PrPSc in lymphoid and CNS tissue samples by using IHC. Positive and negative control samples of sheep brain and tonsils were provided by the Animal Health and Veterinary Laboratories Agency (AHVLA, UK. The IHC examination of CNS samples with both monoclonal and polyclonal antibodies confirmed the granular deposition of PrPSc in the neurons of the positive control tissues. However, while the monoclonal antibodies did not produce positive reactions in the negative controls, the polyclonal antibodies showed some non-specific staining. The testing of positive control tonsil samples with polyclonal and monoclonal antibodies identified positive control-specific reactions, whereas the negative control tissues were IHC-negative with all antibodies, although P4 and the polyclonal antibodies produced some background staining. In summary, although the polyclonal antibodies may be more accessible, their use is not advisable because of possible false positive reactions. The polyclonal antibody M52 was able to identify PrPC in brain and spleen samples by WB but other lymphoid tissues were negative.

  4. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei

    2008-01-01

    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  5. [Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody].

    Science.gov (United States)

    Mao, Xia; Zhang, Bing; Bai, Xue-Ling; Liu, Long-Long; Zhang, Dong-Hua

    2012-12-01

    This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

  6. Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

    Science.gov (United States)

    Zhao, Yinli; Li, Guoxi

    2016-01-01

    A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

  7. Development of mass spectrometry based techniques for the identification and determination of compositional variability in recombinant polyclonal antibody products.

    Science.gov (United States)

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

    2010-09-01

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

  8. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting

    OpenAIRE

    Doria-Rose, Nicole A.; Altae-Tran, Han R.; Roark, Ryan S.; Schmidt, Stephen D.; Sutton, Matthew S.; Louder, Mark K.; Chuang, Gwo-Yu; Bailer, Robert T.; Cortez, Valerie; Kong, Rui; McKee, Krisha; O’Dell, Sijy; Wang, Felicia; Abdool Karim, Salim S.; Binley, James M.

    2017-01-01

    Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction c...

  9. Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

    Institute of Scientific and Technical Information of China (English)

    Liu; Xueguang; Zheng; Huaidong; Guo; Xinshuo; Luo; Jin; Lin; Cuicui; Wang; Qiuyu

    2014-01-01

    [Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

  10. Preparation of Polyclonal Antibody and Expression Analysis of GR in Tomato

    Science.gov (United States)

    Xie, Yuanhong; Zhu, Benzhong; Luo, Yunbo; Chen, Xiangning; Zhang, Hongxing

    The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional.

  11. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting.

    Science.gov (United States)

    Doria-Rose, Nicole A; Altae-Tran, Han R; Roark, Ryan S; Schmidt, Stephen D; Sutton, Matthew S; Louder, Mark K; Chuang, Gwo-Yu; Bailer, Robert T; Cortez, Valerie; Kong, Rui; McKee, Krisha; O'Dell, Sijy; Wang, Felicia; Abdool Karim, Salim S; Binley, James M; Connors, Mark; Haynes, Barton F; Martin, Malcolm A; Montefiori, David C; Morris, Lynn; Overbaugh, Julie; Kwong, Peter D; Mascola, John R; Georgiev, Ivelin S

    2017-01-01

    Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1.

  12. Generation and characterization of a polyclonal antibody against human high mobility group box 4.

    Science.gov (United States)

    Yang, Fen; Li, Runsheng; Hong, Aizhen; Duan, Fei; Li, Yuhua

    2013-11-01

    A human high mobility group box 4 (hHMGB4) expression construct (pET‑28a/hHMGB4) was generated by cloning the hHMGB4 full‑length cDNA in the expression vector pET‑28a(+). The hHMGB4 fusion protein with His6‑Tag was prepared using E.coli BL21 (DE3) transformed with pET‑28a/hHMGB4 and purified via preparative SDS‑PAGE plus electroelution. Immunization of rabbits with the purified hHMGB4 generated polyclonal antibodies. The titer of the antiserum was determined to be 1:102,400 by ELISA analysis. Western blotting analysis showed that the antibody specifically recognized the recombinant hHMGB4 protein and also the endogenous hHMGB4 protein in prostate cancer cells. In addition, immunohistochemical staining analysis using the prepared antibody revealed marked hHMGB4 staining in the nuclei of the human prostate tissue. These data demonstrate that the anti‑hHMGB4 polyclonal antibody may be a useful reagent for the functional study of hHMGB4.

  13. Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax.

    Science.gov (United States)

    Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie A; Dai, Zhongdong; Kubler-Kielb, Joanna; Agulto, Liane; Leppla, Stephen H; Purcell, Robert H

    2015-08-01

    The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10(4) following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines.

  14. Prokaryotic expression, purification, and production of polyclonal antibody against novel human serum inhibited related protein I (SI1).

    Science.gov (United States)

    Ma, Mingxing; Ma, Jie; Shi, Yinghui; Wu, Hong; Zhao, Wenxiu; Huang, Weiwei; Jiao, Yang; Tan, Deyong

    2010-02-01

    A novel serum inhibited related gene (SI1) has been cloned in our lab by using mRNA differential display analysis of U251 cells in the presence or absence of serum, the expression of SI1 was dramatically inhibited by the addition of serum to serum starved cells. Previous reports suggested the potential significance of SI1 in regulating the cell cycle. In this study, the plasmid construction, protein expression and purification, as well as the generation of anti-SI1 polyclonal antibody are described. A full-length cDNA of Si1 was inserted in a prokaryotic expression plasmid pET28-b(+) and efficiently expressed in E. coli Rosetta (DE3) strain after induction by isopropyl-b-D: -thiogalactoside. The expressed 6His-tagged SI1 fusion protein was purified by Ni(+) affinity column and then used to immunize Balb/C mice, and the anti-SI1 polyclonal antibody was purified by protein A column. To determine the sensitivity and specificity of the antibody against SI1, a cell lysate of pEGFP-N2-SI1 plasmid transiently transfected Hela cell was identified by anti-GFP monoclonal antibody and anti-SI1 polyclonal antibody. Both the GFP-SI1 fusion protein and endogenous SI1 protein in Hela cell can be recognized by the anti-SI1 polyclonal antibody. The anti-SI1 polyclonal antibody will provide a useful tool for further characterization of SI1.

  15. Polyclonal antibody against the DPV UL46M protein can be a diagnostic candidate

    Directory of Open Access Journals (Sweden)

    Jia Renyong

    2010-04-01

    Full Text Available Abstract Background The duck plague virus (DPV UL46 protein (VP11/12 is a 739-amino acid tegument protein encoded by the UL46 gene. We analyzed the amino acid sequence of UL46 using bioinformatics tools and defined the main antigenic domains to be between nucleotides 700-2,220 in the UL46 sequence. This region was designated UL46M. The DPV UL46 and UL46M genes were both expressed in Escherichia coli Rosetta (DE3 induced by isopropy1-β-D-thiogalactopyranoside (IPTG following polymerase chain reaction (PCR amplification and subcloning into the prokaryotic expression vector pET32a(+. The recombinant proteins were purified using a Ni-NTA spin column and used to generate the polyclonal antibody against UL46 and UL46M in New Zealand white rabbits. The titer was then tested using enzyme-linked immunosorbent assay (ELISA and agar diffusion reaction, and the specificity was tested by western blot analysis. Subsequently, we established Dot-ELISA using the polyclonal antibody and applied it to DPV detection. Results In our study, the DPV UL46M fusion protein, with a relative molecular mass of 79 kDa, was expressed in E. coli Rosetta (DE3. Expression of the full UL46 gene failed, which was consistent with the results from the bioinformatic analysis. The expressed product was directly purified using Ni-NTA spin column to prepare the polyclonal antibody against UL46M. The titer of the anti-UL46M antisera was over 1:819,200 as determined by ELISA and 1:8 by agar diffusion reaction. Dot-ELISA was used to detect DPV using a 1:60 dilution of anti-UL46M IgG and a 1:5,000 dilution of horseradish peroxidase (HRP-labeled goat anti-rabbit IgG. Conclusions The anti-UL46M polyclonal antibody reported here specifically identifies DPV, and therefore, it is a promising diagnostic tool for DPV detection in animals. UL46M and the anti-UL46M antibody can be used for further clinical examination and research of DPV.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. [Preparation of polyclonal antibody against sAPRIL and analysis of function in suppressing sAPRIL-mediated lymphocyte proliferation].

    Science.gov (United States)

    Du, Ben-Jun; Gao, Quan-Sheng; Lan, Zhi; Fan, Jun-Wen; Ding, Lu-Jing; Li, Min; Qi, Yuan-Yuan; Kong, Wei

    2011-08-01

    This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.

  19. Human polyclonal antibodies produced in transchromosomal cattle prevent lethal Zika virus infection and testicular atrophy in mice.

    Science.gov (United States)

    Stein, Derek R; Golden, Joseph W; Griffin, Bryan D; Warner, Bryce M; Ranadheera, Charlene; Scharikow, Leanne; Sloan, Angela; Frost, Kathy L; Kobasa, Darwyn; Booth, Stephanie A; Josleyn, Matthew; Ballantyne, John; Sullivan, Eddie; Jiao, Jin-An; Wu, Hua; Wang, Zhongde; Hooper, Jay W; Safronetz, David

    2017-09-08

    Zika virus (ZIKV) is rapidly spreading throughout the Americas and is associated with significant fetal complications, most notably microcephaly. Treatment with polyclonal antibodies for pregnant women at risk of ZIKV-related complications could be a safe alternative to vaccination. We found that large quantities of human polyclonal antibodies could be rapidly produced in transchromosomal bovines (TcB) and successfully used to protect mice from lethal infection. Additionally, antibody treatment eliminated ZIKV induced tissue damage in immunologically privileged sites such as the brain and testes and protected against testicular atrophy. These data indicate that rapid development and deployment of human polyclonal antibodies could be a viable countermeasure against ZIKV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  20. 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

    in the kinetics of viral infection. Sequence analysis of the recovered viruses did not suggest antibody-induced viral escape. Conclusion: Polyclonal anti-HCV antibodies isolated from a chronic HCV patient can protect against an in vivo challenge with different HCV genotypes. However, the in vivo protective...... HCV infection still remains elusive. We have previously shown that purified polyclonal antibodies isolated from plasma obtained in 2003 from a chronic HCV patient (Patient H) can protect human liver chimeric mice from a subsequent challenge with the autologous HCV strain isolated from Patient H...... in 1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross-genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different...

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

    Directory of Open Access Journals (Sweden)

    ADRIANA CRISTE

    2013-12-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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. Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces.

    Science.gov (United States)

    Czerny, C P; Eichhorn, W

    1989-06-01

    Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.

  4. Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium

    Science.gov (United States)

    Zarei, Omid; Irajian, Gholam Reza; Zarnani, Amir Hassan; Chamani-Tabriz, Leili; Emami, Shaghayegh; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2011-01-01

    Mycoplasma genitalium (M.genitalium) is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications. PMID:23408484

  5. 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.

  6. Expression of Catalytic Domain of Protein Tyrosine Phosphatase 1B and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study focuses on the expression of human protein tyrosine phosphatase 1B(PTP1B) catalytic domain (△PTP1B) and preparation of polyclonal antibody against △PTP1B. △PTP1B gene was PCR amplified with the cDNA of human PTP1B as the template, and cloned into the pT7 expression vector. The recombinant pT7-△PTP1B was expressed in E. coli Rosetta( DE3 ) host cells and purified. The antiserum was prepared by immunizing rabbit with purified recombinant △PTP1B. The polyclonal antibody against △PTP1B was purified by PVDF immobilized antigen affinity chromatography. △PTP1B was correctly cloned, expressed, and purified as confirmed by PCR, DNA sequence ratio) and 0. 1 ng, respectively. This study provides an important basis for further studying the biological function of PTP1B and its relationship with human diseases.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  9. Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

    Science.gov (United States)

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

    2013-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Przewodowski, Włodzimierz; Przewodowska, Agnieszka

    2017-01-01

    The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents.

  11. Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus

    Science.gov (United States)

    Przewodowska, Agnieszka

    2017-01-01

    The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents. PMID:28068400

  12. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    Science.gov (United States)

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  13. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

    Science.gov (United States)

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

    2015-03-01

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

  14. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,wi...

  15. Anti-miroestrol polyclonal antibodies: a comparison of immunogen preparations used to obtain desired antibody properties.

    Science.gov (United States)

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Yusakul, Gorawit; Tanaka, Hiroyuki; Putalun, Waraporn

    2016-04-01

    Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

  16. Modulation of innate immune responses by influenza-specific ovine polyclonal antibodies used for prophylaxis.

    Directory of Open Access Journals (Sweden)

    Catherine Rinaldi

    Full Text Available In the event of a novel influenza A virus pandemic, prophylaxis mediated by antibodies provides an adjunct control option to vaccines and antivirals. This strategy is particularly pertinent to unvaccinated populations at risk during the lag time to produce and distribute an effective vaccine. Therefore, development of effective prophylactic therapies is of high importance. Although previous approaches have used systemic delivery of monoclonal antibodies or convalescent sera, available supply is a serious limitation. Here, we have investigated intranasal delivery of influenza-specific ovine polyclonal IgG antibodies for their efficacy against homologous influenza virus challenge in a mouse model. Both influenza-specific IgG and F(ab'2 reduced clinical scores, body weight loss and lung viral loads in mice treated 1 hour before virus exposure. Full protection from disease was also observed when antibody was delivered up to 3 days prior to virus infection. Furthermore, effective prophylaxis was independent of a strong innate immune response. This strategy presents a further option for prophylactic intervention against influenza A virus using ruminants to generate a bulk supply that could potentially be used in a pandemic setting, to slow virus transmission and reduce morbidity associated with a high cytokine phenotype.

  17. Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies.

    Science.gov (United States)

    Chen, Xueran; Li, Hao; Wang, Hongzhi; Yang, Haoran; Ye, Fang; Liang, Chaozhao; Fang, Zhiyou

    2016-04-01

    Human Supervillin isoform 4 (SV4), a bigger splicing isoform of Supervillin, contains extra coding exons 3, 4 and 5 (E345), compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton, regulated cell migration and cell survival, its functions are still largely unknown. To broaden our understanding, SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins, which contained SV4 specific domain E345, were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum, and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA, western blot, immunoprecipitation, and immunofluorescence. Taken together, our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function.

  18. Antigenic differentiation of avian pneumovirus isolates using polyclonal antisera and mouse monoclonal antibodies.

    Science.gov (United States)

    Collins, M S; Gough, R E; Alexander, D J

    1993-09-01

    Avian pneumovirus (AVP) isolates F83, CC220 and 1260 from Great Britain and 1556, 657/4, 2119 and 872/S from France, Hungary, Italy and Spain, respectively, were compared in ELISA and virus neutralization (VN) tests for reactions with chicken polyclonal sera against each of the viruses and monoclonal antibodies (MAbs) against two British isolates. ELISA test results using the polyclonal antisera indicated that all seven viruses were antigenically related, but some variation between strains could be detected, especially when antigens were prepared from infected cells using Nonidet P40 (NP40) rather than freezing and thawing. In VN tests results also showed that all viruses tested were related but there was evidence that the three British isolates showed closer relationships with each other than with the viruses from Italy, Hungary and Spain. In ELISA tests, isolates F83 and 1556 bound all 11 MAbs and 1260 reacted with 10/11 MAbs. Isolate CC220 showed reaction with all the MAbs but for 8/11 MAbs the optical density differences were low. Isolates 2119 and 872/S both reacted only with MAb 4 and none of the MAbs reacted with 657/4.

  19. Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization.

    Science.gov (United States)

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

    2012-08-01

    A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.

  20. Serological Differentiation of Plant-parasitic Nematode Species with Polyclonal and Monoclonal Antibodies.

    Science.gov (United States)

    Schots, A; Gommers, F J; Bakker, J; Egberts, E

    1990-01-01

    Although several attempts have been made to differentiate nematode species with polyclonal antisera, these efforts thus far have met with limited success because of extensive crossreactivities of the sera. Since the hybridoma technique offers the opportunity to develop more specific serological reagents, some research groups have recently started to apply this technology to the problem of species identification in nematology. Monoclonal antibodies (MA) that differentiate the potato-cyst nematodes Globodera rostochiensis and G. pallida, as well as MA specific for Meloidogyne species, have been developed. The possibilities of developing serodiagnostic tools for identification of nematodes recovered from soil samples and the implications of such monitoring of nematode infestations in view of integrated control of plant-parasitic nematodes are discussed.

  1. Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-hong; ZHANG Xi-zhen; ZHAO Dong-hai; SHI He-liang; YU Yong-hui; WU Yong-ge; YU Xiang-hui; KONG Wei

    2008-01-01

    Survivin,a novel member of inhibitor ofapoptosis(IAP) protein family,is aberrantly expressed in cancer but undetectable in normal,differentiated adult tissues.The cancer-specific expression of survivin,coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment.Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B.The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21,and the relative molecule mass(Mr) of expressed fusion protein was approximately 21000.The recombinantprotein was purified through Ni2~ affinity chromatography column and characterized by SDS-PAGE and Western blot.The purified recombinant protein was then injected into rabbits,and antisurvivin polyclonal antibody with a high titer was obtained.

  2. Prokaryotic expression of soluble Arabidopsis protein AtERF1 and preparation of its polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    ZHANG Yu

    2013-08-01

    Full Text Available AtERF1 encodes a member of the ERF subfamily B-3 of ERF/AP2 transcription factor family.It has been demonstrated almost every member of the B3 subgroup of AP2/ERF genes is involved in defense responses in Arabidopsis.Codon usage within a gene is a critical determinant of achievable protein expression level in E.coli. Gene optimization,therefore,is an effective method for synthetic genes with the aim of enhancing soluble expression,particular in heterologous hosts.In this paper,the AtERF1 protein of Arabidopsis thaliana was expressed in Escherichia coli using its optimized DNA sequence for E.coli. and yielded high level of soluble AtERF-1 protein in recombinant E.coli. The AtERF1 protein was used as an antigen to immune rabbits and obtains high titer antibodies.The immunological specificity of the polyclonal antibodies to AtERF1 was confirmed by Western blot assay.The prepared antibody in this work would facilitate the further functional investigation of AtERF1 in biochemical levels in Arabidopsis anther development.

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

    Science.gov (United States)

    Dugast, Anne-Sophie; Chan, Ying; Hoffner, Michelle; Licht, Anna; Nkolola, Joseph; Li, Hualin; Streeck, Hendrik; Suscovich, Todd J; Ghebremichael, Musie; Ackerman, Margaret E; Barouch, Dan H; Alter, Galit

    2014-01-01

    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.

  4. Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

    Institute of Scientific and Technical Information of China (English)

    Mallampati SARADHI; Biji KRISHNA; Gauranga MUKHOPADHYAY; Rakesh K TYAGI

    2005-01-01

    Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. Thefull-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21 (DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

  5. Expression of Cytochrome P450nor in Escherichia coli, Purification and Preparation of Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    周建刚; 张山; 陈舒丽; 江月; 刘德立

    2004-01-01

    Cytoehrome P450nor gene was cloned into the expression vector pET-28 to yield the recombinant expression plasmid pET-P450nor, which could direct the synthesis of a eukaryotic derived protein in Escherichia coli BL21. The vector allows overproduction and single-step purification of (His)6-tagged cytoehrome P450nor by the facifitation of metal (Ni2+ )chelate afl]nity chromatography. The expression level of cytoehrome P450nor was high at 30℃ after IPTG induction. SDSPAGE and Western blot analysis showed a specific band (about 43 kDa). The overproduced cytochrome P450nor was purified to eleetrophoretic homogeneity within 2.5 h and about 20.8 mg purified protein was obtained from 2 L cell culture. The proliferation of SSMC-7721 cell line could be inhibited by cytoehrome P450nor. Rabbit polyclonal antibodies (titer over 64 000) were produced against recombinant cytoehrome P450nor and proved to be very useful for immunoblotting study. Availability of this expression system and specific antibodies should facilitate characterization of the role of cytochrome P450nor in the metabolism of NO.

  6. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

    Science.gov (United States)

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

    2014-02-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

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

    Directory of Open Access Journals (Sweden)

    Zhang Shunchuan

    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. Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig.

    Science.gov (United States)

    Bao, Y Y; Li, L; Zhang, H; Gao, C Y; Xiao, C B; Li, C L

    2015-12-29

    Porcine β-defensin 2 (pBD2) is an antimicrobial peptide in pigs that plays an important role in the immune system by preventing bacterial invasion. To produce an anti-pBD2 antibody, which is not commercially available, we expressed and purified a soluble, his-tagged version of pBD2 (his-pBD2). Purified pBD2 was injected into New Zealand white rabbits to generate polyclonal antiserum. Anti-pBD2 antibodies were purified by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose ion-exchange chromatography. The purified polyclonal antibody showed high sensitivity, with a titer as high as 204,800 by enzyme-linked immunosorbent assay, and it also showed high specificity for both his-pBD2 and native pBD2, as assessed by western blotting. Furthermore, immunohistochemistry analysis using the purified antibody revealed that pBD2 protein is distributed in the tongue, liver, kidney, small intestine, and large intestine of pigs. These results indicate that the prepared polyclonal antibody will be a useful tool for further studies of the function and mechanism of pBD2.

  11. 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.

  12. 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....... The 'functionality' assay displayed concentration dependent sensitivity to interference for ammonium sulphate and Tris(hydroxymethyl)-amino-methane, but was essentially unaffected by all other salts and buffer combinations tested. The immunoturbidimetric assays described here are generically applicable to polyclonal...... antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation....

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

    Science.gov (United States)

    Sheng, Jian-Wu; He, Miao; Shi, Han-Chang; Qian, Yi

    2006-07-21

    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 microg L(-1), the 50% inhibition concentration (IC50) for MC-LR was 0.63+/-0.06 microg L(-1) and the quantitative detection range was from 0.17 to 2.32 microg 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.

  14. Development of polyclonal antibodies for the detection of Tribolium castaneum contamination in wheat grain.

    Science.gov (United States)

    Krizkova-Kudlikova, Iva; Hubert, Jan

    2008-09-10

    Two polyclonal antibodies (Pab) were developed for the detection of Tribolium castaneum, which is a stored product pest of medical and economical importance. Selected Pab anti- T. castaneumK51 showed low cross-reactivity to other stored product arthropods but revealed high reactivity to T. destructor, T. confusum, and partly to Tenebrio molitor. PTA-ELISA was used to detect adults, larva, and feces of T. castaneum in artificially contaminated grain samples. Calibration methods were applied to determine detection limits for each type of contaminants. Anti- T.castaneumK51 enabled detection of T. castaneum in grain samples; detection limits reached 60 and 640 individuals/kg of grain for larvae and adults, respectively, and 4 mg of feces/kg of grain. After recalculation, the detection limit for feces enables detection of 30 larvae after 5 days of feeding in optimal conditions. The main advantage of the developed assay is traceability of T. castaneum contamination, especially when the adults and larvae are removed from contaminated material, based on the detection of feces that persist in the grain.

  15. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16.

    Science.gov (United States)

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

    2015-03-01

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

  16. Characterization of Polyclonal Antibody Induced by Autoantibody TPO (Thyroidperoxidase) From Autoimmune Thyroid Disease (AITD) Serum with ELISA and Western Blotting

    OpenAIRE

    Maulidya Aulia Fiqriyana; Aulanni'am Aulanni'am; Anna Roosdiana

    2013-01-01

    Autoantibody TPO is a potential marker for early detection of autoimmune thyroid disease (AITD). Autoantibody TPO has a specifity and a sensitivity ranging from 82% to100% in comparison to other AITD serology markers. Concentration of autoantibody TPO in sera had a positive correlation with activities of chronic AITD. This research have been conducted to investigate the characteristic of polyclonal antibody TPO induced by autoantibody TPO from serum of AITD patients. The autoantibody TPO was ...

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

    Directory of Open Access Journals (Sweden)

    Z Meshkat

    2008-12-01

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

  18. Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis.

    Science.gov (United States)

    Xu, Chuang; Chen, Yuanyuan; Chang, Qiaocheng; Xia, Cheng; Yang, Wei; Zhang, Hongyou

    2015-08-01

    MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28a-MAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDS-PAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund's adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis.

  19. Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.

    Science.gov (United States)

    Finkelsztein, Eli J; Diaz-Soto, Juan C; Vargas-Zambrano, Juan C; Suesca, Elizabeth; Guzmán, Fanny; López, Manuel C; Thomas, M Carmen; Forero-Shelton, Manu; Cuellar, Adriana; Puerta, Concepción J; González, John M

    2015-03-01

    Trypanosoma cruzi's trypomastigotes are highly active and their incessant motility seems to be important for mammalian host cell infection. The kinetoplastid membrane protein-11 (KMP-11) is a protein expressed in all parasite stages, which induces a cellular and humoral immune response in the infected host, and is hypothesized to participate in the parasite's motility. An N-terminal peptide from KMP-11, termed K1 or TcTLE, induced polyclonal antibodies that inhibit parasitic invasion of Vero cells. The goal of this study was to evaluate the motility and infectivity of T. cruzi when exposed to polyclonal anti-TcTLE antibodies. Rabbits were immunized with TcTLE peptide along with FIS peptide as an immunomodulator. ELISA assay results showed that post-immunization sera contained high titers of polyclonal anti-TcTLE antibodies, which were also reactive against the native KMP-11 protein and live parasites as detected by immunofluorescence and flow cytometry assays. Trypomastigotes of T. cruzi were incubated with pre- or post-immunization sera, and infectivity to human astrocytes was assessed by Giemsa staining/light microscope and flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled parasites. T. cruzi infection in astrocytes decreased approximately by 30% upon incubation with post-immunization sera compared with pre-immunization sera. Furthermore, trypomastigotes were recorded by video microscopy and the parasite's flagellar speed was calculated by tracking the flagella. Trypomastigotes exposed to post-immunization sera had qualitative alterations in motility and significantly slower flagella (45.5 µm/s), compared with those exposed to pre-immunization sera (69.2 µm/s). In summary, polyclonal anti-TcTLE serum significantly reduced the parasite's flagellar speed and cell infectivity. These findings support that KMP-11 could be important for parasite motility, and that by targeting its N-terminal peptide infectivity can be reduced.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sheng Jianwu [Environmental Simulation and Pollution Control (ESPC), State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084 (China); He Miao [Environmental Simulation and Pollution Control (ESPC), State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084 (China)]. E-mail: hemiao@tsinghua.edu.cn; Shi Hanchang [Environmental Simulation and Pollution Control (ESPC), State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084 (China); Qian Yi [Environmental Simulation and Pollution Control (ESPC), State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084 (China)

    2006-07-21

    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 {mu}g L{sup -1}, the 50% inhibition concentration (IC{sub 50}) for MC-LR was 0.63 {+-} 0.06 {mu}g L{sup -1} and the quantitative detection range was from 0.17 to 2.32 {mu}g L{sup -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.

  1. Production of Polyclonal Antibody of Morphine and Determination of Morphine in Urine by Capillary Electrophoresis Immunoassay with Laser-induced Fluorescence Detection

    Institute of Scientific and Technical Information of China (English)

    Jian Qiu MI; Xiao Hua QI; Xin Xiang ZHANG; Wen Bao CHANG

    2004-01-01

    N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals.With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine the free morphine in urine of abusers.The detection limit was calculated to be 40 ng/mL.Simulated urine samples were analyzed with good recoveries, which showed the feasibility of its application in specific morphine determination in urine of morphine abusers.

  2. 抗莱克多巴胺多克隆抗体的制备%Preparation for Ractopamine Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    张红琳; 陈昌云; 周红霞; 于小莲; 杨剑波; 石国庆

    2011-01-01

    为了制备抗莱克多巴胺多克隆抗体,试验通过连接剂butane-1,4-diol diglyciydyl ether把莱克多巴胺和载体蛋白BSA和0VA偶联成免疫抗原和包被抗原,免疫新西兰大白兔,饱和硫铵沉淀方法纯化抗体,间接ELISA法检测抗血清效价.结果通过免疫兔获得了抗莱克多巴胺的多克隆抗体.经ELISA测定,其效价为1:12800.%The RCT (Ractopamine) was coupled with BSA and OVA to produce immunogen and envelope antigen by using coupling agent butane-l, 4-diol diglyciydyl ether, respectively. The polyclonal antibodies was obtained by immuning New Zealand rabbits with BSA-RCT and purified by saturated ammonium sulfate precipitation. The indirect ELISA method was used to detect its antiserum titer. The results showed that the polyclonal antibodies against RCT had been obtained from immunized rabbits and the titer of the polyclonal antiserum was 1:12 800.

  3. 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.

  4. Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Yi-shu YANG; Ze-lin LI; Yi ZENG

    2008-01-01

    To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 Vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E. coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

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

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

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

  6. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    Science.gov (United States)

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  7. Production of anti-fullerene C60 polyclonal antibodies and study of their interaction with a conjugated form of fullerene

    Science.gov (United States)

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

    2011-09-01

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

  8. Generation of polyclonal catalytic antibodies against cocaine using transition state analogs of cocaine conjugated to diphtheria toxoid.

    Science.gov (United States)

    Basmadjian, G P; Singh, S; Sastrodjojo, B; Smith, B T; Avor, K S; Chang, F; Mills, S L; Seale, T W

    1995-11-01

    Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenylphosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA-I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and titer of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.

  9. Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purification, and preparation of a polyclonal antibody.

    Science.gov (United States)

    Zheng, H L; Li, H; Sun, Y S; Yang, Z Y; Yu, Q

    2014-08-25

    Parathyroid hormone-related peptide (PTHrP) plays important roles in promoting cancer occurrence and in the development of bone metastases. To increase our knowledge of the biological functions of PTHrP, the prokaryotic expression vector pET-PTHrP was successfully constructed and the His-PTHrP fusion protein was expressed in Escherichia coli. Anti-PTHrP polyclonal antibody was then prepared from rabbits. Finally, the goat tissue expression profile of PTHrP was analyzed by Western blot with the anti-PTHrP polyclonal antibody. The results showed that the expression of PTHrP in goat mammary glands was significantly higher than that in other organs. This indicates that PTHrP may play important roles in the goat mammary gland. The antibody prepared will be a useful tool for detecting PTHrP and will be valuable in future studies investigating the role of PTHrP in calcium metabolism in the goat model.

  10. Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

    Institute of Scientific and Technical Information of China (English)

    WANG,Wen-Jun; LING,Yun; XU,Ting; GAO,Hong-Bin; SHENG,Wei; LI,Ji

    2007-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on polyclonal antibody for the estrogen diethylstilbestrol (DES) was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol (DES-CP) and mono-O-carboxymethyldiethylstilbestrol (DES-CM) with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carrier proteins bovine serum albumin (BSA) by mixed-anhydride method for immunogen and conjugated with ovalbumin (OVA) by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection (LLD) of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.

  11. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    Xiao-quan Li; Shu-lin Zhang; Li-hua Zhong; Jun Cheng; Yuan Hong; Meng-dong Lan; Xiao-bin Chen; Cheng-fu Sun

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

  12. Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    A.J.A. Barbosa

    1991-03-01

    Full Text Available The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160, whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.O método daperoxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquimicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixação. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana ámazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5 µm de espessura. Os antígenos de T. cruzi foram corados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluições entre 1:1.000 e 1:2.000. Diferentemente, os antígenos dç Leishmania foram revelados pelos soros anti- Leishmania somente em baixas diluições, ou seja, entre 1:60 e 1:160 enquanto que os soros

  13. Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Hiroaki Matsushita; Akiko Sano; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/− , bIGHM...

  14. Triple Immunoglobulin Gene Knockout Transchromosomic (Tc) Cattle: Bovine Lambda Cluster Deletion and its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Matsushita, H.; Sano, A.; Wu, H.; J. Jiao; Kasinathan, P.; Sullivan, E. J.; Wang, Zhongde; Kuroiwa, K

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML...

  15. Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein.

    Science.gov (United States)

    Sharma, Susheel Kumar; Kumar, P Vignesh; Baranwal, Virendra Kumar

    2014-10-01

    A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

  16. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

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

    Science.gov (United States)

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

    2017-05-01

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

  18. Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

    Directory of Open Access Journals (Sweden)

    Monica Moro

    Full Text Available The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs. We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1 the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.

  19. 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.

  20. Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins.

    Science.gov (United States)

    Arnaoty, Ahmed; Gouilleux-Gruart, Valérie; Casteret, Sophie; Pitard, Bruno; Bigot, Yves; Lecomte, Thierry

    2013-08-01

    The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

  1. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.

    Science.gov (United States)

    Chen, Yujian; Liu, Yong; Guo, Jiayu; Tang, Tao; Gao, Jian; Huang, Tao; Wang, Bin; Liu, Shaojun

    2016-06-17

    Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.

  2. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    Science.gov (United States)

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K.; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A.; Carroll, Miles W.

    2016-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  3. Immunological cross-reactivity and neutralisation of European viper venoms with the monospecific Vipera berus antivenom ViperaTAb.

    Science.gov (United States)

    Casewell, Nicholas R; Al-Abdulla, Ibrahim; Smith, David; Coxon, Ruth; Landon, John

    2014-08-19

    Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom, ViperaTAb®, to cross-react with, and neutralise lethality induced by, a variety of European vipers. Using ELISA and immunoblotting, we find that ViperaTAb® antibodies recognise and bind to the majority of toxic components found in the venoms of the Vipera species tested at comparably high levels to those observed with V. berus. Using in vivo pre-clinical efficacy studies, we demonstrate that ViperaTAb® effectively neutralises lethality induced by V. berus, V. aspis, V. ammodytes and V. latastei venoms and at much higher levels than those outlined by regulatory pharmacopoeial guidelines. Notably, venom neutralisation was found to be superior to (V. berus, V. aspis and V. latastei), or as equally effective as (V. ammodytes), the monospecific V. ammodytes "Zagreb antivenom", which has long been successfully used for treating European snake envenomings. This study suggests that ViperaTAb® may be a valuable therapeutic product for treating snakebite by a variety of European vipers found throughout the continent.

  4. Immunological Cross-Reactivity and Neutralisation of European Viper Venoms with the Monospecific Vipera berus Antivenom ViperaTAb

    Directory of Open Access Journals (Sweden)

    Nicholas R. Casewell

    2014-08-01

    Full Text Available Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom, ViperaTAb®, to cross-react with, and neutralise lethality induced by, a variety of European vipers. Using ELISA and immunoblotting, we find that ViperaTAb® antibodies recognise and bind to the majority of toxic components found in the venoms of the Vipera species tested at comparably high levels to those observed with V. berus. Using in vivo pre-clinical efficacy studies, we demonstrate that ViperaTAb® effectively neutralises lethality induced by V. berus, V. aspis, V. ammodytes and V. latastei venoms and at much higher levels than those outlined by regulatory pharmacopoeial guidelines. Notably, venom neutralisation was found to be superior to (V. berus, V. aspis and V. latastei, or as equally effective as (V. ammodytes, the monospecific V. ammodytes “Zagreb antivenom”, which has long been successfully used for treating European snake envenomings. This study suggests that ViperaTAb® may be a valuable therapeutic product for treating snakebite by a variety of European vipers found throughout the continent.

  5. Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    P. Zhang

    2008-06-01

    Full Text Available Mouse PNAS-4 (mPNAS-4 has 96% identity with human PNAS-4 (hPNAS-4 in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data, it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2 and colon carcinoma cells (CT26 of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3. The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4 was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Koji, T. (Nagasaki Univ. (Japan). School of Medicine)

    1982-09-01

    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 dealing 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.

  10. Preparation of Polyclonal Antibodies of Rubisco Large and Small Subunits and Their Application in the Functional Analysis of the Genes

    Institute of Scientific and Technical Information of China (English)

    Peng-Da MA; Tian-Cheng LU; Xiao-Fu ZHOU; Xiao-Juan ZHU; Xing-Zhi WANG

    2004-01-01

    Spinach Rubisco (ribulose-l,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 μg antigens were used and 0.3 ml anti-sera with titer of 1:5000were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthaniana was performed. The expression level ofrbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.

  11. Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

    Science.gov (United States)

    Guo, Ting-Ting; Su, Juan; Ma, Zhi-Yu; Ma, Jun-Xiao; Jin, Meng-Meng; Li, Xiang; Lei, Zhi-Hai

    2015-06-10

    Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.

  12. AID activity in B cells strongly correlates with polyclonal antibody affinity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans.

    Science.gov (United States)

    Khurana, Surender; Frasca, Daniela; Blomberg, Bonnie; Golding, Hana

    2012-09-01

    The role of Activation-Induced Cytidine Deaminase (AID) in somatic hypermutation and polyclonal antibody affinity maturation has not been shown for polyclonal responses in humans. We investigated whether AID induction in human B cells following H1N1pdm09 vaccination correlated with in-vivo antibody affinity maturation against hemagglutinin domains in plasma of young and elderly individuals. AID was measured by qPCR in B cells from individuals of different ages immunized with the H1N1pdm09 influenza vaccine. Polyclonal antibody affinity in human plasma for the HA1 and HA2 domains of the H1N1pdm09 hemagglutinin was measured by antibody-antigen complex dissociation rates using real time kinetics in Surface Plasmon Resonance. Results show an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination. Levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlated with increase in polyclonal antibody affinity to the HA1 globular domain (but not to the conserved HA2 stalk). In the younger population, significant affinity maturation to the HA1 globular domain was observed, which associated with initial levels of AID and fold-increase in AID after vaccination. In some older individuals (>65 yr), higher affinity to the HA1 domain was observed before vaccination and H1N1pdm09 vaccination resulted in minimal change in antibody affinity, which correlated with low AID induction in this age group. These findings demonstrate for the first time a strong correlation between AID induction and in-vivo antibody affinity maturation in humans. The ability to generate high affinity antibodies could have significant impact on the elucidation of age-specific antibody responses following vaccination and eventual clinical efficacy and disease outcome.

  13. Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor.

    Science.gov (United States)

    Allard, Bertrand; Priam, Fabienne; Deshayes, Frédérique; Ducancel, Frédéric; Boquet, Didier; Wijkhuisen, Anne; Couraud, Jean-Yves

    2011-09-01

    Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.

  14. 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 seru

  15. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    OpenAIRE

    Laman, J. D.; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J. P.; Claassen, E.; Boersma, W J

    1992-01-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The ai...

  16. An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum.

    Science.gov (United States)

    Chhatre, Sunil; Francis, Richard; Bracewell, Daniel G; Titchener-Hooker, Nigel J

    2010-11-15

    The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.

  17. Preparation of a polyclonal antibody that recognizes a unique galactoseβ1-4fucose disaccharide epitope.

    Science.gov (United States)

    Takeuchi, Tomoharu; Nishiyama, Kazusa; Saito, Saori; Tamura, Mayumi; Fuwa, Takashi J; Nishihara, Shoko; Takahashi, Hideyo; Natsugari, Hideaki; Arata, Yoichiro; Kasai, Ken-ichi

    2015-08-14

    Galactoseβ1-4fucose (Galβ1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as a non-self glycomarker. In order to clarify its biological function, we made a polyclonal antibody using (Galβ1-4Fuc)n-BSA as the antigen, which was prepared by crosslinking Galβ1-4Fuc-O-(CH2)2-SH and BSA. The binding specificity of the antibody was analyzed by frontal affinity chromatography, and it was confirmed that it recognizes naturally occurring N-glycans containing the Galβ1-4Fuc unit linked to the reducing-end GlcNAc via α1-6 linkage. By western blotting analysis, the antibody was also found to bind to (Galβ1-4Fuc)n-BSA but not to BSA or asialofetuin, which has N-glycan chains containing Galβ1-4GlcNAc. Western blotting experiments also revealed presence of stained proteins in crude extracts of C. elegans, the parasitic nematode Ascaris suum, and the allergenic mite Dermatophagoides pteronyssinus, while those from Drosophila melanogaster, Mus musculus, and the allergenic mites Dermatophagoides farinae and Tyrophagus putrescentiae were negative. This antibody should be a very useful tool for research on the distribution of the Galβ1-4Fuc disaccharide unit in glycans in a wide range of organisms.

  18. 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

  19. DNA Vaccine-Generated Duck Polyclonal Antibodies as a Postexposure Prophylactic to Prevent Hantavirus Pulmonary Syndrome (HPS)

    Science.gov (United States)

    Brocato, Rebecca; Josleyn, Matthew; Ballantyne, John; Vial, Pablo; Hooper, Jay W.

    2012-01-01

    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 is the

  20. [Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody].

    Science.gov (United States)

    Bao, Yong-Mei; Liu, Yong-Hui; Xu, Dong-Qing; Huang, Ji; Wang, Zhou-Fei; Wang, Jian-Fei; Zhang, Hong-Sheng

    2010-09-01

    Membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl- maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins. OsNPSN11 is a member of Qb-SNARE gene family isolate from rice. The cDNA of OsNPSN11 was subcloned into pET-30a and fusion to the 6 × His tag. Induced by 0.5 mmol/L IPTG for four hours, the recombinant protein was highly expressed in Escherichia coli, which was purified by Ni2+ -NTA His-bind resin affinity chromatography column to be used as an antigen to raise the antibody in New Zealand rabbits. Western blotting analysis showed that the antibody can specifically recognize the expressed antigen and the OsNPSN11 in plasma membrane protein from various rice tissues. This indicated that the antibody can be used for expres-sion analysis in transgenic rice.

  1. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  2. Expression, purification and polyclonal antibody generation of p23,an Hsp90 cochaperone, in the amphioxus Branchiostoma belcheri

    Institute of Scientific and Technical Information of China (English)

    ZHAO Bosheng; ZHANG Shicui; PANG Qiuxiang; LIU Zhenhui; LIANG Yujun

    2006-01-01

    The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX-6P-1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, polyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST-p23 from induced E. coli cells, purified GST-p23 and p23 protein, but also reacted with the total protein extracted from the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.

  3. ANTIBODY POLYCLONAL PRODUCTION ON RABBIT ANTI-OVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN (Rabbit anti-ovPAG

    Directory of Open Access Journals (Sweden)

    E.T. Setiatin

    2014-10-01

    Full Text Available The aim of the study was to produce polyclonal antibody (rabbit anti-ovPAG which could detectPAG in the urine of pregnant ewes. Twelve rabbits were immunized against ovPG DEAE-TrisHCl (DT,DEAE-NaCl 20mM (DN2, DEAE-NaCl 40mM (DN4, DEAE-NaCl 80mM (DN8, DEAE-NaCl160mM (DN16, DEAE-NaCl 320mM (DN32 and DEAE-NaCl 1M (DN1 and NaCl 0.9 % as aplacebo. The 0.5 ml of isolate (purified from ovine cotyledon was emulsified in equal volume withcomplete and incomplete Freud’s adjuvant. The mixture of each isolate and adjuvant was injected atmutiple sites along the dorsal area of rabbits by subcutaneous route. Blood were collected from marginalear vein, starting before first injection (baseline and every 14 days. Rabbit anti-ovPAG were measuredusing Modified ELISA Technique. By using Western Blot Technique, DN32 showed the best immuneresponse among others and also could differenciate ovPAG in the urine of pregnant ewes It could beconcluded that ovPAG DN32 is a specific source of rabbit anti-ovPAG production. Protein of ovPAG atmolecular weight 31 kDa is a pregnancy protein marker of garut sheep and could be developed as amajor protein for producing antibodi.

  4. Inhibition of Staphylococcus epidermidis biofilm formation by rabbit polyclonal antibodies against the SesC protein.

    NARCIS (Netherlands)

    Shahrooei, M.; Hira, V.; Stijlemans, B.; Merckx, R.; Hermans, P.W.M.; Eldere, J. van

    2009-01-01

    Several well-studied proteins with defined roles in Staphylococcus epidermidis biofilm formation are LPXTG motif-containing proteins. Here, we investigate the possible use of the LPXTG motif-containing protein SesC (S. epidermidis surface protein C; accession no. NP_765787) as a target for antibodie

  5. Polyclonal and Specific Antibodies Mediate Protective Immunity against Enteric Helminth Infection

    NARCIS (Netherlands)

    McCoy, Kathy D.; Stoel, Maaike; Stettler, Rebecca; Merky, Patrick; Fink, Katja; Senn, Beatrice M.; Schaer, Corinne; Massacand, Joanna; Oderrnatt, Bernhard; Oettgen, Hans C.; Zinkernagel, Rolf M.; Bos, Nicolaas A.; Hengartner, Hans; Macpherson, Andrew J.; Harris, Nicola L.

    2008-01-01

    Anti-helminth immunity involves CD4(+) T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasit

  6. Human antibody responses to the polyclonal Dryvax vaccine for smallpox prevention can be distinguished from responses to the monoclonal replacement vaccine ACAM2000.

    Science.gov (United States)

    Pugh, Christine; Keasey, Sarah; Korman, Lawrence; Pittman, Phillip R; Ulrich, Robert G

    2014-06-01

    Dryvax (Wyeth Laboratories, Inc., Marietta, PA) is representative of the vaccinia virus preparations that were previously used for preventing smallpox. While Dryvax was highly effective, the national supply stocks were depleted, and there were manufacturing concerns regarding sterility and the clonal heterogeneity of the vaccine. ACAM2000 (Acambis, Inc./Sanofi-Pasteur Biologics Co., Cambridge, MA), a single-plaque-purified vaccinia virus derivative of Dryvax, recently replaced the polyclonal smallpox vaccine for use in the United States. A substantial amount of sequence heterogeneity exists within the polyclonal proteome of Dryvax, including proteins that are missing from ACAM2000. Reasoning that a detailed comparison of antibody responses to the polyclonal and monoclonal vaccines may be useful for identifying unique properties of each antibody response, we utilized a protein microarray comprised of approximately 94% of the vaccinia poxvirus proteome (245 proteins) to measure protein-specific antibody responses of 71 individuals receiving a single vaccination with ACAM2000 or Dryvax. We observed robust antibody responses to 21 poxvirus proteins in vaccinated individuals, including 11 proteins that distinguished Dryvax responses from ACAM2000. Analysis of protein sequences from Dryvax clones revealed amino acid level differences in these 11 antigenic proteins and suggested that sequence variation and clonal heterogeneity may contribute to the observed differences between Dryvax and ACAM2000 antibody responses.

  7. Preparation and specificity analysis of sildenafil polyclonal antibody%西地那非多克隆抗体的制备及特异性分析

    Institute of Scientific and Technical Information of China (English)

    熊波; 吴胜泽; 苏焕斌; 张燕; 刘辉

    2015-01-01

    目的:为了研究开发一种具有高灵敏度、高特异性的西地那非(sildenafil, SD)多克隆抗体。方法本文通过人工合成西地那非半抗原(SDH)和西地那非抗原(SDH-BSA),然后经免疫兔子,制备了 SD 多克隆抗体,并对制得的抗体的特异性进行了分析。结果免疫后产生了SD多克隆抗体,抗体的效价为3.8×105,线性检测范围为0.02~3.8 ng/mL,抗体除与瓦地那非具有较高的交叉反应率外,与红地那非、他地那非等结构类似物交叉反应率均较低。结论采用本方法制备的SD多克隆抗体具有较高的特异性和灵敏度,可用于SD快速检测试剂盒的开发。%Objective To prepare high sensitivity and specificity polyclonal antibody against sildenafil (SD). Methods Sildenafil hapten (SDH) and Sildenafil antigen (SDH-BSA) were prepared by artificial methods. Then, through immunized rabbits the SD polyclonal antibody was prepared. Results After immunity, the polyclonal antibody against SD was generated. The titer of the antibody was 3.8×105. The linear detection of the polyclonal antibody was ranged from 0.02 ng/mL to 3.8 ng/mL. The polyclonal antibody had a high cross reaction rate to vardenafil, but had a low response rate to hongdenafil and calais. Conclusion SD polyclonal antibody had a high specificity and sensitivity, and it could be applied to the development of SD rapid detection kit.

  8. 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

    We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immumoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rP...... 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....

  9. 单克隆抗体与多克隆抗体配对ELISA方法比较%Study of the Optimum Pairing of Polyclonal Antibodies and Monoclonal Antibodies with Sandwich ELISA

    Institute of Scientific and Technical Information of China (English)

    张小兵; 邸禄芹; 吴萌; 闫静辉

    2009-01-01

    Using human chorionic gonadotrophin( HCG) as the antigen,polyclonal antibodies and monoclonal antibodies(McAbs) specific for HCG were generated, characterized and highly purified. Then these antibodies were labeled with horseradish peroxidase (HRP). By a double antibody sandwich enzyme-linked immunosorhent assay, several problems of the optimum pairing of polyclonal antibodies and monoclonal antibodies were discussed. The results showed that with monoclonal antibody as trapping antibody and polyclonal antibody labeling with enzyme as testing antibody which should be diluted with animal sera as diluent,the sandwich ELISA had a highly specificity and sensitivity to detect antigen.%以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.

  10. 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.

  11. Expression, purification of recombinant human mitochondrial transcription termination factor 3 (hMTERF3) and preparation of polyclonal antibody against hMTERF3.

    Science.gov (United States)

    Xiong, Wei; Luo, Yonghui; Zhang, Cuixiang; Tan, Deyong; Zuo, Shaoyuan

    2012-08-01

    In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.

  12. 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.

  13. 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.

  14. Efficacy of the Rabbit Polyclonal Anti-leptospira Antibody against Homotype or Heterotype Leptospira Infection in Hamster

    Science.gov (United States)

    Ding, Zhuang; Wang, Hai; Wu, Dianjun; Xie, Xufeng; Lin, Tao; Fu, Yunhe; Zhang, Naisheng; Cao, Yongguo

    2016-01-01

    Leptospirosis, caused by Leptospira, is one of the most important of neglected emerging zoonotic diseases that has important impacts on public health worldwide. Polyclonal antibody (pcAb) therapy is a potential method to process a series of pathogens for which there are limited determination of treatment, such as leptospirosis. First, we evaluated the efficacy of pcAb, derived from the sera of rabbits inoculated with Leptospira, against homotype (Leptospira interrogans serovar Lai) or heterotype (Leptospira interrogans serovar Autumnalis) Leptospira infection in a lethal hamster model. The pcAb treatment improved survival compared to the controls. The histopathology’s of the infected kidney, liver and lung were also examined by hematoxylin and eosin staining. Using real-time quantitative PCR, we determined that most of the leptospires in the primary organs were almost completely removed by pcAb. In the second experiment, treatments, including antibiotic, pcAb, and combination, were started immediately after occurrence of the first serious sickness mouse in any group. No significant difference in survival rate between pcAb group and antibiotic group was found, but the combination therapy group significantly improved survival rate compared to the others (Phamster, and combination therapy improved survival compared to antibiotic group in the late treatment of homotype leptospirosis. PMID:28027297

  15. Evolutionarily Successful Asian 1 Dengue Virus 2 Lineages Contain One Substitution in Envelope That Increases Sensitivity to Polyclonal Antibody Neutralization.

    Science.gov (United States)

    Wang, Chunling; Katzelnick, Leah C; Montoya, Magelda; Hue, Kien Duong Thi; Simmons, Cameron P; Harris, Eva

    2016-03-15

    The 4 dengue virus serotypes (DENV-1-4) cause the most prevalent mosquito-borne viral disease of humans worldwide. DENV-2 Asian 1 (A1) genotype viruses replaced the Asian-American (AA) genotype in Vietnam and Cambodia, after which A1 viruses containing Q or M at envelope (E) residue 160 became more prevalent than those with residue 160K in both countries (2008-2011). We investigated whether these substitutions conferred a fitness advantage by measuring neutralizing antibody titer against reporter virus particles (RVPs) representing AA, A1-160K, A1-160Q, and A1-160M, using patient sera from Vietnam and a well-characterized Nicaraguan cohort. Surprisingly, we found that A1-160Q and A1-160M RVPs were better neutralized by heterologous antisera than A1-160K. Despite this, Vietnamese patients infected with A1-160Q or A1-160M viruses had higher viremia levels than those infected with A1-160K. We thus found that independent lineages in Vietnam and Cambodia acquired a substitution in E that significantly increased polyclonal neutralization but nonetheless were successful in disseminating and infecting human hosts.

  16. An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody.

    Science.gov (United States)

    Cui, Jianlan; Zhang, Kun; Huang, Qiuxin; Yu, Yiyi; Peng, Xianzhi

    2011-02-28

    A specific polyclonal anti-norfloxacin antibody was obtained, and a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for determining trace amounts of norfloxacin in various waters. Good linearity was achieved in the range from 0.1 to 10 μg L(-1). The average IC(50) value was determined to be 2.2 μg L(-1) and the limit of detection was 0.016 μg L(-1) at a signal-to-noise ratio of 3 in phosphate-buffered saline buffer. Recoveries of norfloxacin at various spiking levels ranged from 74 to 105% in groundwater, surface water, treated and untreated wastewater samples, with relative standard deviations of 3-5%. The assay was applied for determining norfloxacin in municipal wastewater, surface water, and groundwater collected in a metropolis of China. Raw wastewater samples were only submitted to filtration and pH adjustment while the other water samples were pre-concentrated by solid phase extraction prior to the icELISA assay. Good agreement of the results obtained by the icELISA and liquid chromatography tandem mass spectrometry further confirmed the reliability and accuracy of the icELISA for rapid detection of norfloxacin in waters. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP (19+28) Fusion Protein

    Institute of Scientific and Technical Information of China (English)

    Qing-yu CHENG; Xiao-lin MENG; Jin-ping XU; Wei LU; Jian WANG

    2007-01-01

    We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV,using colloidal gold as an indicator.The fusion protein,VP (19+28),was expressed in E.coli,purified and used to prepare polyclonal antibodies.The purified anti-VP (19+28) IgG were conjugated with colloidal gold.Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes.After assembly,three groups (5 individual animals in each group) of shrimp samples were tested which included healthy,moribund and dead shrimps.For each group,three different tissues (body juices,gills and hepatopancreas) were tested at the same time.In parallel,all the samples were also analyzed using PCR for comparison.Out of 45 samples tested,30 were detected as positive while 15 were classified as negative.The results of LFIA correlate with those obtained by the PCR analysis,indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.

  18. Polyclonal neural cell adhesion molecule antibody prolongs the effective duration time of botulinum toxin in decreasing muscle strength.

    Science.gov (United States)

    Guo, Yan; Pan, Lizhen; Liu, Wuchao; Pan, Yougui; Nie, Zhiyu; Jin, Lingjing

    2015-11-01

    This study aimed to investigate if the effective duration time of botulinum toxin A (Btx-A) could be prolonged by polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab). 175 male SD rats were randomly divided into three major groups: control group (n = 25), Btx-A group (n = 25), and P-NCAM-Ab groups. P-NCAM-Ab groups were composed of five sub-groups, with 25 rats each in the dose-response study. Muscle strength of rat lower limbs was determined using a survey system. The expressions of muscle-specific receptor tyrosine kinase (MuSK) and neural cell adhesion molecule (NCAM) were determined by real-time polymerase chain reactions (RT-PCR) and western blotting (WB). The muscle strength was significantly decreased by Btx-A in Btx-A/P-NCAM-Ab groups compared with normal control group. Besides, the muscle strength of P-NCAM-Ab group was significantly decreased compared with the Btx-A group. The recovery time of muscle strength in P-NCAM-Ab group was significantly longer compared with Btx-A group. RT-PCR and WB assay showed that PNCAM-Ab delayed the increase of MuSK and NCAM after Btx-A injection. P-NCAM-Ab prolongs the effective duration time of Btx-A in decreasing muscle strength, which could provide a novel enhancement in clinical application.

  19. 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

  20. Conjugation of ampicillin and enrofloxacin residues with bovine serum albumin and raising of polyclonal antibodies against them

    Directory of Open Access Journals (Sweden)

    B. Sampath Kumar

    2016-04-01

    Full Text Available Aim: The aim of this study is to test the potency of bovine serum albumin (BSA conjugated ampicillin (AMP and enrofloxacin (ENR antigens in eliciting an immune response in rats using indirect competitive enzyme-linked immunosorbent assay (icELISA. Materials and Methods: AMP and ENR antibiotics were conjugated with BSA by carbodiimide reaction using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC as a cross-linker. The successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sprague-Dawley rats were immunized with the conjugates and blood samples were collected serially at 15 days time interval after first immunization plus first booster, second booster, third booster, and the fourth sampling was done 1½ month after the third booster. The antibody titres in the antisera of each antibiotic in all the four immunization cycles (ICs were determined by an icELISA at various serum dilutions ranging from 1/100 to 1/6400. Results: Analysis of antibiotic-BSA conjugates by sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining revealed high molecular weight bands of 85 kDa and 74 kDa for AMP-BSA and ENR-BSA respectively when compared to 68 kDa band of BSA. Both the antibiotic conjugates elicited a good immune response in rats but comparatively the response was more with AMP-BSA conjugate than ENR-BSA conjugate. Maximum optical density 450 value of 2.577 was recorded for AMP-BSA antisera, and 1.723 was recorded for ENR-BSA antisera at 1/100th antiserum dilution in third IC. Conclusion: AMP and ENR antibiotics proved to be good immunogens when conjugated to BSA by carbodiimide reaction with EDC as crosslinker. The polyclonal antibodies produced can be employed for detecting AMP and ENR residues in milk and urine samples.

  1. A feasible enzyme-linked immunosorbent assay system using monoclonal and polyclonal antibodies against glucosyltransferase-B from Streptococcus mutans.

    Science.gov (United States)

    Shinozaki-Kuwahara, Noriko; Hashizume-Takizawa, Tomomi; Hirasawa, Masatomo; Takada, Kazuko

    2012-06-01

    Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

  2. ANTIBODY POLYCLONAL PRODUCTION ON RABBIT ANTI-OVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN (Rabbit anti-ovPAG

    Directory of Open Access Journals (Sweden)

    E.T. Setiatin

    2011-09-01

    Full Text Available The aim of the study was to produce polyclonal antibody (rabbit anti-ovPAG which could detect PAG in the urine of pregnant ewes. Twelve rabbits were immunized against ovPG DEAE-TrisHCl (DT, DEAE-NaCl 20mM (DN2, DEAE-NaCl 40mM (DN4, DEAE-NaCl 80mM (DN8, DEAE-NaCl 160mM (DN16, DEAE-NaCl 320mM (DN32 and DEAE-NaCl 1M (DN1 and NaCl 0.9 % as a placebo. The 0.5 ml of isolate (purified from ovine cotyledon was emulsified in equal volume with complete and incomplete Freud’s adjuvant. The mixture of each isolate and adjuvant was injected at mutiple sites along the dorsal area of rabbits by subcutaneous route. Blood were collected from marginal ear vein, starting before first injection (baseline and every 14 days. Rabbit anti-ovPAG were measured using Modified ELISA Technique. By using Western Blot Technique, DN32 showed the best immune response among others and also could differenciate ovPAG in the urine of pregnant ewes It could be concluded that ovPAG DN32 is a specific source of rabbit anti-ovPAG production. Protein of ovPAG at molecular weight 31 kDa is a pregnancy protein marker of garut sheep and could be developed as a major protein for producing antibodi.

  3. 3个重要酶的多克隆抗体的制备条件优化%Preparation Optimization of Polyclonal Antibodies Against Three Important Enzymes

    Institute of Scientific and Technical Information of China (English)

    吴丽民; 刘美龙; 胡巧云; 侯巧玲

    2014-01-01

    ABSTRACT:OBJECTIVE To prepare high-quanlity polyclonal antibody against chitinase,α-mannosidase and GST.METHODS Corresponding purified proteins with different doses were used to immunize the New Zealand rab- bits of different ages.RESULTS When using 0.3mg· L-1 purified proteins to immunize six-month-old New Zeal-and rabbit,the polyclonal antibodies possessed higher titer and specificity.CONCLUSION Our results establish a dependable basis for the large-scale production of the related polyclonal antibodies.%目的:制备优质的抗几丁质酶、α-甘露糖苷酶、谷胱甘肽转硫酶的多克隆抗体。方法利用纯化的不同免疫剂量的3种酶分别免疫不同兔龄的新西兰兔。结果使用0.3mg纯化蛋白、免疫6月龄的新西兰兔所制备出的抗体效价高、特异性好。结论这一结果为今后规模化制备相关多克隆抗体奠定了坚实的实验基础。

  4. Amplification and polyclonal antibody preparation of M13 phage display library%M13噬菌体肽库扩增及兔抗血清制备

    Institute of Scientific and Technical Information of China (English)

    任晓峰; 马晓微

    2013-01-01

    为制备M13噬菌体多克隆抗体,将噬菌体十二肽原始文库进行大量扩增,作为免疫原制备兔抗M13的多克隆抗体血清并进行间接ELISA鉴定.结果表明,该多抗效价达1∶1 048 576,说明此多抗可与扩增噬菌体文库发生很好的抗原抗体反应.将制备的噬菌体M13多克隆抗体与商业化M13抗体水平比较,制备多抗与商业化M13抗体效果相当.本研究成功制备兔抗M13噬菌体多克隆抗体,为深入研究噬菌体展示技术提供了材料.%In order to generate polyclonal antibodies of the M13 Phage,one M13 of Ph..D.-12TM Phage Display Peptide Library was amplified and used to immunize a rabbit to generate polyclonal antibody.Indirect ELISA analysis showed that the titer of the polyclonal antibody was approximately 1∶1 048 576,showing that the anti-M13 antibody had a high titer.Compared this polyclonal antibody with a Rabbit polyclonal antibody (Rb pAb) to M13 Bacteriophage Coat Proteins (commercialization),the binding activity of the produced polyclonal antibody to the identical target was as good as that of the Rb pAb to M13 Bacteriophage Coat Proteins.In the study,polyclonal antibody to this M13 of phage library was successfully generated and such phage polyclonal antibody is important material for functional analysis with phage.

  5. 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.

  6. An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT™ is functionally superior to Freund's adjuvant.

    Science.gov (United States)

    Stevens, Natalie E; Fraser, Cara K; Alsharifi, Mohammed; Brown, Michael P; Diener, Kerrilyn R; Hayball, John D

    2013-01-01

    Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund's adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund's adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.

  7. Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

    Institute of Scientific and Technical Information of China (English)

    Hao Chanjuan; Xu Chenggang; Zhang Zhiyun; Liang Aihua

    2008-01-01

    An insect excitatory toxin gene from Buthus martensii Karsch (BmKIT) was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Escherichia coli BL21 (DE3) host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.

  8. Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk

    Institute of Scientific and Technical Information of China (English)

    Guo-ying FAN; Ruo-song YANG; Jin-qing JIANG; Xin-yao CHANG; Jun-jie CHEN; Yong-hua QI; Shi-xiu WU; Xue-feng YANG

    2012-01-01

    Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay (icELISA)standard curve was established.This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml,with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml,respectively.The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested,and the IC50 values to enoxacin,ciprofloxacin,and pefloxacin were 3.1,3.4,and 4.1 ng/ml,respectively.It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%.When spiked in milk at 5,20,and 50 ng/ml,the recoveries for NOR,enoxacin,ciprofloxacin,and pefloxacin ranged 90.5%-98.0%,84.0%-95.2%,94.0%-106.0%,and 89.5%-100.0%,respectively.The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

  9. Effects of the Sheep Polyclonal Antibodies Against the Porcine Adipocyte Plasma Membrane Proteins on Porcine Carcass Composition and Meat Quality

    Institute of Scientific and Technical Information of China (English)

    GAO Shi-zheng; HU Hong-mei; LIU Ling-yun; ZHANG Xi; LIU Yong-gang; GE Chang-rong

    2007-01-01

    To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin (ASIg) or sheep nonimmune serum immunoglobulin (NSIg). At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lung weight, dressing percentage,and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate,cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH,meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.

  10. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

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    Hiroaki Matsushita

    Full Text Available Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs in transchromosomic (Tc cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin (Ig heavy-chain (hIGH, kappa-chain (hIGK, and lambda-chain (hIGL germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO. However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK and lambda-chain (bIGL genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ. To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J and constant (C gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5 by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ.

  11. 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).

  12. Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay.

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    Hong-En Lin

    2012-01-01

    Full Text Available BACKGROUND: The envelope (E protein of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217 at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. CONCLUSIONS/SIGNIFICANCE: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.

  13. Relative reactivity of HIV-1 polyclonal plasma antibodies directed to V3 and MPER regions suggests immunodominance of V3 over MPER and dependence of high anti-V3 antibody titers on virus persistence.

    Science.gov (United States)

    Andrabi, Raiees; Choudhary, Alok K; Bala, Manju; Kalra, Rajesh; Prakash, S S; Pandey, R M; Luthra, Kalpana

    2011-10-01

    Antibodies to two crucial regions, the third variable loop (V3) of gp120 and the membrane-proximal external region (MPER) of gp41 are important for HIV-1 neutralization. We here evaluated the relative binding of polyclonal plasma antibodies from 99 HIV-1-infected individuals from India to the consensus-C V3 and MPER peptides and observed immunodominance of V3 over MPER (p antibody correlates with clinical parameters. Our results revealed that anti-V3 antibody titers are significantly lower in patients on ART compared to drug-naive individuals (p antibodies are dependent on persistence of virus in circulation, while antibodies to MPER are probably not.

  14. Generation and Identifi cation of a Polyclonal Antibody to Matripase-2%Matriptase-2多克隆抗体的制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    杨剑峰; 赵赟霄; 左斌; 何杨

    2014-01-01

    Objective To investigate the expression of Matriptase-2 recombinant protein inE.coli and the generation of its polyclonal antibody.Methods Extracellular region (Glu81-Gly228) proximal to transmembrane domain of Matriptase-2 was cloned into pMAL-C2X. The recombinant plasmid was transformed to BL21 (DE3) bacteria. After IPTG induction, the total protein was extracted and the recombinant Matriptase-2 protein was purifi ed with amylose resin. The Matriptase-2 recombinant protein was used to immunize New Zealand Rabbits to prepare serum with anti-Matriptase-2 polyclonal antibody. The antibody was purifi ed from serum by protein G sepharose 4B.Results Recombinant Matriptase-2 protein was successfully expressed inE.coliand anti-serum with high titer was obtained. Western blotting result showed that the antibody specifi cally recognized Matriptase-2. Conclusion It is concluded that the anti-Matriptase-2 polyclonal antibody with high titer and specificity is successfully generated. The antibody provides an useful experimental tool to investigate the biological function of Matriptase-2.%目的:探讨Matriptase-2重组蛋白的表达及多克隆抗体制备。方法 Matriptase-2胞外区近跨膜段(Glu81-Gly228)克隆入pMAL-C2X,重组质粒转化BL21(DE3)大肠杆菌,经IPTG诱导表达,提取细菌蛋白后用直链淀粉树脂纯化柱进行纯化。将纯化后的融合蛋白免疫新西兰大白兔,制备Matriptase-2抗血清;免疫血清用protein G亲和层析柱进行纯化,获得多克隆抗体。结果成功表达了Matriptase-2重组融合蛋白,获得了高效价的抗血清,Western blot结果显示具有抗原特异性识别。结论成功制备了高特异性、高效价的抗人Matriptase-2多克隆抗体,为今后深入研究Matriptase-2的生物学功能提供了有用的实验工具。

  15. Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009 hemagglutinin conserved domain (HA2: brief report

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    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.

  16. Preparation and Application of Polyclonal Antibody with a Peptide EDS1%EDS1多肽抗体的制备和应用

    Institute of Scientific and Technical Information of China (English)

    陈卓; 宋宝安; 刘开兴; 于丹丹; 刘家驹; 王贞超; 李向阳; 毕亮; 胡德禹; 杨松

    2011-01-01

    EDS1 is an important protein with regulation function at SA signal transduction pathway at plant host. In order to acquire polyclonal antibody of high titer and specificity against enhanced disease susceptibility 1 (EDS1), according to the primary structure of information about EDS1 in NCBI GenBank, 3 polypeptides with sequence-specific was obtained by using bioinformatics software contained Blastn,Blastx and Expasy. One polypeptide was synthetized by fmoc solid phase synthesis methods, and determined theirs purity and molecular weight by using HPLC and GC-MS, purity value reaching at 89.37% and molecular weight being at 1978.33. The polypeptide was coupled to keyhole limpet hemocyanin (KLH) to form a complex of Pep-KLH by us EDC. Anti-sera were acquired by immunizing rabbit with Pep-KLH emulsified by Complete Freund' s adjuvant (CFA) and Incomplete Freund' s adjuvant (IFA), and polyclonal antibody was purified by affinity chromatography. The titer and specificity of anti-sera and polyclonal antibody were determined by Indirect-ELISA and Western blotting. The results shown that anti-sera and polyclonal antibody reacted with Pep-KLH and detected a specific band of 70 kD, and the size was agreed with the predicted molecular mass. The EDS1 polyclonal antibody revealed high sensitivity and specificity.%EDS1蛋白是植物寄主SA信号转导通路中具有重要调控功能的蛋白,为获得效价高和选择性强的EDS1抗体,根据NCBI GenBank中报道的EDS1蛋白一级结构信息,采用Blastn、Blastx和ExPASy等生物信息学软件进行序列同源性分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和GC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达89.37%,目的多肽分子量为1978.33.采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原-Pep-KLH,并将其免疫新西兰大白兔以获

  17. Duodenal enteroglucagonoma revealed by differential comparison of serum and tissue glucagon reactivity with Siemens' Double Glucagon Antibody and DakoCytomation's Polyclonal Rabbit Anti-Human Glucagon: a case report

    Directory of Open Access Journals (Sweden)

    Abouljoud Marwan S

    2010-06-01

    Full Text Available Abstract Introduction This case report demonstrates that the differential immunohistochemical reactivities of Siemens' Double Antibody Glucagon compared to DakoCytomation's Polyclonal Rabbit Anti-Human Glucagon allow for pathologic distinction of enteral versus pancreatic glucagonoma. Case presentation A 64-year-old Caucasian man was diagnosed with a duodenal enteroglucagonoma following presentation with obstructive jaundice. He had a low serum glucagon level using Siemens' Double Antibody Glucagon, a clinical syndrome consistent with glucagon hypersecretion. A periampullary mass biopsy proved to be a neuroendocrine tumor, with positive immunohistochemical reactivity to DakoCytomation's Polyclonal Rabbit Anti-Human Glucagon. Conclusions Differential comparison of the immunohistochemical reactivities of Siemens' Double Antibody Glucagon and DakoCytomation's Polyclonal Rabbit Anti-Human Glucagon discerns enteroglucagon from pancreatic glucagon.

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

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    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.

  19. Preparation of the Polyclonal Antibody against Ochratoxin A%抗赭曲霉毒素A多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    高璟瑜; 杨扬; 张红星; 刘艳华; 畅晓辉; 李丹

    2011-01-01

    为了制备赭曲霉毒素A多克隆抗体,采用戊二醛法合成赭曲霉毒素A-BSA人工抗原,通过紫外扫描法鉴定半抗原与载体蛋白偶联效果.合成的人工抗原用弗氏佐剂乳化后免疫Balb/c小白鼠,4次免疫后眼球下缘采血,分离血清.试验结果表明,免疫后产生了抗赭曲霉毒素A-BSA的特异性多克隆抗体,抗体效价为1:8100,赭曲霉毒素A的最低检出限为9ng/mL.抗赭曲霉毒素A多克隆抗体的制备为开发赭曲霉毒素A的ELISA酶联免疫试剂盒奠定了基础.%To prepare polyclonal antibody against ochratoxin A, artificial antigen of ochratoxin A-BSA were coupled by glutaraldehyde method. The couple effection between semiantigen and carrier protein was identified through ultraviolet spectrum scanning method. The synthetical antigen ochratoxin A-BSA was mixed with the same amount adjuvant and then immunized mouse. After the fourth immunize, the mouse blood was collected from heart. and blood sera was seDarated. The eXDeriment results showed that the SDecifiC Dolvclonal antibody against ochratoxin A was generated after immunity. The titer of anti-sera was 1∶8100. The detection limit of ochratoxin A was 9 ng/mL.Preparation of the polyclonal antibody against ochratoxin A established the groundwork for further research of ochratoxin A immunoassay kit.

  20. Preparation of Polyclonal Antibody against Crystalline Inclusion Protein of Entomopathogenic Bacterium%昆虫病原细菌Cip蛋白多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    游娟; 黄建林; 曹莉; 韩日畴

    2013-01-01

    Entomopathogenic Photorhabdus bacteria produce two types of intracellular crystalline inclusion proteins,CipA and CipB,as the nutrient resource for the symbiotic nematode.Based on constructed high-level prokaryotic expression system,recombinant Cip proteins were purified with a warm extract method and used as the antigens to immune New Zealand rabbits,respectively.The polyclonal antibodies with the titers of 1:6400 were raised and monitored by indirect enzyme-linked immunosorbent assay (ELISA).Western blot assay confirmed that the antibodies could specifically recognize the inclusion proteins in Photorhabdus bacteria.Preparation of Cip polyclonal antibodies will be helpful to develop the immunological assay method of these proteins.%昆虫病原发光杆菌属(Photorhabdus)细菌产生2种胞内晶体蛋白CipA和CipB,为其共生线虫的生长发育提供营养.基于Cip蛋白已在大肠杆茵原核表达系统中得到稳定且高水平表达,以纯化的重组Cip蛋白为抗原,免疫新西兰大耳兔制备多克隆抗体,ELISA测定2种蛋白抗血清效价,均可达到1∶6400.Western blot分析显示,制备的抗体均可特异识别发光杆菌的胞内晶体蛋白.Cip蛋白多克隆抗体的制备为建立该类蛋白的免疫学检测方法奠定了基础.

  1. Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77.

    Science.gov (United States)

    Leblanc, Pierre; Moon, Minho; Kim, Woori; Jeong, Inhye; Kim, Chun-Hyung; Kim, Kwang-Soo

    2015-08-17

    The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson's disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

  2. Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9 antibody [v1; ref status: indexed, http://f1000r.es/5ex

    Directory of Open Access Journals (Sweden)

    Peter van Endert

    2015-05-01

    Full Text Available In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222 expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9 protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

  3. Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components.

    Science.gov (United States)

    Nahid, M Abu; Ross, Steven J; Umiker, Benjamin R; Li, Huapeng; Sugii, Sunji; Bari, Latiful

    2016-02-01

    Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity.

  4. Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

    Science.gov (United States)

    Pongkitwitoon, Benyakan; Sakamoto, Seiichi; Tanaka, Hiroyuki; Tsuchihashi, Ryota; Kinjo, Junei; Morimoto, Satoshi; Putalun, Waraporn

    2010-05-01

    Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

  5. The Production of Polyclone Antibody for Indirect Competitive ELISA for Methamidophos Residue%兔抗甲胺磷多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    董国伟; 王沫; 刘贤进; 余向阳

    2001-01-01

    采用水溶性碳化二亚胺法(EDC),将MTP与牛血清蛋白(BSA)、人血清蛋白(HSA )和鸡卵清蛋白(OVA)共价偶联,分别合成免疫抗原MTP?BSA、MTP?HSA和包被抗原甲胺磷 ?鸡卵清蛋白(MTP?OVA),用合成的免疫抗原对新西兰大白兔进行免疫,制得抗血清经鉴定确证为兔抗MTP多克隆抗体(polyclonal antibody,PAB),效价分别为1∶15?000和1∶12? 000.

  6. Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of Alicyclobacillus strains in apple juice.

    Science.gov (United States)

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Guo, Caixia; Wang, Xin; Niu, Chen

    2012-11-01

    A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

  7. Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes.

    Science.gov (United States)

    He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

    2017-07-15

    Bovine β-lactoglobulin (BLG) is the main allergen in cows' milk, and the most commonly used method for detecting BLG is enzyme-linked immunosorbent assay (ELISA). However, antibodies used in commercial ELISA kits do not recognize specifically BLG IgE epitopes. Here, an antibody specific to IgE linear epitopes for BLG was used to develop a sandwich ELISA using a rabbit anti-BLG polyclonal antibody. The linear range for BLG detection was 31.25-8000ng/mL and limit of detection was 1.96ng/mL. BLG content in dairy samples was determined, and there was a good agreement between this immunoassay and reversed-phase high-performance liquid chromatography with high recovery. Additionally, BLG content in food samples had an average recovery of 104.25%. Allergenic residues were also detected in hydrolyzed infant formulas. The method developed could be a practical approach to determine BLG and its allergenic residues in food with a high degree of sensitivity, reliability and recovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system

    Science.gov (United States)

    Gopal, Gopal Jee; Kumar, Awanish; Pal, Jagannath; Mukhopadhyay, Gauranga

    2014-01-01

    Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world’s residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS. PMID:24637488

  9. Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide.

    Science.gov (United States)

    Wang, Jun; Bing, Xin; Yu, Kun; Tian, Hua; Wang, Wei; Ru, Shaoguo

    2015-01-01

    Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17β-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.

  10. Investigation of Neutron Radiation Effects on Polyclonal Antibodies (IgG) and Fluorescein Dye for Astrobiological Applications

    Science.gov (United States)

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

    2009-09-01

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

  11. Preparation of Polyclonal Anti-Sox2 Antibody in Capra hircus%山羊SOX2多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    刘平; 张明; 张昀; 郑喜邦; 李恭贺; 岑小妹; 岳磊磊; 宗自杰; 卢晟盛; 卢克焕

    2012-01-01

    [Objective] The present study was to construct a prokaryotic expression vector of Capra hircus Sox2 gene, pRSET-Sox2, to induce expression and purification of His-Sox2 fusion protein, which was used to immunize New Zeland white rabbits to prepare polyclonal anti-Sox2 antibody. [Method] Removed from plasmid pMD18-Sox2 by double digestion of BamH I and Xho I, Sox2 fragment was subcloned to pRSET-A vector to construct recombinant plasmid pRSET-Sox2. The plasmid was transformed into E. coli BL 21 (DE3), and His-Sox2 fusion protein was induced to expess with 1 mrnol·L-1 IPTG at 37℃ for 4 h, which was identified with SDS-PAGE analysis and Western blotting. In the same way, large volume of expressing culture was prepared to purify His-Sox2 fusion protein with NI-NTA argrose under denaturing condition. The refolded fusion protein in vitro was injected subcutaneously into New Zeland white rabbits for four times at intervals of 2-3 weeks. Seven days after the last injection, blood samples were collected, serum was isolated, and specificity of polyclonal anti-Sox2 antibody was determined by Western blotting assay. [Result] The prokaryotic expression vector pRSET-Sox2 was expressed efficiently in E. coli. BL21. The purified His-Sox2 was qualified for preparation of polyclonal antibody. The polyclonal anti-Sox2 antibody was prepared, and it could bind His-Sox2 fusion protein specifically, which was illustrated by Western blotting assay. [Conclusion] The polyclonal anti-Sox2 antibody with strong specificity was prepared, which will lay a solid biological foundation for study of Sox2, and for its application in detection of Capra hircus iPS cells (induced pluripotent stem cells).%[目的]构建山羊Sox2原核表达载体—pRSET-Sox2,并将诱导表达、纯化的His-Sox2融合蛋白免疫新西兰大白兔,制备Sox2多克隆抗体.[方法]从pMD18T-Sox2载体上以BamHI和XhoI双酶切截取Sox2 片段,然后将其亚克隆到pRSET-A表达载体上,获得pRSET-Sox2

  12. Polyclonal antibodies to light-harvesting CHL-protein of PSII (LHC II) in marine green algae Bryopsis corticulans

    Science.gov (United States)

    Wu, Xiaonan; Zhou, Baicheng; Tseng, C. K.

    1992-06-01

    Polyc·lonal antibodies raised against LHC II isolated from SDS-solubilized Bryopsis corticulans thylakiod membranes by SDS-PAGE, were characterised by double immunodiffusion, Rocket immunoelectrophoresis and antigen-antibody crossed immunoelectro-phoresis assays showed the antibodies had strong cross-reaction with all B. corticulans LHC II components (even with those which were incubated in boiling water) and showed immunological cross-reactivity with LHC II polypeptides of spinach and the marine green alga Codium fragile. The results suggested that LHC II of different species had similar antigenic determinants and also conservation of amino acid sequences of the polypeptides during evolution, and that the antibodies could cross react with apoproteins of D2 proteins (which contain P680) from B. corticulans, spinach and C. fragile, but not with apoproteins of P700 Chl-proteins. Our results indicated some similarities in primary structure between LHC II of different species, and between LHC II and D2 proteins of marine green algae and spinach. Our finding that D2 and P700 Chl-proteins are not immunologically related suggested that P700 Chl-proteins and D2 proteins pass through independent evolutionary pathways.

  13. Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

    Science.gov (United States)

    Prokopenko, P G; Shkoporov, A N; Petrenko, O Yu; Efimov, B A; Negrebetskii, V V; Terent'ev, A A

    2015-11-01

    We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

  14. Relative contribution of dengue prM- and E-specific polyclonal antibodies to neutralization and enhancement.

    Science.gov (United States)

    Rodpothong, P; Boonarkart, Ch; Ruangrung, K; Onsirisakul, N; Kanistanon, D; Auewarakul, P

    Viral surface proteins, premembrane protein (prM) and envelope (E) protein have been shown to induce a production of antibodies that are involved in both enhancement and neutralization. To explore the feasibility of modifying the relative immune responses to prM and E proteins, four DNA constructs were created and administered into groups of Balb/c mice; pPW01 contains prM and E genes of DENV1, pPW02 contains prM and E genes of DENV2, pPW03 contains DENV1 prM and DENV2 E, and pPW04 contains DENV2 prM and DENV1 E. Exchange of either prM or E from a heterologous serotype does not appear to have an effect on the immunogenicity of the proteins. We have proved that the chimeric pPW03 and pPW04 constructs can produce humoral response in mice. Immunized sera were subjected to neutralization and enhancement assays against DENV2. The results showed that only serotype-specific anti-E antibodies conferred protective function, while the cross-reactive anti-E and anti-prM enhanced infection. In addition, the enhancement of DENV2 infection exhibited a serotype-preference for anti-E antibodies while such response was not observed with anti-prM, reflecting a degree of structural conservation of prM. Taken together, neutralization and enhancement appeared to occur at the same time during the course of infection. Successful prevention of severe symptoms of DENV infection depends on the ability to induce high levels of neutralizing antibodies to subdue the effect of enhancing antibodies.

  15. Comprehensive mapping infection-enhancing epitopes of dengue pr protein using polyclonal antibody against prM.

    Science.gov (United States)

    Luo, Yayan; Guo, Xiaolan; Yan, Huijun; Fang, Danyun; Zeng, Gucheng; Zhou, Junmei; Jiang, Lifang

    2015-07-01

    Dengue vaccine development is considered a global public health priority, but the antibody-dependent enhancement (ADE) issues have critically restricted vaccine development. Recent findings have demonstrated that pre-membrane (prM) protein was involved in dengue virus (DENV) infection enhancement. Although the importance of prM antibodies have been well characterized, only a few epitopes in DENV prM protein have ever been identified. In this study, we screened five potential linear epitopes located at positions pr1 (1-16aa), pr3 (13-28aa), pr4 (19-34aa), pr9 (49-64aa), and pr10 (55-70aa) in pr protein using peptide scanning and comprehensive bioinformatics analysis. Then, we found that only pr4 (19-34aa) could elicit high-titer antibodies in Balb/c mice, and this epitope could react with sera from DENV2-infected patients, suggesting that specific antibodies against epitope peptide pr4 were elicited in both DENV-infected mice and human. In addition, our data demonstrated that anti-pr4 sera showed limited neutralizing activity but significant ADE activity toward standard DENV serotypes and imDENV. Hence, it seems responsible to hypothesize that anti-pr4 serum was infection-enhancing antibody and pr4 was infection-enhancing epitope. In conclusion, we characterized a novel infection-enhancing epitope on dengue pr protein, a finding that may provide new insight into the pathogenesis of DENV infection and contribute to dengue vaccine design.

  16. Produção e caracterização de anticorpos policlonais contra Xanthomonas campestris pv. viticola Production and characterization of polyclonal antibodies against Xanthomonas campestris pv. viticola

    Directory of Open Access Journals (Sweden)

    João Sebastião de Paula Araujo

    2005-03-01

    Full Text Available O objetivo deste trabalho foi a produção de anticorpos policlonais contra Xanthomonas campestris pv. viticola e sua caracterização pelo método Elisa indireto. Os resultados apontaram a qualidade dos anticorpos policlonais produzidos, os quais mostraram-se altamente reativos e específicos para o patovar com potencial para ser empregado no diagnóstico da doença e em programas de certificação.The objective of this work was to produce polyclonal antibodies against Xanthomonas campestris pv. viticola and characterize these antibodies through Elisa serological indirect method. Results indicate that polyclonal antibodies produced were highly reactive against bacterial cells, showing specificity at the pathovar level and potential to be used for diagnosis and certification purposes.

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

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    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.

  18. Prokaryotic Expression of Rubber Elongation Factor Gene and Preparation of Its Polyclonal Antibody%橡胶延长因子基因的原核表达及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    陈章权; 吴坤鑫; 梁晓东; 黄震; 陆田田

    2008-01-01

    [Objective] The aim of this study was to prepare the recombination protein of rubber elongation factor and its polyclonal antibodies. [Method] The encoding gene of rubber elongation factor (REF) was amplified by RT-PCR, and cloned into the prokaryotic expression vector pDEST17 to transform into Escherichia coil BI2I-AI. The recombinant protein induced by L-Arabinose was purified by the affinity chromatography. As the immunogen, the recombination protein was used to immunize mice for preparing polyclonal antibodies, while ELISA and Western blot hybridization were used to detect the titers and specificity. [Result] The purified recombination protein of REF with high expression was used to immunize house mice for preparing polyclonal antibodies with high titer and specificity. The western blot hybridization showed that the antibody could recognize the natural REF from latex. [Conclusion] The recombination protein of REF was successfully obtained and the mouse anti REF antibody with high titer and specificity was prepared, which lays a basis for further studies on biological functions of rubber elongation factor and other membrane proteins in rubber particles.

  19. Preparation and identification of the fragment of rabbit's polyclonal antibodies against human ouabain%抗哇巴因多克隆抗体F(ab)2 片段的研制及鉴定

    Institute of Scientific and Technical Information of China (English)

    张明娟; 吕卓人; 袁育康; 贺浪冲; 王颢

    2000-01-01

    Objective Anti-ouabain polyclonal antibody was digested with pepsin to prepare F fragment in order to find the base of reshaped antibody. Methods Anti-ouabain polyclonal antibody was prepared by immunizing rabbits. Then anti-ouabain polyclonal antibody was digested under different conditions. The reactants were analyzed and purified by High Performance Size Ex-clude Chromatography (HPSEC). The immune activities were detected and identified by Enzyme Linked Immunosorbent Assay (ELISA). Results The proper reactive conditions for preparation of anti-ouabain polyclonal antibody F(ab)2 fragment with pepsin were established. The eligible dose of pepsin, by which 100mg anti-onabain polyclonal antibody was digested better, was 2mg. The eligible digesuve time was 18h. The value of pH of reactive system was 3.0. Conclusion The active F fragment of anti-ouabain polyclonal antibody can be obtained with pepsin. The method is simple and feasible.%目的用胃蛋白酶酶解抗哇巴因多克隆抗体,制备出片段,为改型抗体的研究提 供依据。方法免疫家兔制备出抗哇巴因多克隆抗体,并用胃蛋白酶分别在不同的酶解条件下消化抗体,继之用高效液相排阻色谱法(HPSEC)对其进行分析、纯化,采用酶联免疫吸附法(ELISA)检测、鉴定其活性。结果用胃蛋白酶2mg/100mg抗哇巴因多克隆抗体,消化18h,反应体系的pH3.0时,可获得片段。结论用胃蛋白酶酶解抗哇巴因多克隆抗体是研制出有活性的片段的有效方法。

  20. Polyclonal antibodies against the TLA1 protein also recognize with high specificity the D2 reaction center protein of PSII in the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Mitra, Mautusi; Dewez, David; García-Cerdán, Jose Gines; Melis, Anastasios

    2012-04-01

    The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to

  1. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

  2. Human Polyclonal Antibodies Produced in Transchromosomal Bovine Prevent Lethal Zika Virus Infection and Testicular Atrophy in Mice

    Science.gov (United States)

    2017-03-21

    Guillain- Barre syndrome (3-­‐6). There are currently 76 countries with confirmed mosquito-borne ZIKV transmission worldwide, including recent cases...reproductive tract leading to documented cases of sexual transmission (8,   9). Animal models of ZIKV infection have rapidly been established and...a Rh-positive fetus (18). More recently monoclonal antibody approaches have been used to prevent emerging infectious diseases , most notably the

  3. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    Science.gov (United States)

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross

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

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    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

  5. Preparation and Application of Polyclonal Antibody to Butachlor%抗丁草胺多克隆抗体的制备及应用

    Institute of Scientific and Technical Information of China (English)

    郭永恩; 陈家华; 王能东; 张秀; 郭振泉; 金声

    2002-01-01

    Polyclonal antibody to butachlor was prepared from rabbits immunized with butach lor-BSA conjugate,and a competitive ELISA was developed for the detection of th e butachlor (a major component in a herbicide) within the range of 1×10-9 ~100×10-9mg/mL without cross reactions with the structurally similar c hloroac etanilide herbicides,alachlor and acetochlor.This method could also be used to m onitor the butachlor concentration of water samples from rice field.%以牛血清白蛋白-丁草胺免疫抗原免疫家兔获得了抗丁草胺多克隆抗体,并建立了竞争酶联免疫吸附检测丁草胺(除草剂中的一种主要成分)分析方法.该方法的检测范围在1×10-9~100×10-9mg/mL之间,且和化学结构相似的甲草胺和乙草胺无交叉反应.该方法也可用于检测稻田水样中丁草胺的含量.

  6. 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.

  7. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Fang Ding

    Full Text Available 'Candidatus Liberibacter asiaticus' (CaLas, a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB. Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  8. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Science.gov (United States)

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S

    2015-01-01

    'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

  9. Studies on the immune response and preparation of antibodies against a large panel of conjugated neurotransmitters and biogenic amines: specific polyclonal antibody response and tolerance.

    Science.gov (United States)

    Huisman, Han; Wynveen, Paul; Setter, Peter W

    2010-02-01

    We described the production and characterization of antibodies against three important groups of neuro-active haptens, e.g., neurotransmitters and biogenic amines. First, from the tryptophane metabolic pathway: tryptamine, serotonin, 5-hydroxy-indole acetic acid, and melatonin. Secondly, the tyrosine metabolic pathway: tyramine, dopamine, dihydroxyphenyl acetic acid, and norepinephrine. Thirdly, antibodies against excitatory and inhibitory neurotransmitters: glycine, glutamate, glutamine, and GABA. Immunogenic conjugates were prepared after linking haptens to carrier proteins. Most antibodies displayed high specificity against corresponding neuro-active haptens conjugated in vitro and in situ in biological specimens, but not to closely related conjugated metabolites, precursors, pharmaceuticals, agonists, antagonists, or free neuro-active haptens. Conjugated norepinephrine was highly tolerant in different animal species and produced incidentally a short specific antibody response.

  10. Preparation and Evaluation of Polyclonal Antibodies of Apoptin%凋亡素蛋白多克隆抗体的制备及免疫学评价

    Institute of Scientific and Technical Information of China (English)

    王春晖; 王剑松; 王文举; 詹辉; 李鸿钧; 颜汝平; 徐鸿毅

    2011-01-01

    凋亡素由鸡贫血病毒中的VP3基因编码,能诱导多种肿瘤细胞发生凋亡.以真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经Nde Ⅰ/BamH Ⅰ双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清.抗血清效价高的兔子进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1:243 000.用重组腺相关病毒rAAV-VP3感染细胞后对抗体的特异性进行免疫学评价.首先利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质.其次通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合.实验证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础.%Apoptin is coded by the VP3 gene from chicken anemia virus (CAV) , and the protein can induce apoptosis of a large variety of tumor cells. The preparation of polyclonal antibodies specific to VP3 protein was explored. The VP3 gene was obtained from the eukaryotic expression vector pcDNA3.O-VP3. And then,VP3 gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3 was employed to express VP3 protein in E. coli BL21 which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3 protein was applied to immunize the New Zealand rabbits combined with the Freund's complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA

  11. Preparation of polyclonal antibody specific for NOR1 and detection of its expression pattern in human tissues and nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Bo Xiang; Mei Yi; Li Wang; Wei Liu; Wenling Zhang; Jue Ouyang; Ya Peng; Wenjuan Li; Ming Zhou; Huaying Liu; Minghua Wu; Rong Wang; Xiaoling Li; Guiyuan Li

    2009-01-01

    Oxidored-nitro domain containing protein 1 (NORI)gene is a novel nitroreductase gene first isolated from nasopharyngeal carcinoma (NPC). It plays an important role in the formation of chemical carcinogen and the carcinogenesis of NPC for its nitrosation function. Overexpression of the wild-type NOR1 gene in naso-pharyngeal carcinoma cells is effective to inhibit cell growth and proliferation. In this study, for the first time, we generated a highly specific NOR1 antibody and ana-lyzed NOR1 distribution in the human tissues and NPC biopsies. The results showed that NOR1 protein is predominantly expressed in human nasopharynx and tracheal tissues. Human heart, liver, spleen, stomach, colon, kidney, skeletal muscle, thymus, and pancreas are aH deficient of NORI protein. More importantly, we per-formed immunohistochemistry assay of NOR1 protein expression in the NPC tissues, and the result showed that NOR1 protein is frequently down-expressed in NPC. These data shed light on the selectivity of potential physiological functions of NOR1 and provides an indis-pensable reference to the carcinogenesis process of NPC and to identify or validate tissue-specific drug targets.

  12. Biological Activities of Purified HarpinXoo and HarpinXoo Detection in Transgenic Plants Using Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Ming LI; Min SHAO; Xu-Zhong LU; Jin-Sheng WANG

    2005-01-01

    Many harpins have been found in plant pathogen bacteria that can elicit disease and insect resistance in plants, and promote plant growth. In this work, we overexpressed and purified Xanthomonas oryzae pv. oryzae harpin, harpinXoo, in Escherichia coli BL21/pGEX-hpa1. HarpinXoo was fused to the Cterminus of glutathione S-transferase (GST) and purified using the Bulk GST purification module and thrombin cleavage capture kit. Purified harpinXoo protein was sensitive to protease K and stable to heat treatment, and could not induce a hypersensitive response after treatment with various plant metabolic inhibitors; these characteristics were similar to harpinEa of Erwinia amylovora. The purified harpinXoo showed a similar ability to induce tobacco mosaic virus resistance in tobacco as harpinEa. Its antibody worked well in detecting the purified harpinXoo, harpinXoo in the total protein of E. coli BL21/pGEX-hpa1 and an hpal transgenic rice.

  13. Spectra analysis of coating antigen: A possible explanation for difference in anti-AFB1 polyclonal antibody sensitivity

    Science.gov (United States)

    Ye, Yang; Liu, Aiping; Wang, Xiaohong; Chen, Fusheng

    2016-10-01

    For the detection of small hapten molecules, indirect competitive enzyme-linked immunosorbent assay (icELISA) is a preferred method. However, diverse coating antigen might bring different antiserum titer and sensitivity for the identical antiserum. In the present study, four AFB1-protein (aflatoxin B1-carrier protein) conjugates were prepared by activated ester method (AFB1O-BSA/AFB1O-OVA) and mannich method (AFB1-cBSA/AFB1-cOVA), and then applied as coating antigen for titer and sensitivity detection of the identical antiserum obtained from rabbit immunized by AFB1-KLH. Afterwards, the ultraviolet-visible, fluorescence and far-ultraviolet circular dichroism (far-UV CD) spectra were recorded for understanding the difference in titer and sensitivity obtained. Results revealed that AFB1O-BSA/AFB1O-OVA showed a strong intrinsic fluorescence band centered at 450 nm that originated from the emission of AFB1, which differed from AFB1-cBSA/AFB1-cOVA, while the decrease of α-helical and increase of β-sheet in AFB1-cBSA was the most remarkable. This indicated that the better sensitivity obtained by using AFB1O-BSA as coating antigen might be caused by its extended structure, because such structure affect the binding between AFB1 and antibody. The study might offer structural information for understanding the titer and sensitivity difference caused by coating antigen.

  14. Therapeutic effects of antigen affinity-purified polyclonal anti-receptor of advanced glycation end-product (RAGE) antibodies on cholestasis-induced liver injury in rats.

    Science.gov (United States)

    Xia, Peng; Deng, Qing; Gao, Jin; Yu, Xiaolan; Zhang, Yang; Li, Jingjing; Guan, Wen; Hu, Jianjun; Tan, Quanhui; Zhou, Liang; Han, Wei; Yuan, Yunsheng; Yu, Yan

    2016-05-15

    Cholestasis leads to acute hepatic injury, fibrosis/cirrhosis, inflammation, and duct proliferation. We investigated whether blocking receptor of advanced glycation end-products (RAGE) with polyclonal anti-RAGE antibodies (anti-RAGE) could regulate acute liver injury and fibrosis in a rat bile duct ligation (BDL) model. Male Wister rats received 0.5mg/kg rabbit anti-RAGE or an equal amount of rabbit IgG by subcutaneous injection twice a week after BDL. Samples of liver tissue and peripheral blood were collected at 14 days after BDL. Serum biochemistry and histology were used to analyze the degree of liver injury. Quantitative real-time PCR (qPCR) and immunohistochemical staining were used to further analyze liver injury. Anti-RAGE improved the gross appearance of the liver and the rat survival rate. Liver tissue histology and relevant serum biochemistry indicated that anti-RAGE attenuated liver necrosis, inflammation, liver fibrosis, and duct proliferation in the BDL model. qPCR and western blotting showed significant reductions in interleukin-1β expression levels in the liver by treatment with anti-RAGE. Anti-RAGE also significantly reduced the mRNA levels of α1(1) collagen (Col1α1) and cholesterol 7α-hydroxylase, and the ratio of tissue inhibitor of matrix metalloproteinase-1 to matrix metalloproteinases (MMPs) in the liver. In addition, anti-RAGE regulated the transcriptional level of Col1α1 and MMP-9 in transforming growth factor-β-induced activated LX-2 cells in vitro. Anti-RAGE was found to inhibit hepatic stellate cell proliferation in vivo and in vitro. Therefore, anti-RAGE can protect the liver from injury induced by BDL in rats. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Preparation and Identification of the Polyclonal Antibody Against Plant Sweet Protein Mabinlin Ⅱ%植物甜蛋白马宾灵(MabinlinⅡ)多克隆抗体的制备与鉴定

    Institute of Scientific and Technical Information of China (English)

    顾文亮; 夏启玉; 姚晶; 胡新文; 郭建春

    2012-01-01

    马宾灵(MabinlinⅡ)是中国所特有的植物马槟榔种子中的甜味蛋白,将其作为新型甜味剂有着广阔的市场前景.为了给重组马宾灵的基因工程应用提供可靠的蛋白质检测与鉴定的抗体,通过离子交换法从马槟榔(Capparis masaikai Lévl)种子中分离纯化马宾灵,将其作为抗原免疫新西兰大白兔,收集免疫后血清经抗原亲和纯化制备马宾灵多克隆抗体.结果表明,制备的马宾灵多克隆抗体经ELISA检测效价比达到1:256000,Western-blot检测表明其具有良好的反应性和特异性.本研究制备的马宾灵多克隆抗体可用于马宾灵在不同生物反应器中表达的检测,为马宾灵的基因工程研究提供灵敏可靠的免疫学鉴定.%The plant sweet protein Mabinlin Ⅱ is found rich in mature seeds of Capparis masaikai Levl which is particular to China and may have wide application prospects in food sweeteners industry. In this study, in order to provide a reliable testing antibody for engineering applications of recombinant Mabinlin Ⅱ, Mabinlin Ⅱ was separated and purified from the mature seeds of Capparis masaikai Levl by ion exchange chromatography. The purified protein was used as antigen to immunize New Zealand rabbits to prepare polyclonal antibody against the Mabinlin Ⅱ protein. The result of Enzyme-Linked Immunosorbnent Assay (ELISA) showed that the titer of the polyclonal antibody to purified Mabinlin Ⅱ was 1:256000, and the polyclonal antibody against the purified Mabinlin E also had highly reactivity and specialty in western blot analysis. The polyclonal antibody against Mabinlin II could be used for detecting the protein amounts of Mabinlin Ⅱ expressed in various bioreactors, which may serve sensitive and reliable methods for immunology detection in genetic engineering research of Mabinlin Ⅱ .

  16. Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

    Science.gov (United States)

    Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

    2014-10-01

    One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

  17. Expression, Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA%大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    何婷婷; 龚钢明; 高然

    2012-01-01

    [ Objective] This study aimed to clone the E. Coli NrfA gene and construct the pET-28a( + )-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E. Coli NrfA. [ Method] E. Coli NrfA gene was cloned from the E. Coli genome DNA by PCR and inserted into the vector pET-28a( + )to construct prokaryotic expression vector pET-28a( + )-NrfA. E. Coli NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method. The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting. [ Result] The constructed prokaryotic expression vector pET-28a( + )-NrfA was induced by IPTG, the recombinant NrfA protein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1 =204 900. Western Blotting analysis indicated that the obtained polyclonal antibody against E. Coli NrfA protein had high titer and high specificity. [Conclusion] E. Coli NrfA gene was cloned and the prokaryotic expression vector pET-28a( + )-NrfA was constructed successfully, and the polyclonal antibody with high titer and high specificity was prepared, which had laid the foundation for the study of NrfA in different strains of bacteria.%[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+ )-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定.[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+ )-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性.[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好.[结论]成功克隆大肠杆菌的NrfA基

  18. Prokaryotic Expression of the Arabidopsis Transcription Factor DYT1 and Preparation of Its Polyclonal Antibodies%拟南芥转录因子DYT1的原核表达与多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    俞俞; 邢卫锋; 周茜

    2011-01-01

    以拟南芥雄性不育突变体ms157为材料,对其转录因子DYT1的原核表达进行了分析及多克隆抗体的制备.结果表明:构建的原核表达载体转化入BL21菌株后,经IPTG诱导,表达了分子量约为50 kD的重组蛋白.SDS-PAGE检测结果表明,该重组表达蛋白以可融性形式表达.用该蛋白作为抗原注射新西兰兔后,成功获取了多克隆抗体.Western blotting证实其具良好的免疫原性,ELISA结果表明融合蛋白的效价大于1:5 120.%With a male-sterile mutant ms157 of Arabidopsis as material,we analyzed prokaryotic expression of the transcription factor DYT1 and prepared its polyclonal antibodies. The results indicated that the recombination expression plasmid was constructed and transformed into Escherichia coli BL21 strain. The soluble DYT1 protein was induced with IPTG and used as the antigen to immune the rabbits. SDS-PAGE analysis showed that the fusion protein had relative molecular mass of 50 kD. ELISA assay demonstrated that the titer of prepared polyclonal antibody was over 1: 5 120. Additionally,the immunological specificity of polyclonal antibody was validated by Western blotting.

  19. Prokaryotic Expression and Polyclonal Antibody Preparation of SDG711 C-terminal from Rice%水稻SDG711蛋白C末端原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    张志刚; 李超; 林欣欣; 巫光宏; 杜平州; 王玉琪

    2013-01-01

    [目的]对水稻SDG711蛋白C末端进行原核表达,并制备其多克隆抗体.[方法]选取水稻SDG711蛋白抗原决定簇较密集的C末端进行原核表达,通过构建原核表达载体pET28a-711C,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达融合蛋白后进行纯化,再以纯化的融合蛋白为抗原免疫新西兰白兔,制备多克隆抗体,并对其进行Western-blot分析.[结果]试验制备的多克隆抗体能有效地检测抗原的表达.[结论]该研究为进一步深入研究SDG711蛋白的功能奠定了基础.%[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and prepare its polyclonal antibody. [ Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherkhia coli BL21 (DE3) competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained for Western-blot analysis. [Result] The polyclonal antibody prepared could efficiently detect the antigen expression. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein.

  20. Produção e caracterização de anticorpos policlonais contra Xanthomonas campestris pv. viticola Production and characterization of polyclonal antibodies against Xanthomonas campestris pv. viticola

    OpenAIRE

    2005-01-01

    O objetivo deste trabalho foi a produção de anticorpos policlonais contra Xanthomonas campestris pv. viticola e sua caracterização pelo método Elisa indireto. Os resultados apontaram a qualidade dos anticorpos policlonais produzidos, os quais mostraram-se altamente reativos e específicos para o patovar com potencial para ser empregado no diagnóstico da doença e em programas de certificação.The objective of this work was to produce polyclonal antibodies against Xanthomonas campestris pv. vitic...

  1. 登革病毒4型E蛋白的表达与多克隆抗体的制备%Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    李竹石; 俞永新; 李玉华; 林华; 杨健; 杨会强; 曾献武; 赵宇; 刘俐; 刘莉娜; 康庄

    2012-01-01

    目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白Ⅲ区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体.方法 将登革病毒4型E蛋白与E蛋白Ⅲ区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测.分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白Ⅲ区的多克隆抗体,并对其进行Western blot鉴定.结果 登革病毒4型E蛋白与E蛋白Ⅲ区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带.结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础.%Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a ( + ) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody formed bands as expected when reacting with the dengue virus. Conclusions The polyclonal antibody is prepared and may be used in the detection of dengue virus.

  2. 白念珠菌烯醇化酶1的原核表达及多克隆抗体制备%Prokaryotic Expression and Preparation of Polyclonal Antibody of Candida Albicans Enolase-1

    Institute of Scientific and Technical Information of China (English)

    贺政新; 侯天文; 李玮; 王宪灵

    2016-01-01

    目的 构建白念珠菌烯醇化酶1(eno1)基因的原核表达质粒并诱导其表达,制备其多克隆抗体. 方法聚合酶链反应(PCR)获取白念珠菌SC5314的eno1全长基因,克隆入原核表达载体pET30(a),转化感受态细胞E. coli BL21(DE3),经IPTG诱导表达后以亲和层析纯化重组蛋白并以SDS-PAGE和蛋白免疫印迹(Western blot)法鉴定. 用纯化的重组蛋白免疫新西兰大白兔,制备Eno多克隆抗体,间接酶联免疫吸附试验( ELISA)测定抗体效价, Western blot法鉴定其特异性. 结果 成功构建原核表达质粒pET30(a)-eno1,诱导后表达的重组蛋白相对分子质量约为50 000,主要以可溶性上清形式存在. 纯化制备的eno1多克隆抗体效价约为1:12 800 000,可与重组Eno反应. 结论 成功获得白念珠菌eno1重组蛋白并制备了该蛋白的多克隆抗体.%Objective To construct prokaryotic expression plasmids of candida albicans enolase1 (eno1) gene and to induce its expression so as to prepare polyclonal antibody of recombinant enolase-1 of candida albicans. Methods Full-length eno-1 gene of candida albicans SC5314 was acquired using polymerase chain reaction (PCR), and was cloned into the prokaryotic expression vector pET30 ( a ) . The constructed recombinant plasmid pET30 ( a )-eno1 was transformed into E. coli BL21 (DE3) and induced by IPTG. The recombinant protein was purified by affinity chromatog-raphy, and was identified by SDS-polyacrylamide gel electrophoresis ( SDS-PAGE) and Western blot methods. New Zeal-and white rabbits were immunized with purified recombinant proteins and the Eno polyclonal antibody was prepared. The polyclonal antibody titer was determined by indirect enzyme linked immunosorbent assay ( ELISA) , and its specificity was determined by Western blot. Results The pET30(a)-eno1 prokaryotic expression plasmid was successfully constructed. After induction, the recombinant eno1 showed that a relative molecular mass was about 50 000, which

  3. 人内皮高表达脂多糖相关因子1多克隆抗体的制备%Preparation of polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1

    Institute of Scientific and Technical Information of China (English)

    刘月明; 刘海荣; 蔡震; 马兵; 刘漪沦; 张玮

    2010-01-01

    目的 制备人内皮高表达脂多糖相关因子 1(EOLA1)多克隆抗体,检测EOLA1在人脐静脉内皮细胞(HUVEG)中的表达.方法 复性成功表达EOLA1的蛋白样品(样品1和2),二辛丁酸法检测2种蛋白样品浓度,应用肽质量指纹图谱(PMF)分析法对2种样品进行验证.采用与理论肽段符合率高的EOLA1蛋白样品免疫4只小鼠,同时取4只未免疫小鼠作对照.采集各小鼠血液分离血清,应用饱和硫酸铵盐析沉淀法纯化EOLA1多克隆抗体,ELISA法分析效价(数据以吸光度值表示).蛋白质印迹法检测EOLA1在HUVEC中的表达.结果 蛋白样品1和2的浓度分别为0.124 16、0.132 15 mg/mL.PMF分析显示,样品1的理论肽段符合率为32%,样品2的理论肽段符合率为24%.选用蛋白样品1免疫小鼠,经检测抗体效价在1∶10 000以上;应用制备的EOLA1多克隆抗体可以检测到EOLA1蛋白在HUVEC中表达.结论 应用EOLA1原核表达蛋白免疫小鼠可以制备EOLA1多克隆抗体,并可用于检测EOLA1蛋白表达.%Objective To prepare the polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) , and to determine the expression of EOLA1 in human umbilical vein endothelial cell (HUVEC). Methods The protein samples ( sample 1 and 2) expressing EOLA1 were purified and renatured. The protein concentrations were determined with bicinchoninic acid assay. The protein samples were identified with peptide mass fingerprinting (PMF) analysis. Protein sample with higher coincidence rate of amino acid sequence with theoretic protein was used to inoculate 4 mice; another 4 mice inoculated with adjuvant were used as control. Serum was isolated from collected mice blood. Polyclonal antibody of EOLA1 was purified with saturated ammonium sulfate precipitation, and was determined with ELISA for the titer ( data were denoted by absorbance value). The expression of EOLA1 in HUVEC was determined with Western blot. Results The

  4. Preparation of Polyclonal Antibody of Helicoverpa armigera Single-nucleopolyhedrovirus orf80%棉铃虫核型多角体病毒orf80多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    徐利; 吕婷婷; 甘恩宇; 高秋荣; 王敦

    2012-01-01

    从HearNPV G4株基因组中克隆得到orf80基因的全序列,将该基因构建于pMAL-c4E载体并在TB1中融合表达,orf80融合表达蛋白分子质量为73.3 ku,与预测的蛋白一致,Western blot验证正确.纯化融合蛋白后制备抗原并注射新西兰大白兔从而得到orf80的多克隆抗体,经ELISA检测其效价达到2.56×105,表明该抗体具有较高的免疫原性,可用于orf80编码蛋白的检测及蛋白互作等相关研究.%The HearNPV ori80 was cloned from Helicoverpa armigera Single-nucleopolyhedrovirus (HearNPV) G4 genome by PCR and the prokaryotic expression vector pMAL-c4E-Ha80 was constructed. The recombinant plasmid was transformed into the Escherichi coli TB1 to express the MBP-Ha80 fushion protein. Purified MBP-Ha80 protein was injected into rabbits as antigen to prepare the polyclonal antibodies of orf80. The titer of anti-HearNPV orf80 polyclonal antibodies was 2. 56 X 105 detected by ELISA. The prepared antibodies can be used to determine orf80 coding protein in the research of protein detection and protein interaction.

  5. Expression and polyclonal antibody preparation of chitinase gene in Tetranychus urticae Koch%二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    张道伟; 陈静; 张正玲; 曾燕玲; 郭玉双

    2015-01-01

    The sequence of part chitinase gene of Tetranychus urticae was amplified by PCR from cDNA. The sequence length of this domain was 786 bp. Then, the gene was cloned into pET-32a (+) prokaryotic expressive vector, and the constructed recombinant plasmids pET-32a-TuChi was transformed into the host bacteria E. coli BL21 (DE3). About 45 ku fusion protein was abundantly expressed at 4 h after the recombinant vector was induced with 0.6 mmol·L-1 IPTG. The fusion protein was purified on a Ni+affinity column. The purified chitinase protein was used to immunize New Zealand rabbits for preparing polyclonal antibody with specificity as defined by Western Blot. ELISA analysis showed that the titer of the polyclonal antibody was 1��80 000, polyclonal antibody that was prepared had a high titer and specificity, and the results laid the foundation to further study the function of the chitinase in Tetranychus urticae.%以制备的二斑叶螨cDNA为模板,克隆二斑叶螨几丁质酶SeChi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体pET-32a (+)中,获得多克隆原核表达载体pET-32a-TuChi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21(DE3),经0.6 mmol·L-1 IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备TuChi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1��80000,效价较高,为进一步研究二斑叶螨几丁质酶相关功能奠定基础。

  6. Prokaryotic Expression and Polyclonal Antibody Preparation of Chicken MHC Ⅰα andβ2m Genes%鸡MHCⅠα和β2m的原核表达与多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    戴银; 王承志; 刘生杰; 沈学怀; 赵瑞宏; 胡晓苗; 张丹俊

    2016-01-01

    为制备 MHCⅠ基因工程疫苗的基础材料,构建了鸡 MHCⅠ分子重组质粒并进行原核表达,进而制备鸡 MHCⅠ分子的多克隆抗体。应用 PCR 方法,克隆鸡 MHCⅠα和β2m 基因,构建重组载体 pET-MHCⅠα和 pET-MHCⅠβ2m,经 PCR、双酶切和测序鉴定后,将重组质粒在大肠埃希菌 Rosetta 中进行诱导表达,融合蛋白纯化后,接种昆明小鼠制备多克隆抗体,血清稀释后用免疫印迹法(Western blot)分析。结果表明,鸡 MHCⅠα和β2m 基因在大肠埃希菌中成功表达,融合蛋白分子质量分别约为52.1 ku 和33.0 ku;制备的鼠抗鸡 MHCⅠα和β2m 链多克隆抗体,经 Western blot 检测证实抗体特异性较强,可进一步用于鸡 MHCⅠ分子的研究。%To explore MHC Ⅰ function as DNA vaccine,chicken MHC Ⅰ genes were expressed by using Escherichia coli prokaryotic expression system,and polyclonal antibodies against the recombinant protein MHCⅠwere prepared.Chicken MHCⅠαandβ2m genes were cloned,the recombinant plasmid pET-MHCⅠα,pET-MHCⅠβ2m were constructed,and confirmed by PCR amplification,double enzyme digestion and DNA sequencing.Next,the recombinant plasmids were expressed in E.coli Rosetta induced by using IPTG.After purification of the recombinant protein,the polyclonal antibodies against the recombinant pro-tein were prepared in Kunming mice.The reactivity of the prepared polyclonal antibodies were determined by Western blot.The results revealed that the MHCⅠαandβ2m genes were successfully expressed in E. coli,and the fusion proteins were about 52.1 ku and 33.0 ku,respectively.The polyclonal antibodies had the specific reactinogenicity,it was proved by Western blot.All this made it possible to do further studies on chicken MHCⅠmolecule.

  7. Preparation of Polyclonal Antibody of Allophycocyanin Protein in the Cyanobacterium Synechocystis sp.Strain PCC 6803%集胞藻6803藻胆体别藻蓝蛋白多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    陈李萍; 杜玲瑜; 马为民; 王全喜

    2012-01-01

    Phycobilisome is the major accessory light-harvesting supramolecular complexes, which is composed of core and peripheral rods. Furthermore,the core contains several cylindrical protein assemblies that mainly consist of allophycocyanin,and is involved in the transfer of light energy to the reaction centers of photosystems. In this study,the apcA gene was amplified from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. The expression plasmid pET-32a(+)-apcA was constructed and transformed into BL21(DE3)pLysS,and the expression of ApcA protein was induced by IPTG. After purification by His-tag,the recombinant protein pET-ApcA was used to immunize Japanese White Rabbit to obtain the polyclonal antibody. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The titer of polyclonal antibody was found to be up to 1 ∶ 1 025 000,and thus possessed a high specificity. A polyclonal allophycocyanin antibody of Synechocystis 6803 was successfully obtained in this study,and it will further help in understanding the important roles of core of cyanobacterial phycobilisome during light energy transfer by using biochemical strategy.%藻胆体是蓝藻细胞主要的捕光天线色素超分子复合体,主要由核心体和外围的杆两部分组成,核心体主要由别藻蓝蛋白组装而成,参与光能向光合作用反应中心的传递.该研究通过PCR扩增出集胞藻6803别藻蓝蛋白a亚基(ApcA)编码基因apcA,构建表达质粒pET-32a(+)-apcA,并将其转入大肠杆菌BL21(DE3)pLysS菌株中;通过IPTG诱导表达重组蛋白,并利用组氨酸标签将可溶性目的蛋白进行亲和纯化后,免疫日本大耳白兔,从而获得多克隆抗体.间接ELISA法揭示ApcA抗体效价可高达1∶1025000;蛋白免疫印迹确定该抗体具有高度特异性.表明该研究成功制备了集胞藻6803藻胆体别藻蓝蛋白多克隆抗体,为进一步研究藻胆体的核心体在光能

  8. 溶藻弧菌菌体及其外膜蛋白多克隆抗体的研究%Preparation, Purification and Identification of Polyclonal Antibodies of Vibrio alginolyticus and Outer Membrane Proteins of Vibrio alginolyticus

    Institute of Scientific and Technical Information of China (English)

    黄志坚; 何建国

    2006-01-01

    采用常规方法制备抗溶藻弧菌及其外膜蛋白的多克隆抗体(Polyclonal antibody,PcAb).制备的抗溶藻弧菌(Va)及其外膜蛋白的多克隆抗体(Va-PcAb和Va-OMP-PcAb)的效价达到1∶ 3200以上,特异性较好,只与Va菌不同菌株反应,与其他弧菌及不同属的菌株没有交叉.同时建立的ELISA、IFIA等免疫学检测方法可应用于生产实践,适应不同的需要.

  9. Evaluation of an immunohistochemical test with polyclonal antibodies raised against mycobacteria used in formalin-fixed tissue compared with mycobacterial specific culture.

    Science.gov (United States)

    Carabias, E; Palenque, E; Serrano, R; Aguado, J M; Ballestín, C

    1998-03-01

    An immunohistochemical (IH) test (commercially available polyclonal antiserum rabbit anti-Myco-bacterium bovis; DAKO A/S) was used to detect the presence of mycobacteria in 65 formalin-fixed, paraffin-embedded tissue blocks from different organs, showing necrotizing caseous granuloma lesions on hematoxylin and eosin sections from 65 patients. These 65 samples were dyed using an acid-fast fluorescent technique and compared using the immunohistochemical method. Both results were also compared with the mycobacterial cultures. The IH test, compared with the culture, showed a sensitivity (S) of 52%, a specificity (Sp) of 76%, a positive predictive value (PV pos) of 61% and a negative predictive value (PV neg) of 69%. We analyze these data and discuss the possible causes of false-positive and -negative results of the IH test. This rapid test on paraffin embedded tissue seems valuable in the period when waiting for the culture results.

  10. Synthesis of Acrylamide Artificial Antigen and Preparation of Anti-Acrylamide Polyclonal Antibody%丙烯酰胺人工抗原的合成及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    张红星; 高美琴; 张馨如; 刘慧

    2009-01-01

    利用偶联方法合成丙烯酰胺人工抗原,通过免疫方法获得抗丙烯酰胺多克隆抗体;应用戊二醛法将丙烯酰胺与牛血清白蛋白(BSA)进行偶联,并对这两种物质及其复合物进行紫外扫描,并用此复合物免疫兔子,利用间接酶联免疫吸附法测定抗体的效价;应用牛血清白蛋白与丙烯酰胺偶联人工抗体成功,抗体效价大于8100;应用人工抗原免疫成功制备抗丙烯酰胺多克隆抗体.%Artificial antigen of acrylamide was synthetized by couple method, and anti- acrylamide polyclonal antibody were obtained by immunizing method. The Glutaraldehyde method was used to conjugate Acrylamide to Bovine Serum albumin(BSA), and artificial antigen, acrylamide and BSA were respectively taken on UV spectrum scan. Rabbits had been immunized with artificial antigen, and the titer antiserum from those rabbits were testified by indirect enzyme-linked immunosorbent assay. The results showed that artificial antigen was synthesized successfully between Acrylamide and BSA, and titer of anti-acrylamide serum was above 8100; Anti-Acrylamide polyclonal antibody was prepared by immunizing method of artificial antigen.

  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. The binding effectiveness of anti-r-disintegrin polyclonal antibodies against disintegrins and PII and PIII metalloproteases: An immunological survey of type A, B and A+B venoms from Mohave rattlesnakes.

    Science.gov (United States)

    Cantú, Esteban; Mallela, Sahiti; Nyguen, Matthew; Báez, Raúl; Parra, Victoria; Johnson, Rachel; Wilson, Kyle; Suntravat, Montamas; Lucena, Sara; Rodríguez-Acosta, Alexis; Sánchez, Elda E

    2017-01-01

    Snake venoms are known to have different venom compositions and toxicity, but differences can also be found within populations of the same species contributing to the complexity of treatment of envenomated victims. One of the first well-documented intraspecies venom variations comes from the Mohave rattlesnake (Crotalus scutulatus scutulatus). Initially, three types of venoms were described; type A venom is the most toxic as a result of ~45% Mojave toxin in the venom composition, type B lacks the Mojave toxin but contains over 50% of snake venom metalloproteases (SVMPs). Also, type A+B venom contains a combination of Mojave toxin and SVMP. The use of an anti-disintegrin antibody in a simple Enzyme-Linked Immunosorbent Assay (ELISA) can be used to identify the difference between the venoms of the type A, B, and A+B Mohave rattlesnakes. This study implements the use of an anti-recombinant disintegrin polyclonal antibody (ARDPA) for the detection of disintegrins and ADAMs (a disintegrin and metalloproteases) in individual crude snake venoms of Mohave rattlesnakes (Crotalus scutulatus scutulatus) of varying geographical locations. After correlation with Western blots, coagulation activity and LD50 data, it was determined that the antibody allows for a quick and cost-efficient identification of venom types. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Preparation and application of polyclonal antibodiesagainst KSHV v-cyclin.

    Science.gov (United States)

    Xue, Min; Guo, Yuanyuan; Yan, Qin; Qin, Di; Lu, Chun

    2013-09-01

    We prepared rabbit polyclonal antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded v-cyclin (ORF 72) and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypeptides of v-cyclin were designed and synthesized. A fragment of the v-cyclin gene was cloned into a eukaryotic expression vector pEF-MCS-Flag-IRES/Puro to construct a recombinant vector, pEF v-cyclin. Then, pEF v-cyclin was transfected into 293T and EA.hy926 cells to obtain v-cyclin-Flag fusion proteins. Six New Zealand white rabbits were immunized with KLH-conjugated peptides to generate polyclonal antibodies against v-cyclin. The polyclonal antibodies were then characterized by ELISA and Western blotting assays. Finally, the polyclonal antibodies against v-cyclin were used to detect natural viral protein expressed in BCBL-1, BC-3, and JSC-1 cells. The results showed that using the Flag antibody, v-cyclin-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-v-cyclin. Furthermore, ELISA showed that the titer of the induced polyclonal rabbit anti-v-cyclin antibodies was higher than 1:8,000. In Western blotting assays, the antibodies reacted specifically with the v-cyclin-Flag fusion protein as well as the natural viral protein. The recombinant expression vector pEF-v-cyclin was constructed successfully, and the polyclonal antibodies prepared can be used for various biological tests including ELISA and Western blotting assays.

  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. Preparation and Application of Canine Anti-Canine Parvovirus Polyclonal Antibody%犬细小病毒特异性抗血清的制备及应用研究

    Institute of Scientific and Technical Information of China (English)

    薛青红; 李润; 印春生; 支海兵; 夏业才; 李宁

    2014-01-01

    为制备犬细小病毒特异性抗血清,用犬细小病毒DD株免疫健康易感比格犬,无菌采血后分离血清制备犬抗CPV-2血清。通过SN、ELISA、IFA、HI方法对制备的血清进行检测,未检出犬类常见的5种病毒抗体,说明该血清特异性良好。经测定,该血清中和抗体效价为1∶512;HI效价为1∶1280。应用结果表明,该血清对不同犬细小病毒毒株中和能力相当,对CPV-2感染犬有良好的治疗效果。%Sensitive canines were immunized with CPV DD strain for preparation anti-CPV-2 polyclonal antibody. The collected canine antisera were free of 5 kinds of foreign antibody by SN, ELISA, IFA or HI. The titers of the serum were 1 ∶ 512 by SN, 1 ∶ 1280 by HI. The studies showed that the antisera had the same neutralizing capality for different CPV-2 strains,and also may be used for treatment of CPV-2 canine.

  16. Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes.

    Science.gov (United States)

    Su, S B; Mukaida, N; Wang, J; Nomura, H; Matsushima, K

    1996-11-01

    cDNA cloning has revealed the presence of at least three distinct human receptors for macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES: C-C chemokine receptor (CCR) 1, 4, and 5. To clarify the physiological role of CCR1, we prepared specific antibodies to CCR1 by immunizing rabbits with recombinant glutathione-S-transferase (GST) fused with its NH2-terminal portion. The resultant antibodies stained positively 293 cells transfected with CCR1 cDNA but neither parental cells nor cells transfected with CXCR1 [interleukin-8 (IL-8) receptor type A] cDNA, confirming its specificity. Immunofluorescence analysis revealed that peripheral blood lymphocytes and monocytes but not neutrophils express CCR1. Positive staining of transfectants, monocytes, and lymphocytes was inhibited by the GST protein fused with the NH2-terminal portion of CCR1, further indicating that this antibody recognized the NH2-terminal portion of CC CKR1. A majority of CD3+, CD4+, CD8+, or CD16+ peripheral blood lymphocytes but not CD19+ lymphocytes expressed CCR1. Among CD4+ peripheral blood lymphocytes, CD45RO+ cells expressed a larger number of CCR1 compared with CD45RO-. Moreover, CD34+ cells in human bone marrow as well as cord blood were uniformly stained with this antibody. Furthermore, the antibody inhibited calcium mobilization in CCR1 transfectants stimulated with human rMIP-1alpha, suggesting that its NH2-terminal portion is critically involved in ligand binding or signaling. Finally, the antibody partially inhibited monocyte chemotactic activities of human rMIP-1alpha, suggesting that CCR1 is a functional receptor for MIP-1alpha on human peripheral blood monocytes.

  17. Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody

    Science.gov (United States)

    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the a-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA poly...

  18. Prokaryotic Expression of i-type Lysozyme from Periplaneta americana and Preparation of Its Polyclonal Antibodies%美洲大蠊i型溶菌酶的原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    王赟; 翟素珍; 张春林; 王吉平

    2016-01-01

    The objective of this work is to express i-type lysozyme(PaI)from Periplaneta americana in Escherichia coli and prepare its polyclonal antibodies of anti-PaI in mice. The coding gene of mature peptide region was amplified by PCR and prokaryotic expression vector pET28a-PaI was constructed. PaI-His fusion protein was expressed with IPTG induction and purified by Ni-NTA affinity chromatography. Then, Balb/c mice were immunized with the purified recombinant protein, and the titers of antiserum and the specificity of polyclonal antibodies were detected by indirect ELISA and Western blotting respectively. Results were as below. The length of nucleotide sequence encoding the mature peptide was 414 bp that encoded a putative protein with 137 amino acids. The constructed prokaryotic expression vector pET28a-PaI was successfully expressed in Escherichia coli. SDS-PAGE detection indicated that the PaI-His fusion protein was about 18 kD. Indirect ELISA and Western blotting analysis showed that the antiserum from immunized mice had high titer and specificity. In conclusion, the prokaryotic expression of PaI protein was successfully realized, and the anti-PaI polyclonal antibody with high efficiency and specificity were prepared, which laid the foundation for the further researches on the biological function of PaI protein.%旨在获得美洲大蠊 i 型溶菌酶 PaI 的原核表达蛋白,并制备鼠抗 PaI 多克隆抗体。PCR 扩增 PaI 成熟肽编码序列,构建原核表达载体 pET28a-PaI,诱导表达、纯化 PaI-His 融合蛋白,免疫 Balb/c 小鼠,间接 ELISA 法检测抗血清效价,Western blotting 检测多克隆抗体的特异性。结果显示,PaI 成熟肽部分的编码序列长414 bp,由137个氨基酸组成。构建的原核表达载体pET28a-PaI 在大肠杆菌 Rosetta(DE3)中成功表达,SDS-PAGE 检测显示纯化的 PaI-His 融合蛋白的大小约为18 kD,间接 ELISA和 Western blotting 分析表明免疫小鼠产生的抗

  19. Expression of mouse calreticuin protein and preparation of its polyclonal antibodies%小鼠钙网蛋白的原核表达和多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    汪龚泽; 刘朝奇; 杨建林; 聂纪芹

    2011-01-01

    In order to investigate the biological properties of calreticulin(CRT), a protein present in the mammalian cells with highly conservative and multiple biological properties, a short fragment of mouse CRT was amplified through RT-PCR method and then was cloned into prokaryotic expression vector pDET28a( + ). The recombinant protein CRT was induced by IPTG in E.coli BL2KDE3) and the expressed protein was detected by Western blotting. The purified protein was used to immune mouse to prepare polyclonal antibody and the specificity and titer of the antibody were detected by ELISA, Western blotting and FCM. It was demonstrated that the recombinant prokaryotic expression plasmid pET28a( + ) /CRT was successfully con structed and the CRT protein was expressed in E. Coli BL2KDE3) with high efficiency. Western blot assay showed that this re combinant protein was characterized with its antibody. Mouse immunized with the purified protein produced high titer of anti body. Western blot assay displayed that the CRT protein was highly expressed in some of eukaryotic cells and could specifically combine with the antibody. FCM assay displayed that the antibody could also specifically combine with the membrane extracel lular region of CRT. It is evident that the preparation of recombinant CRT and its polyclonal antibodies have a strong specificity to match with the CRT protein from mouse and human.%钙网蛋白(calreticulin,CRT)是存在于哺乳动物细胞具有高度保守性和多种生物学活性的蛋白,为了更好的研究其生物学活性,本课题组通过PCR方法扩增CRT截短基因,将其克隆到原核表达载体PET-28a(+)中,经大肠杆菌表达并纯化CRT蛋白.以纯化的CRT蛋白抗原免疫BALB/c小鼠,制备多克隆的CRT血清.进一步采用Western blot、ELISA、流式细胞术等技术对制备的抗体进行初步鉴定.结果显示:原核表达重组质粒在大肠杆菌中能高效表达CRT蛋白;获得多抗血清应用Western blot鉴定几种

  20. 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.

  1. 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

  2. 早孕因子多克隆抗体的制备及应用%Preparation and application of polyclonal antibody of early pregnancy factor

    Institute of Scientific and Technical Information of China (English)

    杨杰; 张海涛; 翟频; 潘孝青; 邵乐

    2011-01-01

    通过含有家兔早孕因子的重组质粒EPF-pET-32a(+)转化表达宿主菌Escherichia coli Rosetta,IPTG诱导,融合表达硫氧还原蛋白-兔早孕因子Trx-EPF.亲和层析纯化Trx-EPF,并在柱上进行肠激酶酶切,得到EPF蛋白并进行复性.EPF免疫波尔山羊,制备羊抗兔家兔早孕因子抗血清,硫酸铵沉淀纯化多克隆抗体,用于家兔早孕检测.Western-blot结果表明,羊抗兔家兔早孕因子多克隆抗体对家兔早孕的检出率达到100%.%A stable inducible prokaryotic expression system was set up and the protein Trx-EPF was induced by IPTG after recombinant expression plasmid EPF-pET-32a( +) was transformed into host bacteria Escherichia coli Rosetta. The expressed protein was then purified by Ni-NTA HisBind purification kit and renatured. EPF was prepared by the lysis of enterokinase on Ni-NTA affinity chromatography. Goat was immunized with EPF,and serum was pooled. The goat anti-rabbit polycolonal antibody was prepared through ammonium sulfate precipitation from the serum. Early pregnancy of rabbits were detected by goat anti-rabbit polycolonal antibody by Western-blot, with a positive rate of 100%.

  3. Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain

    Science.gov (United States)

    Chen, Li-Ming; Kelly, Michelle L.; Rojas, José D.; Parker, Mark D.; Gill, Harindarpal S.; Davis, Bruce A.; Boron, Walter F.

    2010-01-01

    NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO3− transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting—both of purified peptides representing the initial ~120aa of the transporters and of full-length transporters expressed in Xenopus oocytes—demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of E18 fetal and adult choroid plexus. NCBE protein is present by western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (~150 kDa). PNGase F reduces the MW of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression. PMID:18061361

  4. Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.

    Science.gov (United States)

    Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan

    2015-01-01

    Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.

  5. Preparation and HRP Labeling of Rabbit Polyclonal Antibodies against Seventeen Mammals%十七种哺乳动物二级抗体的制备和辣根过氧化物酶标记

    Institute of Scientific and Technical Information of China (English)

    林树柱; 刘先菊; 杨帆; 张连峰

    2011-01-01

    目的 制备兔抗17种哺乳动物的二级抗体,并进行辣根过氧化物酶标记,为哺乳血清学检测系统的建立提供工具.方法 利用饱和硫酸铵沉淀法粗纯抗体后,再利用protein A或protein G亲和层析的方法进一步纯化哺乳动物的IgG,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗哺乳动物的二级抗体,采用简易过碘酸钠法对纯化的兔抗哺乳动物的二级抗体进行辣根过氧化物酶标记,通过ELlSA方法测定标记抗体的效价,并利用Western blot方法考察标记抗体的特异性.结果 纯化了恒河猴、东北虎、布氏田鼠、黑线姬鼠、斑羚、原驼、果子狸、食蟹猴、梅花鹿、长爪沙鼠、马鹿、骆驼、大仓鼠、豚鹿、熊猴、大耳羊和雪貂等17种哺乳动物的血清IgG,分别免疫大耳白兔制备了这17种哺乳动物的兔抗血清,免疫双扩散法测定兔抗这17种哺乳动物的抗血清效价均达到1:32,并对纯化的兔抗恒河猴IgG、兔抗东北虎IgG、兔抗布氏田鼠IgG、兔抗黑线姬鼠IgG、兔抗斑羚IgG、兔抗原驼IgG、兔抗果子狸IgG、兔抗食蟹猴IgG、兔抗梅花鹿IgG、兔抗长爪沙鼠IgG、兔抗马鹿IgG、兔抗骆驼IgG、兔抗大仓鼠IgG、兔抗豚鹿IgG、兔抗熊猴IgG、兔抗大耳羊IgG和兔抗雪貂IgG等17种哺乳动物的二级抗体进行了辣根过氧化物酶标记,ELISA测定标记抗体的效价达到1:(2000~60000)左右,Western blots显示标记抗体具有很好的特异性.结论 成功制备了HRP标记的兔抗17种哺乳动物的二级抗体,为哺乳动物血清学检测体系的建立提供了工具.%Objective To prepare the rabbit polyclonal antibodies against seventeen mammals and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of mammals. Methods IgGs of mammals were purified by precipitation with ammonium sulfate

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

    Directory of Open Access Journals (Sweden)

    Sadeq Eivazi

    2015-03-01

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

  7. The preparation of anti-hnRNP A2/B1 polyclonal antibody and its potential application in non-small cell lung cancer%抗hnRNPA2/B1多克隆抗体制备及其在非小细胞肺癌中的应用

    Institute of Scientific and Technical Information of China (English)

    Lejie Cao; Yeshan Li; Meiqing Xu; Runsheng Li; Zubao Lei; Xianwu Li

    2008-01-01

    Objective:In order to evaluate potential application for diagnosis and prognosis of non-small celt lung cancer (NSCLC),as well as to determine its role in the pathogenesis of the disease,we prepared anti-human hnRNP A2/B1 polyclonal antibody.Methods:Prokaryotic expression vector of pET28a (+)-hnRNP A2/B1 was constructed and transformed into E.coli BL21.The recombinant protein induced by IPTG was purified and injected to rabbits for antibody preparation.Expression of hnRNP A2/B 1 was examined in 45 tissues of NSCLC and 16 inflammatory pseudotumor tissues of lung by immunohistochemistry with the antibody.The commercial hnRNPA2/B1 monoclonal antibody was used as a control.Results:(t) Polyclonal antibody against hnRNP A2/B1 with high title was obtained.(2) The positive staining in NSCLC tissues was 62.22%,which was substantially higher than that in normal tissues (40%,P=0.035) or inflammatory pseudotumor tissues (31.25%,P=0.033).(3) Expression of hnRNP A2/B1 positively correlated with age and the history of smoking,whereas it negatively correlated with differentiation staging of tumors.(4) Follow-up study showed that the survival time of patients with positive staining was significantly shorter than that of patients without hnRNP A2/B1 expression (P=0.048).Conclusion:It is successful to make the recombinant protein and prepare the polyclonal antibody agonist human hnRNP A2/B1.It may be a valuable marker for the diagnosis and prognosis of NSCLC.Our results provide a basis for further study in clinical application.

  8. Overexpression of Truncated Barley Cellulose Synthase and Preparation of Its Polyclonal Antibody%大麦纤维素合成酶截短体的重组表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    李学俊; 李新宇; 李迟园; 陈鹏

    2011-01-01

    A truncated non-transmembrane domain of barley cellulose synthase A (Hv-cesA)was determined by analysis of the hydrophobicity and prediction of transmembrane domains. The coding region of truncated non-transmembrane domains was obtained by PCR,and cloned into prokaryotic expression vector pET-28a( + ) with his-tag at its N terminal. The identified construct was transformed into E. Coli and overexpressed protein was purified by cobalt chelating chromatography and polyclonal antiserum was raised against rabbit. The results showed the truncated non-transmembrane domain was expressed in E. Coli Rosetta-gami2(DE3)in the form of inclusion bodies,and western blotting a-nalysis showed the raised antibody can specifically react with the antigen. These results laid the detection basis for further research on the expression of Hv-CesA and cellulose synthesis in cell wall.%对大麦纤维素合成酶(Hordeum vulgare CesA,Hv-CesA)的蛋白质序列进行疏水性分析和跨膜区预测,获得截短的亲水性非跨膜区特征序列,采用PCR扩增截短序列编码区,定向克隆入N端带有His标签的pET-28a(+)表达载体中,并转化大肠杆菌进行诱导表达,利用钴离子螯合层析纯化重组表达蛋白,并制备高效价的多克隆抗体.结果表明,截短的Hv-CesA基因在大肠杆菌Rosetta gami2以包涵体的形式高效表达,western blotting显示制备的多克隆抗体能特异识别其对应的抗原.

  9. Sf9细胞PHB2蛋白的原核表达及其抗血清的制备%Prokaryotic expression of Sf-PHB2 and preparation of polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    马国达; 王茜; 于力

    2011-01-01

    To prepared polyclonal antibody of the Sf-PHB2 protein, the sf-phb2 coding region from cDNA of Sf9 cells was amplified by PCR, and cloned into pET-30a to construct the prokaiyotic expression plasmid pET-sf-phb2. The Sf-PHB2 protein was expressed in E. Coli of BL21 as a fusion protein with 6 histidine residues with IPTG induction. Thereafter, the fusion protein was purified by Ni-NTA affinity chromatography under denature condition and the antiserum against Sf-PHB2 was obtained from the mouse immunized with Sf-PHB2. Specificity and reactivity of Sf-PHB2 antibody were examined by western blot. The present study has laid a foundation for functional study of Sf-PHB2.%为了研究Sf9细胞PHB2蛋白(Sf-PHB2)的功能及其对虾白斑综合症病毒(WSSV)极早期基因ie1的转录调控作用,本研究从昆虫细胞Sf9中扩增sf-phb2基因编码区,克隆至表达载体pET30a中,转化到大肠杆菌BL21,IPTG诱导表达6His-Sf-PHB2重组蛋白.SDS-PAGE分析表明,表达的重组蛋白以包涵体形式存在.在变性条件下,用镍离子螯合柱(Ni-NTA)纯化Sf-PHB2重组蛋白,免疫BALB/c小鼠制备抗体,并用western blot方法检测抗体的反应性.本研究制备了Sf-PHB2抗体,为进一步研究该蛋白的功能奠定基础.

  10. Prokaryotic expression and polyclonal antibody preparation of enterovirus 71 3A protein%人肠道病毒71型3A蛋白的原核表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    黄琴琴; 唐瑞; 杨勇波

    2013-01-01

    Objectives To construct a prokaryotic expression vector of the EV71 3a gene and prepare its recombinant protein and polyclonal antibody for subsequent study.Methods The 3a gene was amplified with PCR and cloned into the vector PET28a to yield PET28a-3a for the prokaryotic expression of 3A protein.The recombinant 3A protein was expressed in E.coli BL21 and was subsequently used to immunize mice.The resulting anti-sera were evaluated using immunofluorescence staining and Western blotting.Results The recombinant protein 3A was efficiently produced in E.coli in the form of inclusion bodies in the expressed protein.Western blot analysis indicated that the resulting mouse anti-3A sera reacted with the eukaryotically expressed EGFP-3A fusion protein.Moreover,the antisera positively recognized cells infected with EV71 according to immunofluorescence staining.Conclusion An anti-3A antibody was successfully prepared and may provide a foundation for subsequent study of the 3a gene.%目的 构建人肠道病毒71型3A基因原核表达质粒,制备重组3A蛋白及其抗体. 方法 PCR方法扩增人肠道病毒71型3A基因,构建原核表达质粒PET28a-3A并转化大肠埃希菌,诱导表达3A重组蛋白,免疫小鼠制备3A蛋白抗体.通过免疫荧光和Western blot方法鉴定抗体特异性. 结果 成功构建了PET28a-3A原核表达质粒并表达了3A重组蛋白,3A蛋白以包涵体的形式存在.细胞免疫荧光和Western blot检测显示,制备的鼠源3A蛋白抗体可识别真核表达的EGFP-3A融合蛋白以及EV71感染细胞表达的3A蛋白. 结论 成功构建了人肠道71型3A基因原核表达质粒,利用该质粒表达的重组蛋白成功制备了特异性的3A蛋白的鼠源抗体.

  11. Produção, caracterização e aplicação de anticorpo policlonal contra Azospirillum amazonense estirpe Am15 Production, characterization and application of polyclonal antibodies against Azospirillum amazonense strain Am15

    Directory of Open Access Journals (Sweden)

    Marinete Flores da Silva

    2009-01-01

    Full Text Available O uso de ferramentas moleculares e imunológicas permite a detecção e o monitoramento específico de microrganismos usados como inoculantes agrícolas. O maior exemplo de sucesso com uso de bactérias diazotróficas na agricultura é o caso da inoculação de soja no Brasil. Entretanto, a inoculação de bactérias diferentes de rizóbio não permite a localização de um sítio específico onde ocorra A redução do nitrogênio atmosférico, pois não há a formação de estruturas nodulares. Este trabalho foi realizado na Embrapa-Agrobiologia, e objetivou produzir e caracterizar um anticorpo policlonal a partir da inoculação de células intactas da estirpe Am15 pertencente à espécie Azospirillum amazonense (Anti-Am15 utilizando o método de ELISA indireto. O anticorpo foi usado em sementes peletizadas com turfa contendo a bactéria-alvo e avaliada sua sobrevivência durante 60 dias. Este anticorpo foi capaz de quantificar populações bacterianas em amostras cujo número mínimo celular foi superior a valores de 100.000 células por mL. De acordo com os resultados, o soro policlonal foi especifico contra o antígeno de interesse, sendo possível sua utilização em estudos de quantificação e monitoramento do Azospirillum sp em plantas de milho que receberam inóculo e no controle da qualidade de inoculante turfoso.Molecular and immunological approaches are useful for monitoring the localization of microrganism used as agricultural inoculants. A successful example is the use of a diazotrophic bacteria, as soybean in Brazil. However, as diazotrophic bacterias other than rizobium do not form nodular structures, the localization of the specific site where nitrogen reduction occurs is impossible. In this study, carried out at Embrapa Agrobiology, a polyclonal antibody against Azospirillum amazonense (As-Am15 was to produced and characterized through an indirect Enzyme Linked Immunosorbent Assay (ELISA. Maize seedscoated with peat containing

  12. 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

  13. Production of Polyclonal Antibodies in Rabbits

    Science.gov (United States)

    1995-10-01

    Gattefosse (Westwood, NJ) I Lot No. 3502. 5. Triglyceride Dynasan , a microcrystalline triglyceride was procured from Dynamit Nobel Chemicals (Rockleigh, NJ...accomplished by placing microspheres into a maurumerizer. The sealant (Suppocire-D or Dynasan ) is dissolved in ethylacetate. As the maurumerizer turns, the

  14. Human-based Polyclonal Antibodies to Ricin

    Science.gov (United States)

    2010-05-21

    components are linked by both a disulfide bond and 8 amino acid peptide. The peptide linkage contains a sequence of amino acids that is the substrate for...COHORT DOSE mcg/m2 DOUBLING MODIFIED FIBONACCI 1000 2000 4000 8000 1600 3200 6400 1100 1600 2100 2800 3800 5000 1 4 2 1

  15. Preparation and identification of polyclonal antibody against protein H1b: the variant of major subunit of human ASGPR%抗人ASGPR大亚基异构体蛋白H1b多克隆抗体的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    刘嘉; 丁红晖; 杨燕; 胡斌; 余源; 黄红平; 陆蒙吉; 杨东亮

    2009-01-01

    AIM: To prepare and identify mouse polyclonal antibody against protein Hlb, which is the variant of major subunit of human ASGPR. METHODS: Hlb specific peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH) for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. ELISA assay was used to detect the titer of the antibody. Antibody was also identified by Western blot and immunohistochemistry assays. RESULTS: Mouse antibody against Hlb was prepared after injection of H1bKLH conjugation. The titer of H1b antibody was about 1:10~5.Western blot confirmed its high specificity. This antibody could also be used for immunohistochemistry analysis. CONCLUSION: The successful preparation of the polyclonal antibody against protein H1b, which can discriminate the two variants of the major subunit of ASGPR with high specificity, will provide an efficient reagent for further study of the physiologic functions of H1b and its role in the pathogenesis of human disease.%目的:制备小鼠抗人ASGPR大亚基异构体蛋白H1b的多克隆抗体,并鉴定其特异性.方法:合成H1b特异性多肽,以匙孔血蓝蛋白(KLH)作为载体构建多肽免疫原,通过致敏小鼠,制备小鼠抗人H1b蛋白的多克隆抗体;ELISA法检测抗体效价,通过Western blot和免疫组织化学检测,鉴定抗体的特异性与适用范围.结果:得到小鼠抗人H1b蛋白的多克隆抗体,效价达1:10~5.纯化后的抗体用于Western blot可高特异性识别真核表达的H1b蛋白,并可用于免疫组织化学实验.结论:成功制备小鼠抗人H1b蛋白的多克隆抗体,该抗体具有较高的效价以及特异性,能区分ASG-PR大亚基的两种剪接异构体,从而为进一步研究H1b蛋白的生理功能及其在人类疾病中的意义提供了有利工具.

  16. 定向偶联制备三肽囊素人工抗原及其多克隆抗体的研制%Preparation of Artificial Antigen by Conjugation to the Amino Group on Lysine of Bursin and Production of Polyclonal Antibodies against Bursin

    Institute of Scientific and Technical Information of China (English)

    邓舜洲; 冷闯; 张文波; 许昌满; 胡杨; 邹柳; 邬向东; 谌南辉; 朱芝秀

    2013-01-01

    为研制三肽囊素(Bursin,KHG-NH2,BS)抗体,采用EDC法将三肽囊素分子赖氨酰(K)上的氨基与载体蛋白(BSA、OVA)定向偶联,分别制备免疫抗原(BSA-KHG-NH2)和检测抗原(OVA-KHG-NH2).以BSA-KHG-NH2免疫5只BALB/c小鼠,经4次免疫后,均产生了较高的抗体水平,其中5号小鼠血清抗体效价达1∶125 000.免疫小鼠血清多克隆抗体与检测抗原的结合不仅能被三肽囊素阻断,而且能被GKHG-NH2四肽特异性阻断,但不能被KHGK四肽阻断,表明本试验制备的三肽囊素人工抗原是由载体蛋白的羧基与三肽囊素分子赖氨酰(K)上的氨基定向偶联而成.利用小鼠多克隆抗体建立了三肽囊素间接竞争ELISA检测方法,线性范围为15~1 000 ng/mL,IC50为160 ng/mL.%In order to produce polyclonal antibodies against Bursin, and develop immunological detective methods for Bursin (BS) , the amino group on - Lys of Bursin molecules were conjugated to bovine serum albumin ( BSA) or chicken ovalbumin ( OVA) by EDC method. The titer of anti-Bursin antibodies in one BLAB/c mice serum, immunized by BSA-KHG-NH2, was about 1:125000, in which polyclonal antibodies binding to OVA-KHG-NH2 could be inhibited by free Bursin and GKHG-NH2, could not be inhibited by KH-GK peptide. These results indicated that the immunized mice produced specifical antibodies against bursin, and the amino group on-Lys of Bursin were conjugated to BSA or OVA. A competitive indirect ELISA ( CI -ELISA) with the anti - Bursin polyclonal antibodies for detecting Bursin was developed, in which the linear range of the inhibition curve was 15-1 000 ng/mL, and the IC50 was 160 ng/mL.

  17. PROKARYOTIC EXPRESSION OF ENVELOPE PROTEIN DOMAIN III OF JAPANESE ENCEPHALITIS VIRUS AND PREPARATION OF POLYCLONAL ANTIBODIES%日本脑炎病毒E III结构域蛋白的表达及多克隆抗体的制备与鉴定

    Institute of Scientific and Technical Information of China (English)

    沈强; 魏建超; 史子学; 常维山; 赵凡凡; 蒋春英; 王水明; 马志永

    2013-01-01

    使用原核表达系统融合表达乙型脑炎病毒EIII结构域蛋白,经过纯化后多次免疫BALB/c小鼠,制备了鼠抗JEV E蛋白的多克隆抗体。Western blot结果表明该抗体与重组蛋白能够发生明显反应,与浓缩的JEV反应显出1条分子量大小约为53 kDa的目的条带(JEV E蛋白);间接免疫荧光(indirect immunofluorescence,IFA)显示,多克隆抗体与BHK-21细胞内的JEV作用,在细胞质内产生绿色荧光,而对照组无特异性荧光;空斑减少中和试验测定多抗血清的中和抗体效价(PRNT50)为1:160。以上结果暗示制备的多克隆抗体能与重组蛋白和JEV E蛋白都能发生良好反应,并且该多抗有较高水平的JEV中和抗体效价。%In this study, E protein domain III (EDIII) of JEV was expressed in E.coli and purified with a His column. BALB/c mice were immunized with EDIII and polyclonal antibodies to E protein were prepared. The reactivity of the prepared antibodies was characterized in Western blotting and IFA. Furthermore, the polyclonal antibodies at dilution of 1:160 blocked the entry of JEV into BHK-21 cells in plaque reduction neutralization test. These results demonstrated that the polyclonal antibodies of JEV EDIII possessed good antigenic specificity and could be used for development of a diagnostic method of JEV infection.

  18. Preparation of hemocyanin polyclonal antibody and the identification of 35 kDa hemocyanin fragment in HaHotis diversicolor Reeve%杂色鲍血蓝蛋白多克隆抗体的制备与血蓝蛋白35kDa片段鉴定

    Institute of Scientific and Technical Information of China (English)

    姜敬哲; 韩焘; 王江勇; 杨慧英; 刘金叶

    2012-01-01

    以杂色鲍(Haliotis diversicolor Reeve)为研究对象,通过密度梯度离心方法纯化得到高纯度血蓝蛋白,以其作为抗原皮下注射免疫新西兰大白兔,从而获得高效价的兔源多克隆抗血清。进一步通过ProteinA抗原亲和纯化的方法对该抗血清纯化,最终获得效价更高、检测特异性更好的血蓝蛋白多克隆抗体。应用该抗体进行Western检测发现,鲍血淋巴中存在着多样的血蓝蛋白衍生产物;进一步结合质谱技术对其中35kDa条带进行鉴定发现,其来源于血蓝蛋白I型亚基的H结构域。%Haliotis diversicolor Reeve, was taken as research material. High purity hemocyanin proteins were puri- fied from the hemolymph of abalone using CsC1 density gradient centrifugation method. By injection of the purified antigen to a rabbit, high titer polyclonal anti-serum was acquired. The anti-serum was further processed with the protein A affinity purification to produce purified antibodies. Finally, the purified polyclonal antibodies with higher titer and specificity were acquired. Further Western blotting with these antibodies, plenty of hemocyanin was found as derivates of various molecular weights in abalone hemolymph.

  19. 登革2型病毒贵州分离株NS1部分基因原核表达蛋白及其多克隆抗体制备%Prokaryotic expression and production of polyclonal antibodies against nonstructural protein 1 of the Guizhou strain of the dengue type 2 virus

    Institute of Scientific and Technical Information of China (English)

    朱海东; 左丽; 任丽娟

    2012-01-01

    Objectives To prokaryotically express the nonstructural protein NS1 of the Guizhou strain of dengue virus type 2 and to prepare polyclonal antibodies against NS1. Methods A partial sequence of the NS1 gene of the M strain of dengue type 2 virus was inserted into the prokaryotic vector pET28a( + ) to construct an expression plasmid. NS1 fusion protein was expressed after the expression plasmid was transformed into E. coli BL2KDE3) and induced with IPTG. Purified NSl-poly(His)-tagged fusion protein was obtained from the expressed product using the His Trap Kit. Anti-NS1B antiserum against NS1 was prepared in mice and the antibody titer was determined with ELISA. Results NS1 of the dengue type 2 virus was successfully expressed as a fusion protein. Polyclonal antibodies against NS1 were prepared, and the antibody titer was 1: 800 according to ELISA. After purification, NS1 recombinant protein was combined with His monoclonal antibodies in Western blotting Conclusion The nonstructural protein of a partial sequence of the NS1 gene of the M strain of dengue type 2 virus was expressed prokaryotically, and effective and specific polyclonal antibodies were induced in mice. This provides a basis for further understanding of the structure and function of NS1 of the dengue virus.%目的 原核表达、纯化DENV-2贵州分离株NS1部分序列表达蛋白,并制备其多克隆抗体以研究其功能.方法 合成DENV-2 M株NS1部分基因序列,克隆到原核表达载体pET28(a),转化大肠埃希菌BL21 (DE3);用IPTG诱导表达,Ni柱亲和层析纯化、回收融合蛋白;用纯化融合蛋白免疫BALB/c鼠,制备多克隆抗体,采用ELISA法检测抗体效价.结果 原核表达了NS1部分序列融合蛋白,并获得了其多克隆抗体,ELISA检测抗体效价为1∶800.Western blot检测该蛋白能被His标签抗体识别.结论 DENV-2M株NS1部分序列表达蛋白可诱导小鼠产生较高效价和特异性的多克隆抗体,为进一步研究DENV-2M株NS1

  20. Preparation of the polyclonal antibody of human papillomavirus type HPV33 for E6E7%人乳头瘤病毒 HPV33型 E6 E7蛋白的表达及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    卢钧雄; 段炼; 王若伦; 易俊波

    2016-01-01

    Objective To clone and efficiently express E6E7 proteins of human papilloma virus HPV33 in E.coli.The recombinant proteins were purified and immunized to mice, and polyclonal antibodies were purified.Methods E6 and E7 genes of HPV33 were amplified by PCR using HPV33 human papilloma virus as template, and were sub-cloned into pET28a and pCDNA3.1 /His C vectors.The recombinant plasmids pET28a-HPV33 E6 and pET28a-HPV33 E7 were transformed into Escherichia coli BL21 (DE3) strain.We purified the recombinant proteins and immunized mice for preparing antibodies, and the polyclonal antibodies were purified. Moreover the plasmids pCDNA3.1 /His C-HPV33 E6 and pCDNA3.1 /His C-HPV33 E7 were transfected into 293 cells.The cells were used in immunofluorescence and Western-Blot to detect antibody specificity. Results The recombinant proteins were successfully obtained, and the polyclonal antibody was prepared, which had good specificity. Conclusion The recombinant proteins and antibodies may be used in the clinical detection and pathogenesis research of HPV33 type human papilloma disease.%目的:表达人乳头瘤HPV33型病毒癌蛋白E6E7,并免疫小鼠制备抗体。方法从HPV33型基因组中扩增出E6 E7的基因片段,通过基因测序加以证实;将其克隆至原核表达载体pET28a中,在大肠埃希菌中经IPTG诱导表达,经HIS亲核层析对重组蛋白进行纯化,收集重组蛋白免疫的小鼠阳性血清,经ProteinA/G亲核层析柱纯化得到多克隆抗体,同时将E6 E7克隆至真核表达载体pCDNA3.1/His C中,转染293细胞,分别采用免疫荧光法和Western Blot检测抗体的特异性。结果用得到高纯度的重组蛋白E6、E7制备的多克隆抗体,具有良好的特异性。结论这种高纯度重组E6、E7蛋白和抗体有望用于HPV33型人乳头瘤疾病的临床检测及发生机制的研究。

  1. Antibody

    Science.gov (United States)

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

  2. Polyclonal clustering algorithm and its convergence

    Institute of Scientific and Technical Information of China (English)

    MA Li; JIAO Li-cheng; BAI Lin; CHEN Chang-guo

    2008-01-01

    Being characteristic of non-teacher learning, self-organization, memory, and noise resistance, the artificial immune system is a research focus in the field of intelligent information processing. Based on the basic principles of organism immune and clonal selection, this article presents a polyclonal clustering algorithm characteristic of self-adaptation. According to the core idea of the algorithm, various immune operators in the artificial immune system are employed in the clustering process; moreover, clustering numbers are adjusted in accordance with the affinity function. Introduction of the recombination operator can effectively enhance the diversity of the individual antibody in a generation population, so that the searching scope for solutions is enlarged and the premature phenomenon of the algorithm is avoided. Besides, introduction of the inconsistent mutation operator enhances the adaptability and optimizes the performance of local solution seeking. Meanwhile, the convergence of the algorithm is accelerated. In addition, the article also proves the convergence of the algorithm by employing the Markov chain. Results of the data simulation experiment show that the algorithm is capable of obtaining reasonable and effective cluster.

  3. Purification of soybean major allergen Gly m Bd 28K and preparation of its polyclonal antibodies%大豆主要过敏原GlymBd28K的分离纯化及多抗血清的制备

    Institute of Scientific and Technical Information of China (English)

    席俊; 高学梅; 闫慧丽; 陆启玉

    2015-01-01

    The 28K(Gly m Bd 28K)was isolated and purified from defatted soybean flour. Then it was used to prepare the rabbit polyclonal antisera. 28K protein was extracted with an alkali solution and acid formulation. 28K crude protein was attended by ammonium sulfate precipitation method, and then DEAE–Sepharose CL–6B further purified 28K,28K was detected purity by SDS–PAGE electrophoresis,prepared the rabbit polyclonal antiserum,tested antisera titer by indirect ELISA. In the study,28K was successfully purified,the titer of rabbit antisera obtained was up to 1∶51200,and rabbit antisera were prepared. That might provide a basis for the study on major allergens of soybean protein 28K monoclonal antibody and 28K IgG determine.%该文从脱脂大豆粉中分离纯化28K(Gly m Bd 28K)蛋白,并制备多抗血清。方法用碱溶酸提法提取28K蛋白,盐析法制得28K粗蛋白,DEAE–Sepharose CL–6B层析纯化28K蛋白, SDS–聚丙烯酰胺凝胶电泳检测蛋白的纯度,制备兔抗血清,用间接ELISA法检测效价。该实验纯化出28K蛋白,制备了抗28K的多抗血清,效价为1∶51200。为研究大豆主要过敏原蛋白28K的单克隆抗体制备及28K的IgG结合表位的确定奠定了基础。

  4. Involvement of allelopathy in the formation of monospecific colonies of ferns.

    Science.gov (United States)

    Kato-Noguchi, Hisashi

    2015-05-01

    Some fern species often dominate plant communities by forming large monospecific colonies. However, the potential mechanism for this domination of the ferns remains obscure. Many plants secrete a wide range of compounds into the rhizosphere and change the chemical and physical properties of the rhizosphere soil. Through the secretion of compounds, such as allelopathic substances, plants inhibit the germination and growth of neighboring plants to compete more effectively for the resources. Ferns contain a variety of secondary metabolites and some of those compounds are released from the ferns into the rhizosphere soil, either as exudates from living ferns or by decomposition of fern residues in sufficient quantities to affect the germination and growth of neighboring plants as allelopathic substances. Therefore, allelopathic chemical interaction of the ferns with neighboring plants may play an important role in the formation of the monospecific colonies of the ferns.

  5. 蓝氏贾第鞭毛虫α-4贾第素特异性多克隆抗体的制备及免疫电镜定位研究%Preparation of a polyclonal antibody specific for α-4 giardin and its immunoelectron microscopic localization

    Institute of Scientific and Technical Information of China (English)

    王洋; 王沂; 杨文思; 李冀; 余源; 沈海娥; 刘青; 田喜凤

    2012-01-01

    Objectives To prepare specific polyclonal antibody against α-4 giardin and detect its subcellular localization using im-munogold electron microscopy. Methods Synthetic antigenic peptides designed using bioinformatics were coupled to KLH, an immunogen, and then administered to rabbits. Antibodies were purified using Protein A affinity-chromatography. The affinity and specificity of antibodies were determined in Western blotting using recombinant α-4 giardin protein and Giardia lamblia trophozoite lysate, respectively. The subcellular localization of cr4 giardin was determined with immunogold electron microscopy using the antibody with the highest specificity. Results Three potential antigenic peptides were synthesized and coupled to KLH. Three highly purified antibodies were obtained from the sera of immunized rabbits using affinity-chromatography. All three antibodies had a high affinity for recombinant α-4 giardin but only anti-P2 had the highest specificity. Immunogold electron microscopy performed using anti-P2 showed that α-4 giardin was located at the base and outer surface of the flagella and occasionally in the cytoplasm. Conclusion The prepared polyclonal antibody is highly specific for or4 giardin. Immunogold electron microscopy performed using anti-P2 showed that α-4 giardin is mainly located in the flagella of Giardia lamblia trophozoites.%目的 制备针对蓝氏贾第鞭毛虫α-4贾第素的特异性抗体,免疫胶体金电镜观察该贾第素的亚细胞定位.方法 生物信息学分析并合成α-4贾第素抗原肽,与钥孔血蓝蛋白(KLH)偶联后免疫新西兰白兔,Protein A亲和层析纯化抗血清,采用α-4贾第素重组蛋白和贾第虫滋养体全虫体裂解物通过Western blot验证抗血清结合力和结合特异性,选择高特异性抗体通过免疫胶体金电镜观察α-4贾第素的亚细胞定位. 结果 筛选出3段可能的抗原肽,合成的抗原肽与KLH偶联后免疫

  6. PRODUCCIÓN DE LA PROTEÍNA DE CHOQUE TÉRMICO HSC70 RECOMBINANTE EN Escherichia Coli BL21(DE3 PARA GENERAR ANTICUERPOS POLICLONALES PRODUCTION OF RECOMBINANT HEAT SHOCK PROTEIN HSC70 IN Escherinchia coli BL21(DE3 TO OBTAIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    Rocio Cely Castro

    2006-07-01

    Full Text Available Antecedentes. Los organismos vivos responden al estrés aumentando la síntesis de proteínas. El estrés por choque térmico ha sido el más estudiado y las proteínas que se inducen, se han denominado genéricamente como proteínas de choque térmico. Objetivo.En este trabajo se establecieron las condiciones óptimas de producción de la proteína HSC70, expresada en E.coli BL21 (DE3 y de los anticuerpos policlonales que permitan identificarla. Material y métodos. Mediante varios ensayos se establecieron las concentraciones óptimas del agente inductor isopropil- -D-thiogalacto­piranosido (PTG, del inóculo bacteriano, de empleo de los plásmidos de expresión pET-3a y pET-28a (+; y el método más eficiente para la recuperación de las formas soluble o insoluble de la proteína y de anticuerpos policlonales que la identifiquen. Resultados. Encontramos que el inóculo de cinco colonias con IPTG (2mM en tubos con cinco mililitros de medio modificado e incubadas por 24 horas a 37°C con agitación constante (200 r.p.m y tratamiento de sonicación, produce el mejor rendimiento de HSC70. La calidad de la proteína inducida se estableció mediante “Western blotting”. Conclusión. La proteína recombinante así obtenida permitió, generar anticuerpos policlonales que a su vez permiten detectar la proteína HSC70 natural en la membrana citoplasmática de diferentes células por inmunofluorescencia, en ELISA, en Western Blot y en pruebas de bloqueo de infección de rotavirus.Background. Live organisms respond against stressing conditions raising the production of stress proteins. The heat shock stress is a very well kwon condition that produces heat shock proteins. Objective. In this work we determined the optimum conditions to produce HSC70 protein in E. coli BL12 (DE3 under stress conditions, and the best way to obtain its polyclonal antibodies. Materials and methods. The best concen­trations for isopropyl- ß -D

  7. Prokaryotic Expression of the Soluble Cry2Ab Protein from Bacillus thuringiensis and Preparation of the Polyclonal Antibody Against Cry2Ab%苏云金杆菌Cry2Ab可溶蛋白的原核表达及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    邵恩斯; 林莉; 关雄

    2013-01-01

    Cry2Ab toxin of Bacillus thuringiensis is a toxic protein, which is wildly used in controlling lepidopteran pest in agricultural production. In this research, the cry2A b gene (1 914 bp) was amplified from total DNA of B. Thuringiensis WB9 strain by a pair of primer designed by the full-length sequence of published crylAb gene. Then, cry2Ab was ligated with linearized pGEX-KG vector by restriction enzyme BamH Ⅰ and Xho Ⅰ for the construction of cry2Ab-pk expression vector. The soluble Cry2Ab-GST fusion protein (approximately 90 kD) was obtained after transferring Cry2Ab-PK expression vector into Escherichia coli BL21 (DE3) and then inducing by 0.8 mmol/L IPTG at 16℃ for 36 h. Total soluble protein was purified by batch purification and GST tag was removed by the use of prescission protease to obtain soluble Cry2Ab protein (approximately 65 kD). Polyclonal antibody against Cry2Ab was produced by immunizing the purified Cry2Ab to New Zealand white rabbit (Oryctolagus cuniculus) after three times of intramuscular injection and one time of intravenous injection. The titter of antibody was over 1:150 000, measured by indirect ELISA. Specificity of the prepared antibody was determined by Western blot, showing that the polyclonal antibody against the Cry2Aa or Cry2Ab protein was positive and the antibody against Cry1Ab or Cry3Aa protein was negative. These results indicated that antibody against Cry2Ab protein can specifically identify Cry2A protein but cannot identify other three domains Cry protein including CrylAb and Cry3Aa. These results will provide technical support for further study of Cry2A toxins mechanism and the interaction between Cry2A toxins and its receptors.%苏云金杆菌Cry2Ab蛋白是一类对鳞翅目昆虫有特异性毒性作用的毒素蛋白,已广泛应用于针对鳞翅目害虫的防治之中.依据苏云金杆菌cry2Ab基因序列设计一对全长引物,从苏云金杆菌(Bacillus thuringiensis WB9菌株总DNA中克隆出cry2Ab

  8. 抗酮洛芬多克隆抗体的制备及其间接竞争ELISA检测方法的建立%Preparation of polyclonal antibody and establishment of indirect competitive ELISA method for detection of ketoprofen

    Institute of Scientific and Technical Information of China (English)

    王晓敏; 庄惠生

    2012-01-01

    分别将酮洛芬与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制得免疫原和包被原,经过免疫新西兰白兔制备多克隆抗体,抗体经纯化后效价为1:128000.使用自制的抗体,建立了测定酮洛芬的间接竞争酶联免疫吸附(ic-ELISA)新方法.ic-ELISA的线性范围为0.010 ~ 10.0μg/L,IC50为0.2351g/L,最低检测限为0.0040μg/L,线性回归方程为y=- 22.97p+104.5(R2=0.980),与布洛芬、双氯酚酸的交叉反应率均小于4%,方法可用于水体中酮洛芬的检测.%Ketoprofen (KET) was conjugated with albumin bovine (BSA) and albumin egg (OVA) to prepare immunogen and coating antigen, respectively. Polyclonal antibody was generated by immunizing the New Zealand white rabbit. The titer of the antibody after purified was 1:128000. The indirect competitive ELISA for ketoprofen was developed by using the homemade antibody. For the indirect ELISA the calibration range was 0. 010 ~10. 0μg/L, an average IC50 was 0. 235μg/L and the detection limit was 0.004μg/L. The equation was y = - 22. 97 ρ + 104. 5 ( R2 = 0. 980 ). The cross reactivity with diclofenac and ibuprofen was less than 4% . The sensitivity and specificity of the method were high, which can be used for the detection of the ketoprofen in water possible.

  9. Determination of oxytocin in biological samples by isocratic high-performance liquid chromatography with coulometric detection using C18 solid-phase extraction and polyclonal antibody-based immunoaffinity column purification.

    Science.gov (United States)

    Kukucka, M A; Misra, H P

    1994-03-04

    A specific high-performance liquid chromatographic (HPLC) method is described for the reliable quantitation of oxytocin using culture media supernatants. The procedure employs solid-phase extraction, antibody-based immunoaffinity purification and isocratic HPLC with dual channel coulometric detection (ED). The lower limit of detection for this cyclic nonapeptide was 40 pg (40 fmol). Due to its relative simplicity, specificity and precision, the HPLC-ED of oxytocin is an accurate and attractive alternative to many existing quantitative methods.

  10. Polyclonal light chains in cerebrovascular disease

    Directory of Open Access Journals (Sweden)

    Patrizia Fiori

    2010-08-01

    Full Text Available Patrizia Fiori1, Maria Giannetti Luigi2, Linda Iurato1, Carminantonio Tammaro3, Gigliola Esposito3, Antonio Monaco11Central Operative Unit of Neurology (Dir. A Monaco, 2Infantile Neuropsychiatry and Social Service (Dir. LM Giannetti, 3Laboratory (Dir. CA Tammaro, ASL AV, Civil Hospital of Ariano Irpino, University of Naples, ItalyAbstract: Altered membrane permeability is a hallmark of inflammation and ischemia with systemic spreading. Renal dysfunction is a risk factor for cardiovascular, cerebrovascular, and metabolic diseases. The aim of the present study was to assess proteinuria and urinary polyclonal light chains in acute stroke and chronic cerebrovascular disease compared with other neurologic diseases. Our results showed significantly increased levels of urinary polyclonal light chains in cerebrovascular disease compared with other neurologic diseases. The highest values of urinary polyclonal κ chains were found in acute stroke compared with chronic cerebrovascular disease and other neurologic diseases, while the level of λ chains was mainly increased in chronic cerebrovascular diseases. The shift to chronic renal failure seems to be signaled by a decreased polyclonal light chain/creatinemia ratio. The absence of a significant correlation with blood pressure and other seric parameters suggests that polyclonal light chains are an early marker of reversible vascular impairment with renal dysfunction before progression to irreversible renal failure and need for dialysis and/or intensive care.Keywords: polyclonal light chains, cerebrovascular disease, renal failure

  11. 斑马鱼心脏发育候选基因Pygo1多克隆抗体的制备及检测%Preparation and Detection of Polyclonal Antibody of Heart Development Candidate Genes Pygo1 in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    唐雄卓; 江志钢; 赵碧峰; 李发祥; 谢华平; 邓云; 吴秀山

    2011-01-01

    Pygol is a candidate gene for heart development,which might play an important role in Zebrafish heart development.Hydrophilic antigen region of Pygol is selected by bioinformatic method.Pygol fragment was amplified by PCR and purified.The fragment was inserted into the prokaryotic expression vector pGEMT-4T-l.This new recombined plasmid was transformed into E.Coli and expression of the GST fusion protein was induced with IPTG.After purified by GST affinity chromatography,GST-Pygol fusion protein was used to immune the New Zealand white rabbits to prepare polyclonal antibody.The antibody titer and specificity was identified by Western blot.%Pygo1是心脏发育候选基因,可能在斑马鱼心脏发育过程中起着重要的调控作用.利用生物信息学选择斑马鱼Pygo1基因抗原亲水区,将扩增出的PCR片段克隆到原核表达pGEMT-4T-1载体中,转入E.coli中后通过IPTG(Isopropyl β-D-thiogalactoside)分别诱导表达GST融合蛋白.蛋白经纯化分离后用于免疫新西兰大白兔制备多克隆抗体.用Western Blot检测抗体的效价和特异性.

  12. Expression,purification and polyclonal antibody preparation of recombinant E.coli outer membrane protein OmpC%重组大肠埃希菌外膜蛋白 C的表达、纯化及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    俱雄; 陈春琳; 刘祥; 王成祥; 王令; 吴三桥; 张涛

    2015-01-01

    E.coli outer membrane protein ( OmpC) could oppose bacterium infection by inducing body immunologic mechanism, and had important perspective in vaccine development.The OmpC protein expres-sion strain was obtained by molecular clone;OmpC was purified by the way of SDS-PAGE gel extraction and urea gradient renaturation, and was used to immunize mice to prepare the polyclonal antibody.The OmpC an-tibody titer was 1∶6 400 by ELISA, and Western blotting proved that the antiserum had good specificity. OmpC homology analysis by DNAMAN showed that different bacteria had high homology;Phylogenetic analysis of OmpC by MEGA proved the close relationship between intestinal tract pathogenic bacteria.These studies laid the groundwork for the immunological and functional research of OmpC.%采用分子克隆方法获得大肠埃希菌外膜蛋白OmpC表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得OmpC蛋白,免疫小鼠制备OmpC蛋白多克隆抗体。 ELISA法证实OmpC抗血清滴度达1∶6400倍,Western blotting发现OmpC抗血清具有很好的特异性。通过DNAMAN软件对OmpC序列同源性分析显示不同细菌间存在较高同源性,采用MEGA软件对OmpC的系统进化分析发现肠道致病菌亲缘关系更近。为OmpC蛋白免疫学功能研究奠定基础。

  13. Purification and polyclonal antibody preparation of Cd-MT from Pteria penguin%企鹅珍珠贝镉金属硫蛋白的分离纯化及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    吴晓萍; 廖爱琳; 章超桦; 廖艳; 卢虹玉; 杨捷

    2011-01-01

    Heavy metals in three wastes are discharged into water by a variety of means with the industry development such as mine, smelting and electroplating, which lead to pollution of aquatic environments and threaten human health and biological survival seriously. Cadmium(Cd) is one of the main marine pollutants. As Cd can accumulate in different tissues of shellfish through food chains, which threaten the quality and safety of shellfish foods, it is extremely necessary to research and monitor conditions of shellfish contamination with Cd. Cadmium-metallothioneins (Cd-MTs) are important biomarker used for indicating cadmium pollution in aquatic environment. The research on these Cd-MTs not only is propitious to establish Cd-MT immunoassay method, but also provides the theoretical basis for technology development in monitoring heavy metal pollution. Cd-MT was induced to synthesize by injecting different concentrations of CdCl2(0 -0.8 mg/L)in Pteria penguin and was isolated and purified from whole tissues of P. penguin by tissue homogenization, freezing centrifugation, heat treatment and Sephadex G-50 gel column chromatography. The characteristic absorption of Cd-MT in crude extract by ultraviolet spectrophotometry was the strongest at 0.8 mg/L Cd2 +. The results showed that the content of Cd-MT in P. penguin increased with concentration of Cd2 +. The results from SDS-PAGE assay showed that molecular weight of purified Cd-MT and its dimer were 9 ku and 18 ku respectively. Then Cd-MT was coupled to bovine serum albumin (BSA) with glutaraldehyde and polyclonal antiserum by immunizing rabbits with Cd-MT-BSA was prepared. The titer of antiserum was measured by indirect ELISA assay and reached 1:12 800. IgG ( molecular weight is about 35 ku) in the antiserum was obtained by methods including saturated ammonium persulfate precipitation and DEAE-Sephadex A50 column chromatography. ELISA assay showed that the IgG from antiserum could react to Cd-MT of P. martensi as well as Cd

  14. 应用新型抗胎儿血红蛋白抗体富集母体循环中胎儿有核红细胞%Enrichment of circulating fetal nucleated red blood cell for non-invasive prenatal diagnosis with a new polyclonal antibody specific to fetal hemoglobin

    Institute of Scientific and Technical Information of China (English)

    汤冬玲; 周新; 李艳; 郑芳; 李从荣; 荣媛

    2008-01-01

    目的 利用多肽合成技术制备抗胎儿血红蛋白(fetal hem-oglobin,HBF)γ链的抗体,探讨其用于检测孕妇外周血中胎儿有核红细胞(nucleated red blood cell,NRBC)进行无创性产前基因诊断的可行性.方法 针对胎儿血红蛋白的特异性抗原表位,选定第69~78位HbF-γ特异的11个氨基酸残基的肽段为免疫原,将人工合成的胎儿血红蛋白γ链的多肽与载体蛋白(KLH)偶联,佐剂乳化后免疫羊,制备羊抗人胎儿血红蛋白的抗血清,经蛋白G纯化,HbF特异性抗体标记、识别、显微操作法富集32例孕周为22~39周的孕妇外周血中的胎儿有核红细胞,引物延伸预扩增后,利用9个短串联重复序列(short tandem repeat,STR)的复合扩增方法对富集的阳性细胞进行扩增,用于孕妇外周血中富集的HbF阳性细胞胎儿来源的遗传学鉴定.结果 经HbF多克隆抗体标记,32名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC,并具有鲜明的形态学特征,光学显微镜下可见NRBC细胞质呈棕黄色,核浆比例较低,苏木素复染后胞核呈蓝色,明显区别于其他细胞,每份样本出现NRBC 0.6~1.8个/ml,共计183个,平均为1.3个/ml,经STR多态性基因位点鉴定,准确率为90.6%.结论 利用多肽合成技术制备的抗胎儿血红蛋白γ链的抗体能有效识别母血中的胎儿有核红细胞,可应用于无创性产前基因诊断,具备良好的应用前景.%Objective To investigate the feasibility of a new polyclonal antibody specific to fetal hemoglobin (HbF) and its application in enrichment of circulating fetal nucleated red blood cell(NRBC) for non-invasive prenatal diagnosis. Methods A polyclonal antibody against a synthetic peptide comprising residues 69-78 of the γ-chain of HbF was prepared and conjugated to carrier protein KLH as the immunogen according to the specific antigenic determinant. The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and

  15. Size variation within monospecific plant canopies under enhanced UV-B radiation

    Energy Technology Data Exchange (ETDEWEB)

    Searles, P.S.; Flint, S.D.; Caldwell, M.M. [Utah State Univ., Logan, UT (United States)] [and others

    1995-06-01

    Monospecific stands often exhibit distinct size distributions with respect to individual plant size and morphology. Previous work has shown that enhanced ultraviolet-B (UV-B) radiation (280-320 nm; simulating a 20% stratospheric ozone depletion) can change the mean response of individual plants and plant parts within dense monospecific stands of annual dicots and monocots in a glasshouse experiment. Further analysis of the data reveal that individual plant variability may also be altered by UV-B. When analyzed across all (12) species, the coefficient of variation (CV) for fraction of biomass in tillers or branches and leaf length was reduced under enhanced UV-B compared to a no UV-B control. Number of leaves exhibited a decrease in the CV to a lesser degree. No difference in CV was seen for total plant biomass. The results suggest increased solar UV-B associated with ozone depletion has the potential to reduce size variability in plant populations and thus may alter intraspecific competition.

  16. 曼氏血吸虫虫卵白细胞介素-4诱导因子的原核表达及多克隆抗体的制备%Prokaryotic Expression of IL-4-inducing Principle of Schistosoma Eggs of Schistosoma mansoni and Preparation of Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    赵娜; 李世杰; 徐晓芳; 吴威; 范大为; 宫鹏涛; 李建华; 张西臣

    2011-01-01

    目的 原核表达曼氏血吸虫虫卵白细胞介素-4诱导因子(IL-4-inducing principle of schistosoma eggs,IPSE),并制备小鼠多克隆抗体.方法 人工合成IPSE基因,采用PCR技术克隆其成熟体开放阅读框,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-IPSE.转化E coli BL21(DE3),IPTG诱导表达.表达产物经SDS-PAGE和Western blot分析后,应用组氨酸标签蛋白纯化柱纯化包涵体蛋白,免疫BALB/c鼠,制备多克隆抗体.结果 克隆的IPSE基因与GenBank中登录的基因序列同源性为100%;重组表达质粒pET-28a-IPSE经PCR及双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约为16 000,以包涵体形式表达,表达量约占菌体总蛋白的30%,可与鼠抗His单抗特异性结合;用纯化的包涵体蛋白免疫BALB/c小鼠后,获得的特异性抗血清效价为2× 10-4,该血清可与IPSE蛋白发生特异性反应.结论 已原核表达、纯化了曼氏血吸虫IPSE蛋白,并制备了高滴度的特异鼠抗血清,为进一步从日本血吸虫中筛选相似蛋白及研究其功能奠定了基础.%Objective To expression the IL-4-inducing principle of schistosoma eggs(Lpse) of Schistosoma mansoni in prokary otic cells and prepare mouse polyclonal antibody. Methods IPSE gene was synthesized artificially, of which the open reading frame (ORF) was cloned by PCR and inserted into expression vector Pet-28a. The constructed recombinant plasmid Pet-28a-IPSE was transformed to E. Coli BL21 (DE3) for expression under induction of IPTG. The expressed protein was identified by SDS-PAGE and Western blot, then purified by His GraviTrap protein purification column chromatography and used for immunization of BALB/c mice to prepare polyclonal antibody. Results Sequencing result showed a homology of 100% of the cloned IPSE gene to that reported in GenBank. Both PCR and restriction analysis proved that recombinant plasmid Pet-28a-IPSE was constructed correctly. The

  17. Preparation of Polyclonal Antibodies Against MHC Ⅱα and MHC Ⅱβ of Mangrove Red Snapper (Lutjanus argentimaculatus)%紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    王天燕; 常虹; 余时琛; 陈璐; 蔡中华

    2013-01-01

    目的:制备紫红笛鲷主要组织相溶性复合体MHC Ⅱα和MHC Ⅱβ多克隆抗体,为蛋白水平研究紫红笛鲷MHCⅡ分子提供理论和实践依据.方法:从已有的紫红笛鲷cDNA文库菌中分别克隆其MHC Ⅱα和MHC Ⅱ3分子的部分开放阅读框,与PQE-30构建表达载体,转入大肠杆菌E.coli M15以IPTG诱导表达;纯化得到的重组蛋白与弗氏佐剂混合乳化后注射新西兰大白兔制备多克隆抗体,再以酶联免疫吸附(ELISA)和免疫印迹(Western blot)检测所获抗血清的效价及效果.结果:①重组表达和纯化得到紫红笛鲷MHC Ⅱα和MHC Ⅱβ部分肽链.②制备的紫红笛鲷MHC Ⅱα和MHC Ⅱβ兔抗血清效价都大于1:25600,达到预期水平.③以获得的紫红笛鲷MHC Hα和MHC Ⅱβ兔抗血清分别与紫红笛鲷头肾巨噬细胞蛋白进行免疫印迹,显示两种抗血清能分别杂合出各自的目标蛋白,说明制备的多克隆抗体实际应用效果良好.结论:紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗血清制备成功.%Objective: To prepare the polyclonal antibodies against MHC Ⅱα and MHC Ⅱβ of mangrove red snapper. Methods: A partial of MHC Ⅱα and MHC Up chain was cloned from the cDNA library of mangrove red snapper, respectively. The PCR products were inserted into the expression vectors pQE30 and transformed into the E. coli M15. By inducing of IPTG, the recombinant proteins of MHC Ⅱα and MHC Ⅱβ fragments were purified, respectively. The proteins were thoroughly mixed with Freund's adjuvant, and injected rabbit. The antiserums were detected by ELISA and Western blot. Results: ① The recombinant proteins of MHC Ⅱα and MHC Ⅱβ fragments were purified successfully. ② The antiserums against MHC Ⅱα and MHC Ⅱβ both had a high titer above 1:25600. ③The western blot of head kidney macrophages showed the specification of MHC Ⅱα and MHC Ⅱβ antiserums, respectively. Conclusions: The high titer and specific polyclonal

  18. 猪盖他病毒衣壳蛋白的原核表达、纯化和多克隆抗体的制备%Prokaryotic Expression and Purification of the Capsid Protein of Porcine Getah Virus and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    姜焱; 何丹妮; 张小敏; 周斌; 陈溥言

    2013-01-01

    原核表达猪盖他病毒(Getah virus)衣壳蛋白(Cap)并制备多克隆抗体.设计一对特异性引物,从含有Cap基因的pT Cap质粒中扩增全长Cap基因,克隆至携带有His标签的原核表达载体pCold Ⅰ中,通过PCR、酶切鉴定和序列测定后,重组质粒pCold-Cap转化大肠杆菌Rosetta 2,IPTG诱导后SDS-PAGE和Western blot鉴定融合蛋白;蛋白经镍柱纯化后切胶免疫Balb/c小鼠,制备多克隆抗体.实验表明:经终浓度0.1 mmol/L IPTG 15℃诱导24 h后,Cap基因在Rosetta 2中获得高效表达,表达量占菌体蛋白的40.2%,SDS-PAGE显示融合蛋白相对分子质量为32.3 kD.Western blot显示制备的鼠源抗血清可以与融合蛋白发生反应,有明显的特异性条带.猪盖他病毒衣壳蛋白原核表达成功,制备的多抗可以识别Cap蛋白.%Based on a pair of specific primers,a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold Ⅰ which carried the His tag,this recombinant plasmid was then determined by enzyme digestion,PCR and DNA sequencing.This recombinant plasmid pCold-Cap was transformed into E.coli Rosetta 2,and PGETV Cap fusion protein was expressed through IPTG induction.The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0.lmmol/L IPTG under 15℃ for 24h,the expression quantity was 40.2 %.The product had a molecular mass about 32.3kD as expected.The target protein was separated in gel slices and used to immunize Balb/c mice.The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained.The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.

  19. 利用多克隆抗体有效识别中草药中抗表皮生长因子抑制剂%The Novel Application of Polyclonal Antibodies in Recognizing Anti-EGFR Inhibitors Directly from Herb

    Institute of Scientific and Technical Information of China (English)

    朱丽荔; 徐筱杰

    2003-01-01

    用一种已知的抗表皮生长因子受体抑制剂 (piceatannol) 作为半抗原与载体牛血清白蛋白 (BSA) 连接后免疫制备相应的多克隆抗体 (PcAb).利用该多克隆抗体来模拟酶制成亲和色谱柱,从一种藏药粗提物中将包括该半抗原在内的几种结构不同的抗表皮生长因子受体抑制剂识别出来 .研究采用前沿亲和色谱质谱联用技术对样品进行分析,可以直接从中药复杂体系中识别出有效成分并进行鉴定,实现中药有效成分的筛选与鉴定一体化技术 .%A polyclonal antibody (PcAb) prepared with piceatannol,a known inhibitor against the epidermal growth factor receptor (EGFR),was adopted as a stationary phase in the chromatographic system.Using the antibody to mimic the enzyme,several anti-EGFR inhibitors were recognized directly from a crude extract of Tibetan herb.Frontal affinity chromatography (FAC) was used here for analyzing molecular interactions between the analytes and an immobilized ligand (in this case the PcAb conjugated to Sepharose CL-4B) by calculating the extent of retardation of the elution front.By combining FAC with mass spectrometry (MS) in the current study,a very efficient and straightforward procedure was developed for analyzing the binding properties of different inhibitors.The novel effective screening of anti-EGFR inhibitors using PcAb from natural resources affords us a new feasible approach for the discovery of lead compounds.

  20. 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

  1. 禽白血病病毒p27蛋白在大肠杆菌中的表达和多克隆抗体的制备%Expression of p27 Protein of Avian Leukosis Virus in Escherichia coli and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    毛娅卿; 王嘉; 吴涛; 王哲; 蒋桃珍

    2014-01-01

    p27 protein gene from avian leukosis virus was cloned and constructed with plasmid pET-28a(+) . The recombinant protein p27 was expressed in an E. coli strain BL21(DE3) with IPTG induction and detected by Western blot with horseradish peroxidase labeled rabbit-anti-p27 antibodies. After that,the protein was purified to above 95% of purity by affinity chromatography with Ni-NTA agarose column. The anti-sera against p27 protein was obtained by immunizing rabbit with the purified recombinant p27 protein. The specificity of polyclonal antibody was about 1 ∶ 256000 after detected by ELISA. The results show that the p27 protein expressed by E. coli has good antigenicity and can replace the purified virus protein for detection of avian leukosis virus.%将禽白血病病毒p27基因克隆并构建重组表达质粒pET28a-p27,在大肠杆菌BL21中经IP TG诱导后产生可溶性表达蛋白。表达的蛋白用Western blot进行活性检测,其能与辣根过氧化物酶标记的兔抗p27抗体发生特异性反应;采用金属螯和层析对表达蛋白进行纯化,其纯度约为95%;用纯化的表达蛋白p27免疫家兔制备抗血清,ELISA抗体效价可达1∶25600。研究结果表明,大肠杆菌表达的p27蛋白具有良好的抗原性,可以替代纯化病毒蛋白用于禽白血病病毒检测。

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

    African Journals Online (AJOL)

    2013-09-11

    Sep 11, 2013 ... Tecnológico, Universidade Federal de Pelotas, Brazil, 96010-900, Pelotas, RS, - P.O. Box 354, ... However, both methods have several limitations including technical ... cell production, and consequently increases tissue oxy-.

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

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... Full Length Research Paper. High-level expression ... resistance severely compromises effective therapeutic options. ... In the present study, we first report the expression of the oprD ... databases of National Center for Biotechnology Information (NCBI) ..... assembly of the head of bacteriophage T4. Nature.

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

    African Journals Online (AJOL)

    user

    2011-03-14

    Mar 14, 2011 ... Key Laboratory for Space Biosciences and Biotechnology, Faculty of Life Sciences, ... microtubule actin cross-linking factor 1 (MACF1), plays a key role in ... into prokaryotic expression vector pQE-80L for the expression of.

  5. 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.

  6. Host-feeding behaviour of Dermacentor reticulatus and Dermacentor marginatus in mono-specific and inter-specific infestations.

    Science.gov (United States)

    Buczek, Alicja; Bartosik, Katarzyna; Zając, Zbigniew; Stanko, Michał

    2015-09-17

    Given the sympatric occurrence in some regions of Europe and the great epidemiological significance of D. reticulatus and D. marginatus species, we investigated the behaviour of these ticks during inter-specific and mono-specific host infestations. The investigations were conducted on rabbits at 20 ± 3 °C and humidity of 38 ± 1 %. The inter-specific infestations groups consisted of 20 females and ten males of D. marginatus and 20 females and ten males of D. reticulatus on each host, whereas mono-specific infestations involved 40 females and 20 males of each species. The investigations have demonstrated competition between the two tick species resulting in modification of the behaviour on the host and the feeding course in D. marginatus females by the presence of D. reticulatus. In the inter-specific group, D. marginatus females attached for a longer time (mean 2.74 ± 1.12 h) than in the mono-specific group (mean 1.24 ± 0.97 h) (p feeding period of these females was shorter (9.45 ± 1.30 days) than in the mono-specific group (13.15 ± 2.53 days) (p feeding rates between the mono-specific and inter-specific groups. The differences in the behaviour of the females from both species during co-feeding reflect physiological adaptation to environmental conditions, which enables them to ingest blood and reproduce. During co-feeding of D. reticulatus and D. marginatus on the same host, two inter-specific systems with different physiological features are formed, which may influence the transmission of tick-borne pathogens.

  7. Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (Corvus brachyrhynchos

    Directory of Open Access Journals (Sweden)

    Wu Ping

    2007-05-01

    Full Text Available Abstract Background Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos. Our objectives were to determine 1 the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC diagnosis of WNV infection in free-ranging American crows, 2 which organ(s is/are most suitable for IHC-based diagnosis of WNV, and 3 how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection. Methods Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed. Results Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72

  8. 凡纳滨对虾精氨酸激酶的多克隆抗体制备及组织特异性表达分析%Preparation of polyclonal antibody and analysis of tissue specific expression of arginine kinase protein in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    姚翠鸾; 冀培丰; 孔鹏; 王志勇

    2009-01-01

    Arginine kinase (ATP: arginine N-phosphotransferase, AK) in invertebrates catalyzes the reversible phosphorylation of arginine by MgATP to form phosphoarginine and MgADP. The conventional view is that phosphoarginine functions as ATP buffers, permitting maintenance of high ATP values during periods in burst of cellular activity in invertebrates. In this paper, the open reading frame (ORF) was cloned from shrimp, Litopenaeus vannamei, based on the full-length cDNA sequence that was obtained in our previous study. The ORF encoding 356 amino acids of AK was cloned and inserted to a prokaryotic expression vector pGEX-4T-2. The recombinant protein was expressed as glutathione S-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. The anti-AK polyclonal antibody was prepared by immunization of mice using this purified AK protein. The specific recognition of anti-AK antibody was further verified. The AK protein expression in various tissues was then analyzed using this newly prepared antibody by Western-blotting. The results revealed a strong expression of AK in heart, nerve, hemocyte, stomach and muscle, weak expression in eyestalk, hepatopancrease, gill and skin. The present study may be very useful for studying the function of AK in shrimp.%精氨酸激酶(arginine kinase,AK)在调节无脊椎动物磷酸精氨酸与ATP之间能量平衡的过程中具有重要作用.在前期工作基础上,设计特异引物,以凡纳滨对虾肌肉组织cDNA为模版,克隆得到了1071 bp的凡纳滨对虾精氨酸激酶的完整开放阅读框;将它克隆到原核表达载体pGEX-4T-2上,转化BL21(DE3)菌株,经诱导后表达出GST-AK融合蛋白.以纯化的融合蛋白作为抗原免疫小鼠,制备多克隆抗血清,经检测该抗原与抗体识别良好.利用得到的抗体对凡纳滨对虾AK在蛋白质水平上的特异性表达进行分析发现,精氨酸激酶在对虾的肌肉、心

  9. Prokaryotic Expression of Major Antigenic Epitope Region of African Swine Fever Virus VP73 Protein and Preparation of Polyclonal Antibody%非洲猪瘟病毒VP73蛋白主要抗原表位区的原核表达及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    李洪利; 王志亮; 王君玮; 张维; 吴晓东; 赵永刚; 李慧; 李娟; 包静月; 曹金山

    2012-01-01

    protein. The polyclonal antibody built the foundation for immunology research of VP73 and serological diagnosis of African swine fever.

  10. 暗纹东方纯CD8α基因克隆、原核表达与多克隆抗体制备%CD8α gene in Takifugu fasciatus: cloning, expression and preparation of polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    邵爱华; 杜建; 陈葵; 朱江

    2012-01-01

    In the fish species with T cellular immune function, CD8 molecules are important immune acceptor molecules. The cDNA sequence and its corresponding genomie sequence were cloned from the thymus of Tak- ifugufasciatus. The eDNA of Takifugufasciatus CD8α was found to be 1 061 nucleotides long, the open reading frame (ORF) 657 bp long, and its corresponding genomic sequence 1 533 bp. The CD8α gene consisted of six exons separated by five introns and encoded by 218 amino acid residues with an estimated molecular weight of 22 Kda. The predicted primary structure of CD8α contained a signal peptide, an IgV-domain, a hinge region, a transmembrane region and the cytoplasmic tail. The two cysteine residues were involved in the V-domain of T. fasciatus CD8α capable of forming an intra-ehain disulphide bond, and the other two cysteines in the hinge re- gion are conserved in most of fishes responsible for CD8α's ability of forming both homodimers and heterodimers. The protein structure of the T. fasciatus CD8α gene was similar to other fishes, especially to those of Pleuronecti- formes. In order to further investigate the biological function of CD8, the CD8α fragment constructing the extra- cellular region of mature peptide was sub-cloned into pET28a (+) expression veetor after digestion with a combi- nation of Hind III and Xho I. The protein was expressed in baeteria by inducing and was purified. The anti- CD8α polyclonal antibody was prepared by immunization of rabbit with the purified fusion protein. The specific recognition anti-CD8α polyclonal antibody was further verified by ELISA analysis of the serum from pre- immu- nized and post-immunized rabbit. The preparation of CD8α antibody will greatly faeilitate the identification and understanding of CD8 in fish lymphocyte populations and adaptive immunity.%CD8是与Ⅰ型主要组织相容性复合体(MHCI)结合,是T细胞表面受体(TCR)的共受体.也是T淋巴细胞表面的重要标志

  11. Antibodies against antibodies: immunogenicity of adalimumab as a model

    NARCIS (Netherlands)

    van Schouwenburg, P.A.

    2012-01-01

    Upon repeated adalimumab exposure part of the patients start to produce ADA. The antibody response is polyclonal and consists mainly of antibodies of IgG1 and IgG4 isotype. In the majority of ADA positive patients ADA are already produced within the first 28 weeks of treatment and in part of the pat

  12. 水稻Os着-c op 1基因的原核表达及多克隆抗体制备%Prokaryotic Expression and Polyclonal Antibody Preparation of Osε-c op 1 in Rice (Oryz a s ativ a L.)

    Institute of Scientific and Technical Information of China (English)

    杨加伟; 周玲艳; 庄楚雄

    2013-01-01

      着-COP蛋白是真核生物分泌途径中COP玉有被小泡的一个亚基。本研究利用PCR技术扩增水稻着-c op基因(Os着-c op 1)的ORF(开放阅读框),并克隆到原核表达载体pET-23d上,将表达载体pET-Os着-cop1转入大肠杆菌BL21(DE3),以1.0 mmol/L的IPTG (isopropylβ-D-thiogalactoside)诱导表达重组蛋白,然后以重组蛋白作为抗原免疫家兔,制备多克隆抗体。SDS-PAGE电泳分析结果表明,成功诱导表达了分子量约为35 kD的重组蛋白,Western blot检测表明,免疫家兔的抗血清与水稻幼穗总蛋白杂交信号较好。Os着-COP1抗体的制备有助于研究该基因及COPI小泡在水稻中的功能。%Theε-COP is one of the subunits of COPⅠvesicle in secret pathway of eukaryotic cells. The ORF (open reading frame) ofε-cop gene in rice (Osε-cop1) was obtained by PCR and cloned into the prokaryotic expres-sion vector pET-23d. The resulting plasmid was then introduced into E. coli strain BL21 (DE3) and induced to ex-press the recombinant Osε-COP1 protein by 1.0 mmol/L isopropyl?-D-thiogalactoside (IPTG). Afterwards, the re-combinant Osε-COP1 protein was used as antigen to immune rabbits. The results demonstrated a 35 kD protein was expressed successfully inducing by IPTG. Western blot analysis showed that the antiserum immunological recog-nized rice Osε-COP1 protein with high specificity, indicating that the polyclonal antibody was prepared successful-ly. This work would contribute to study the function of Osε-cop1 gene and COPⅠvesicle in rice.

  13. How do tree competition and stand dynamics lead to spatial patterns in monospecific mangroves?

    Directory of Open Access Journals (Sweden)

    M. N. I. Khan

    2013-01-01

    Full Text Available Information on mangrove stand development is rare because long-term monitoring data is often lacking. Such information is important in order to plan management measures effectively. Novel approaches are required to bridge this gap of knowledge based on existing data sets. This study uses a unique combination of field data analyses with simulation experiments in order to demonstrate how information on mangrove dynamics can be extracted if data are sparse. The paper provides a~baseline characterization of stand development in a monospecific pioneer mangrove stand of Kandelia obovata. Point pattern analyses revealed that in the young stage, self-thinning has started but has not yet lead to a regularity of spatial tree distribution in the entire stand, and trees located in smaller clumps hinder each other in growth but do not lead to a significant size class differentiation. However, after ca. 2 decades the self-thinning and the size class differentiation start to become more visible. A mutual inhibition of growth was observed within 2 m circular distance (r in the young stage and within 3 m distance after two decades of stand development as confirmed by the negative values of mark correlation function. As a stand grows older the spatial pattern of individuals become more regular from a clustered pattern. In order to understand and predict the future stand development, simulation experiments were carried out by means of the individual-based model KiWi.

  14. Preparation of polyclonal antibodies against pneumococcal serotype 1 capsular polysaccharide and development a sandwich ELISA in determination of concentration for type 1 capsular polysaccharide%肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA 法建立及其在多糖浓度测定中的应用

    Institute of Scientific and Technical Information of China (English)

    刘威; 廖红梅; 谢谦; 黄放; 邹莎莎; 马诚; 江山; 崔长法

    2013-01-01

    Objective To prepare polyclonal antibodies against capsular polysaccharide of pneumococcal serotype 1, and use the antibodies to develop a sandwich ELISA in determination of concentration for pneumococcal capsular polysaccharide of serotype 1 in the process of fermentation and purification .Methods High titer serum of anti-capsular polysaccharide se-rotype 1 was obtained after immunizing rabbits by using of inactivated streptococcus pneumoniae cells of serotype 1 for 6 weeks.IgG was purified through affinity chromatography and used as a coating antibody .Polysaccharide samples , alone with in house polysaccharide standard , were then added followed by using biotinylated antibody as a signal antibody .The optimal linear range of the standard curve , specificity, accuracy and precision of the developed method were validated accordingly.Results The antibody titer of rabbit immune serum reached to 1 ∶32, the standard PS1 linear detection range was 1.56 ng/mL to 50 ng/mL, detection limit was 3.13 ng/mL.Standard polysaccharide was mixed with polysaccha-rides from different serotypes or culture media before asssay , the recovery ratio was 102%and 108%respectively .Intra-as-say precision and inter precision of the method were 6.08%and 7.01%.Conclusion High titer rabbit anti-serum against type 1 capsular polysaccharide of streptococcal pneumoniae was prepared .The developed sandwich ELISA method showed high specificity , good accuracy and precision .This method can be used in specific detection of concentration for pneumo-coccal polysaccharide serotype1 .%目的:利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法( Enzyme-linked immunosorbent assay ,ELISA),用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖

  15. Delayed hemolytic transfusion reaction with multiple alloantibody (Anti S, N, K and a monospecific autoanti-JK b in intermediate β-thalassemia patient in Tabriz

    Directory of Open Access Journals (Sweden)

    Roya Dolatkhah

    2013-01-01

    Full Text Available It appears that delayed hemolytic transfusion reactions may occur several days after the administration of donor red cells is true even though they have been shown to be compatible in cross match tests by the antiglobulin technique. A specific case was observed in our center, which confirms the fact. The patient was a 37-year-old male suffering from intermediate β-thalassemia. He had a history of two previous transfusions, with unknown transfusion reaction. In the last transfusion, laboratory data showed: Hb 7.8 g/dL and Hematocrit (Hct 24.2%. The patient received two units of cross matched, compatible concentrated red blood cells (RBCs. After eight days a severe reaction was observed with clinical evidence of tachycardia, fatigue, fever, back pain, chest discomfort, jaundice, nausea and anorexia. Accordingly delayed hemolytic transfusion reaction was suspected, and anti-RBC antibodies were tested. Laboratory tests revealed the presence of three alloantibodies: Anti-N, anti-S, anti-K, and a monospecific autoanti-JK b .

  16. The tropical African legume Scorodophloeus clade includes two undescribed Hymenostegia segregate genera and Micklethwaitia, a rare, monospecific genus from Mozambique

    DEFF Research Database (Denmark)

    Mackinder, B. A.; Saslis-Lagoudakis, H.; Wieringa, J. J.

    2013-01-01

    Legume subfamily Caesalpinioideae accommodates approximately 2250 species in 171 genera which traditionally are placed in four tribes: Caesalpinieae, Cassieae, Cercideae and Detarieae. The monophyletic tribe Detarieae includes the Amherstieae subclade which contains about 55 genera. Our knowledge......, and the previously unsampled rare monospecific genus Micklethwaitia from Mozambique. Zenkerella is suggested as a possible sister genus to the Scorodophloeus clade. © 2013 South African Association of Botanists....

  17. Biomass in monospecific and mixed stands of eucalyptus and black wattle and corn in an agroforestry system

    Directory of Open Access Journals (Sweden)

    Márcio Viera

    2011-06-01

    Full Text Available This study aimed at quantifying the production and distribution of aboveground biomass from the plants in monospecific and mixed stands of eucalyptus (hybrid E. urophylla x E. grandis and black wattle (Acacia mearnsii and, of corn (Zea mays in agrosilvicultural systems. The biomass evaluation (leaf, branch, bark and wood from the forest species at 6 and 18 months of age were performed at the treatments: 100E (100% of eucalyptus + corn; - 100A (100% of black wattle + corn; - 50E:50A (50% of eucalyptus + 50% of black wattle + corn. The corn biomass evaluation (stem, leaves, straw, cob and grains was performed at treatments 100E; 100A; 50E:50A; 75E:25A (75% of eucalyptus + 25% of black wattle + corn; and - 25E:75A (25% of eucalyptus + 75% of black wattle + corn. The biomass production from eucalyptus and from the black wattle, in both monospecific and mixed planting, did not differ in any of the assessed ages but, when evaluated by plants compartments, it was verified an interspecific competitive interaction from the eucalyptus on the black wattle, reducing the formation of crown biomass. The total production of corn biomass in agrosilvicutural systems with eucalyptus and with black wattle in monospecific or mixed plantings did not differ in the studied treatments.

  18. Resistant prolactinoma: Is it monoclonal or polyclonal?

    Directory of Open Access Journals (Sweden)

    K. V. S. Hari Kumar

    2013-01-01

    Full Text Available Prolactinomas are solitary benign neoplasms and resistance to dopamine agonists occur in a small percentage of prolactinomas. Multiple pituitary adenomas are reported in less than 1% of pituitary adenomas and rarely result in resistant prolactinoma. We recently encountered an interesting patient of hyperprolactinemia with multiple pituitary microadenomas. Dopamine agonist use resulted in prolactin normalization and subsequent pregnancy resulted in drug withdrawal. Repeat evaluation after delivery showed a macroprolactinoma and dopamine agonist therapy resulted in biochemical cure without reduction in tumor size. We report the case for its presentation with multiple microadenomas progressing to macroprolactinoma suggesting polyclonal in origin.

  19. Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

    Institute of Scientific and Technical Information of China (English)

    Faiz MMT Marikar; Dingyuan Ma; Jianqiang Ye; Bo Tang; Weijuan Zheng; Jing Zhang; Min Lu; Zichun Hua

    2008-01-01

    The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichla coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refoided and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD不得 protein was allowed the production of high titre polycional antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.Cellular & Molecular Immunology. 2008;5(6):471-474.

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

    Science.gov (United States)

    Levesque, Marc C; Moody, M Anthony; Hwang, Kwan-Ki; Marshall, Dawn J; Whitesides, John F; Amos, Joshua D; Gurley, Thaddeus C; Allgood, Sallie; Haynes, Benjamin B; Vandergrift, Nathan A; Plonk, Steven; Parker, Daniel C; Cohen, Myron S; Tomaras, Georgia D; Goepfert, Paul A; Shaw, George M; Schmitz, Jörn E; Eron, Joseph J; Shaheen, Nicholas J; Hicks, Charles B; Liao, Hua-Xin; Markowitz, Martin; Kelsoe, Garnett; Margolis, David M; Haynes, Barton F

    2009-07-07

    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.

  1. 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.

  2. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Science.gov (United States)

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  3. ABZYME ACTIVITY OF POLYCLONAL IMMUNOGLOBULINS IN SPONDYLOARTHROPATHIES

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    E. V. Kunder

    2009-01-01

    Full Text Available Abstract. Polyclonal immunoglobulins G (subclasses 1, 2, 4 from sera of 255 patients and 69 healthy persons were studied by a combined approach using rivanol treatment and affinity chromatography. Enzymatic reactions were carried out according to the methods that we have previously developed and validated for evaluation of abzyme activity in the patients with different disorders. The levels of DNase, proteolytic BAPNA-amidase (benzoylarginine-p-nitroanilide amidase, and superoxyde dismutase abzyme activity in spondyloarthropathies proved to be significantly higher (p = 0.001, as compared with a control group. Catalase activity of immunoglobulines in the disorders studied was compatible to control levels (p > 0.05. Analysis of relations between abzyme activity and clinical and laboratory signs of the diseases has revealed some significant correlations. Prevalence of abzyme DNAse activity is found in the patients with psoriatic arthritis, as compared to reactive arthritis and ankylosing spondilitis (p < 0.001.

  4. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  5. Polyclonal IgG Response of Balb/c Mice to the 23 kDa Antigen of Entamoeba histolytica

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    Herbert J. Santos

    2008-06-01

    Full Text Available Entamoeba histolytica is one of the most significant protozoan pathogens found in developing countries like the Philippines. This intestinal parasite causes the disease amebiasis, which has a yearly average mortality of about 100,000 people worldwide. Thus, it is essential to develop new diagnostic markers and possible treatment against this disease. The crude cell extract of E. histolytica, was used to induce polyclonal antibody response in mice. Balb/c mice were given immunizations of the prepared crude E. histolytica antigens for a period of twelve weeks. Indirect fluorescent antibody test showed the specificity of polyclonal IgG in recognizing the cytosolic components of E. histolytica trophozoites. Enzyme-linked immunosorbent assay was performed to determine the antibody titers in sera collected at various time intervals. Antibody titers for the mouse serum taken 10 and 20 days after the third booster immunization were known to be 16,384 and 4,096 respectively. SDS-PAGE profile of the crude E. histolytica antigens revealed three bands with molecular weights of 23, 41, and 47 kDa. Western immunoblot results indicated that the polyclonal IgG produced by mice targets the potentially novel 23 kDa antigen from an axenic E. histolytica culture.

  6. [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.

  7. Get effective polyclonal antisera in one month

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks. In general,high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis.It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.

  8. 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

  9. Development of Antibody Against Sulfamethazine

    Institute of Scientific and Technical Information of China (English)

    LIZi-ying; XUWen-ge; LIUYi-bing; ZHANGLi-ling; GUOWei-zheng; HANShi-quan

    2003-01-01

    Polyclonal antibodies(PcAbs) against sulfamethazine(SMT) are obtained by immunizing rabbits with SMT-conjugated bovine serum albumin(BSA). The affinity constants (Ka) of the PcAbs are higher than 1×108 and the cross-reactivities with sulfadiazine(SD), sulfaquinoxaline (SQX) are lower than 0.05% (R/A).

  10. Why Does the Molecular Structure of Broadly Neutralizing Monoclonal Antibodies Isolated from Individuals Infected with HIV-1 not Inform the Rational Design of an HIV-1 Vaccine?

    Directory of Open Access Journals (Sweden)

    Marc H V Van Regenmortel

    2015-05-01

    Full Text Available It is commonly assumed that neutralizing Mabs that bind to the HIV-1 Env glycoprotein are more specific reagents than anti-HIV-1 polyclonal antisera and that knowledge of the structure of these Mabs facilitates the rational design of effective HIV-1 vaccine immunogens. However, after more than ten years of unsuccessful experimentation using the structure-based reverse vaccinology approach, it is now evident that it is not possible to infer from the structure of neutralizing Mabs which HIV immunogens induced their formation nor which vaccine immunogens will elicit similar Abs in an immunized host. The use of Mabs for developing an HIV-1 vaccine was counterproductive because it overlooked the fact that the apparent specificity of a Mab very much depends on the selection procedure used to obtain it and also did not take into account that an antibody is never monospecific for a single epitope but is always polyspecific for many epitopes. When the rationale of the proponents of the unsuccessful rational design strategy is analyzed, it appears that investigators who claim they are designing a vaccine immunogen are only improving the binding reactivity of a single epitope-paratope pair and are not actually designing an immunogen able to generate protective antibodies. The task of a designer consists in imagining what type of immunogen is likely to elicit a protective immune response but in the absence of knowledge regarding which features of the immune system are responsible for producing a functional neutralizing activity in antibodies, it is not feasible to intentionally optimize a potential immunogen candidate in order to obtain the desired outcome. The only available option is actually to test possible solutions by trial-and-error experiments until the preset goal is perhaps attained. Rational design and empirical approaches in HIV vaccine research should thus not be opposed as alternative options since empirical testing is an integral part of a so

  11. Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers

    DEFF Research Database (Denmark)

    Martel, Cyril Jean-Marie; Aasted, Bent

    2009-01-01

    immunoglobulins, we identified and characterized polyclonal antibodies towards ferret IgG, IgM and IgA. We also identified 22 monoclonal antibodies (mAbs) raised mostly against human CD markers which cross-reacted with ferret leukocytes. These antibodies were originally specific against human CD8, CD9, CD14, CD18...

  12. The impact of polyclonal neural cell adhesion molecule antibody on the potency of botulinum toxin%多克隆神经细胞粘附分子抗体对肉毒毒素生物学效应的影响

    Institute of Scientific and Technical Information of China (English)

    郭艳; 靳令经; 刘务朝; 郑玉果; 管强; 潘丽珍; 聂志余

    2013-01-01

    Objective To investigate the impact of polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab) on the potency of botulinum toxin A (BTX-A).Methods Ninety male Sprague-Dawley rats were randomly divided into 3 equal groups:a normal control group,a BTX-A group and a P-NCAM-Ab group.The rats in the normal control group were injected with 100 μl of saline solution in their right gastrocnemius,while those in the BTX-A and P-NCAM-Ab groups were injected with 100 μl of BTX-A (0.5 U).In addition,the rats in the P-NCAM-Ab group were also injected with 100 μl of P-NCAM-Ab (the dosage was 20 U) at the same site on the 3rd day after the BTX-A injection.The rats' gastrocnemius muscle strength was evaluated with a self-made system for evaluating neuromuscular function before and after the toxin injection,on the 3rd day,as well as 1,2,4,6,8,10 and 12 weeks after the BTX-A injection.Any wet weight changes in the muscles were observed,and immunochemistry methods were employed to observe any structural changes in the motor endplates and nerve fibers at the different time points.Results After the saline injection,the average gastrocnemius muscle strength of the control group increased with time,while strength in the BTX-A and P-NCAM-Ab groups demonstrated a decrease in strength followed by a gradual increase.The average gastrocnemius muscle strength of the rats in the BTX-A and P-NCAM-Ab groups was significantly lower than that of the control group at all time points.Compared with the BTX-A group,the muscle strength of the P-NCAM-Ab group rats decreased further.Strength recovery in the BTX-A and P-NCAM-Ab groups was significantly slower than in the control group.The wet weight percentage in the BTX-A and P-NCAM-Ab groups at first decreased and then recovered with time.After the BTX-A injection,the average wet weight percentage of the P-NCAM-Ab group rats was significantly lower than that of the BTX-A group after 3 days,and 1,2 and 4 weeks.Karnovsky-Roots AchE staining showed

  13. Phage display for the generation of antibodies for proteome research, diagnostics and therapy.

    Science.gov (United States)

    Schirrmann, Thomas; Meyer, Torsten; Schütte, Mark; Frenzel, André; Hust, Michael

    2011-01-10

    Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  14. Phage Display for the Generation of Antibodies for Proteome Research, Diagnostics and Therapy

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2011-01-01

    Full Text Available Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  15. Shifts in the bacterial community composition along deep soil profiles in monospecific and mixed stands of Eucalyptus grandis and Acacia mangium.

    Science.gov (United States)

    Pereira, Arthur Prudêncio de Araujo; Andrade, Pedro Avelino Maia de; Bini, Daniel; Durrer, Ademir; Robin, Agnès; Bouillet, Jean Pierre; Andreote, Fernando Dini; Cardoso, Elke Jurandy Bran Nogueira

    2017-01-01

    Our knowledge of the rhizosphere bacterial communities in deep soils and the role of Eucalyptus and Acacia on the structure of these communities remains very limited. In this study, we targeted the bacterial community along a depth profile (0 to 800 cm) and compared community structure in monospecific or mixed plantations of Acacia mangium and Eucalyptus grandis. We applied quantitative PCR (qPCR) and sequence the V6 region of the 16S rRNA gene to characterize composition of bacterial communities. We identified a decrease in bacterial abundance with soil depth, and differences in community patterns between monospecific and mixed cultivations. Sequence analysis indicated a prevalent effect of soil depth on bacterial communities in the mixed plant cultivation system, and a remarkable differentiation of bacterial communities in areas solely cultivated with Eucalyptus. The groups most influenced by soil depth were Proteobacteria and Acidobacteria (more frequent in samples between 0 and 300 cm). The predominant bacterial groups differentially displayed in the monospecific stands of Eucalyptus were Firmicutes and Proteobacteria. Our results suggest that the addition of an N2-fixing tree in a monospecific cultivation system modulates bacterial community composition even at a great depth. We conclude that co-cultivation systems may represent a key strategy to improve soil resources and to establish more sustainable cultivation of Eucalyptus in Brazil.

  16. Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

    Directory of Open Access Journals (Sweden)

    Debalina Sarkar

    2014-01-01

    Full Text Available Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft versus host disease in bone marrow transplantation. Here, we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e., hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express green fluorescent protein–marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 100-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within 2 weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

  17. Fraction of absorbed photosynthetically active radiation for a semi-deciduous forest and Vochysia divergens monospecific forest in Brazil

    Science.gov (United States)

    Sanches, L.; Nogueira, J. D.; Pinto Junior, O. B.

    2013-12-01

    Ecophysiological model employ the fraction of photosynthetically active radiation (fAPAR) absorbed by the plant canopy such as critical input to diagnostic seasonal patterns in the vegetation dynamics. Here, this study focuses on the patterns of variation of transmittance, reflectance and fAPAR for two forests with different canopy structures, a forest located in a climatic transition between Amazonian rain forest (a semi-deciduous forest) and savanna and a V. divergens monospecific forest. Transmittance (t), reflectance (r) and fAPAR were calculated with meteorological data measured from the tower installed in each forest. Our results indicate the annual average fAPAR for the semi-deciduous and Vochysia divergens was 92.3 and 84.8%, respectively. Measurements indicate that the fAPAR for the V. divergens was 9% less than that for the semi-deciduous forest. The values and variation of r, t and fAPAR was due to the fraction of canopy openness, and was influenced by the fraction of canopy openness, solar zenith angle and amount of diffuse radiation.

  18. Novel polyclonal-monoclonal-based ELISA utilized to examine lupine (Lupinus species) content in food products.

    Science.gov (United States)

    Holden, Lise; Moen, Lena Haugland; Sletten, Gaynour B G; Dooper, Maaike M B W

    2007-04-04

    Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.

  19. Production and characterisation of polyclonal antisera against feline IgE.

    Science.gov (United States)

    Gilbert, S; Halliwell, R E

    1998-05-29

    Cats, naturally or experimentally infected with Toxocara cati were immunised with dinitrophenylated ascaris antigen (DNP-Asc). All cats developed immediate skin reactivity to DNP coupled to bovine serum albumin (DNP-BSA) and the sera of the nine cats had a heat labile homocytotropic antibody detectable by homologous Prausnitz-Küstner (PK) tests. Reagin-rich fractions were prepared from these sera and used for the preparation of polyclonal antisera in rabbits. Resultant antisera were passed through a immunoabsorbent column of Sepharose 4B coupled to heated normal cat serum. An immunoabsorbent column prepared with the resultant antisera removed the PK reactivity from the cat sera, and the activity was recovered following acid elution. The antiserum failed to detect any recognised immunoglobulin in cat sera, but precipitated with a heat labile protein with gamma-1 electrophoretic mobility in the sera of parasited cats. These findings support the contention that the antisera are specific for feline IgE.

  20. Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes

    DEFF Research Database (Denmark)

    Gunnarsson, Anders Patrik Alexander; Christensen, Rikke; Prætorius, Jeppe

    2016-01-01

    unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive......Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been...... that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges...

  1. Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

    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 W H I; 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.

  2. Postembryonic development of Loxosceles intermedia Mello-Leitão, 1934, L. laeta (Nicolet, 1849 and L. gaucho Gertsch, 1967 (Araneae; Sicariidae breeding under conditions of monospecific diet

    Directory of Open Access Journals (Sweden)

    Emanuel Marques da Silva

    2005-05-01

    Full Text Available The influence of monospecific feeding on the post-embryonic development of L. intermedia, L. laeta and L. gaucho was evaluated. Two hundred and ten spiderlings were individualized in containers (120 ml, 105 being fed with Tenebrio molitor larvae and 105 with Pycnoscelus surinamensis nymphs. In the three species, the number of molts varied according to the diet. The number of spiders that reached maturity was lower in L. gaucho. In L. intermedia, the duration of the post-embryonic period was greater when larvae are fed. Mortality was higher in the second instar in the three species, the highest frequency being registered for L. gaucho. The data provided evidence that monospecific feeding influenced the post-embryonic development of the studied species. This influence might intensified by specific characteristics such as origin, habits and habitats.

  3. Response of soil mite abundance and diversity to a monospecific timber Tectona grandis plantation in Ivory Coast

    Directory of Open Access Journals (Sweden)

    Julien Kouadio N’DRI, Henri Marc ANDRE, Jan LAGERLÖF, Jérôme Ebagnérin TONDOH,Thierry HANCE

    2013-10-01

    Full Text Available This study aims to assess the impact of monospecific Tectona grandis forest plantation on the soil mite abundance and diversity. To achieve these objectives, two sites situated in Ivory Coast were investigated. The first, a primary forest was characterized by a very weak human activities whereas the second, a teak plantation was characterized by a high disturbance performed during the planting. After extracting, sorted and description, 116 mite species were described in the two sites. Mite densities were lower in teak plantation and also higher in the litter and decreased to the depth in both sites. Species richness recorded in teak plantation (52 species was significantly lower compared to primary forest (98 species. The same trend was observed for Oribatida but not for Gamasida. The lower Oribatida (5 vs. 17 and higher Oribatida (24 vs. 41 were recorded respectively in teak plantation and primary forest. Mite Shannon index and evenness were significantly different between sites. High Jaccard index values and the appearance of exclusive species in both habitats showed that the sites are very distinct. Total number of species recorded corresponded to 58%–63% of the total number of species estimated by ACE and Chao 1&2 estimators, indicating that the sampling effort was not sufficient. Mite abundance and diversity varied depending on the characteristics of habitats. Chemical element (Corg, Ctot, Ntot, and SOM values were lower in teak plantation (disturbed habitat and significantly different to primary forest in the topsoil. Apart from litter height, soil depth, pH and C/N ratio, others variables were strongly correlated to mite abundance and diversity [Current Zoology 59 (5: 633–643, 2013].

  4. Preparation of Monospecific Serum against Brucella A and M Epitopes%布鲁氏菌A、M因子血清的研制

    Institute of Scientific and Technical Information of China (English)

    张金亚; 毛开荣; 程君生; 冯宇; 丁家波; 王楠; 皮向成; 吴竞; 张东东; 朱良全

    2014-01-01

    In order to identify different smooth Brucella strains, we prepared the monospecific serum against B.abortus O-chain A epitope and B.melitensis O-chain M epitope. Hyperimmune serum was collected from five rabbits, which were inoculated with heat-inactivated B.abortus (2308 strain) and heat-inactivated B.melitensis (16M strain) respectively. The hyperimmune serum was treated by serum antigen cross-absorption to get mono-specific antiserum. Monospecific serum against B. abortus O-chain A epitopes were obtained which at a 1/320 dilution agglutinated the homologous antigen and at a 1/2. 5 dilution did not agglutinated the the heterologous antigen. Monospecific serum against B. melitensis O-chain M epitopes were obtained which at a 1/160 dilution agglutinated the homologous antigen and at a 1/2.5 dilution did not agglutinated the the heterologous antigen. Then the monospecific serum was lyophilized and stored at-20℃. The slide agglutination test showed that monospecific serum against B. abortus O-chain A epitope and B. melitensis O-chain M epitope had no agglutination with homologous antigen. The results showed that the obtained monospecific serum were of high specificity and high titer, which could assist the identification of smooth Brucella species.%研制牛种布鲁氏菌A因子血清和羊种布鲁氏菌M因子血清,用于光滑型布鲁氏菌特异性种属鉴定。将灭活牛种布鲁氏菌2308株及羊种布鲁氏菌16M株免疫1.5~2 kg家兔各5只制备高免血清。对高免血清采用抗原交叉吸附的方法,制得A、M因子血清,分装冻干后置-20℃保存。微量试管凝集试验结果显示:研制的A因子血清对16M抗原在1∶2.5时无凝集现象,对2308抗原凝集价为1∶320;研制的M因子血清对2308抗原在1∶2.5时无凝集现象,对16M抗原凝集价为1∶160。玻片凝集试验结果显示:研制的A、M因子血清对异种抗原无凝集现象。试验表明,研制的A、M因子血清具

  5. 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

    . 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...

  6. Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing.

    Science.gov (United States)

    Hagemann, Sascha; Faude, Alexander; Rabenstein, Monika; Balzer-Geldsetzer, Monika; Nölker, Carmen; Bacher, Michael; Dodel, Richard

    2010-08-15

    Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Cloning of buffalo growth differentiation factor 9 mature peptide cDNA sequence and preparations of the recombinant protein and polyclonal antibody%水牛生长分化因子9成熟肽基因克隆及重组蛋白质和多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    刘玉鹏; 彭中友; 郭日红; 雷明明; 于建宁; 陈瑞爱; 施振旦

    2013-01-01

    为了获得水牛生长分化因子9(Growth differentiation factor 9,GDF9)重组蛋白质和抗GDF9抗体,根据GDF9基因编码区序列(GenBank:FJ529501.1)设计1对引物,用水牛基因组DNA为模板扩增水牛GDF9成熟肽基因序列,并与其他反刍动物的GDF9成熟肽基因序列进行同源性比较.将该序列克隆到表达载体pRSET-A的BamH Ⅰ和HindⅢ酶切位点之间以构建重组表达载体,并将重组表达载体转化大肠杆菌E.coli BL21(DE3),转化菌经IPTG诱导表达重组蛋白质.经Ni-NTA凝胶纯化后的重组蛋白质与矿物油佐剂混合免疫新西兰兔制备抗GDF9抗血清,使用ELISA方法检测血清抗体效价,从抗血清中进一步纯化GDF9抗体.结果显示,成功获得了水牛GDF9成熟肽基因序列,该序列在牛和其他反刍动物之间高度同源.成功构建了重组表达载体并转化大肠杆菌E.coli BL21 (DE3),E.coli BL21(DE3)经IPTG诱导后表达了分子量为1.96× 104的GDF9重组蛋白质,其最高表达量达到菌体蛋白质总量的30%左右.成功制备了抗GDF9抗血清,血清效价达到1∶51 200,抗血清经纯化后得到了高纯度的GDF9抗体.GDF9重组蛋白质和抗GDF9抗体有望应用于提高羊繁殖性能和促进动物胚胎发育的研究和技术开发.%In order to obtain recombinant GDF9 (growth differentiation factor 9 ) protein and anti-GDF9 antibody, a pair of primers designed according to the buffalo GDF9 gene mature peptide sequence ( GenBank; FJ529501. 1) was used to amplify the cDNA sequence of water buffalo GDF9 mature peptide with water buffalo ge-nomic DNA as the template, and the alignment was made between the cDNA sequence and this region of other ruminant. The cDNA sequence was cloned into the BamH Ⅰ and Hind Ⅲ sites of plasmid pRSET A to generate the expression vector pR-GDF9, which was further transformed into bacteria Escherichia. coli BL21 (DE3). The transformed bacteria were induced to produce a recombinant GDF9 protein by IPTG

  8. 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...... chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward...

  9. Polyclonal gammopathy related to renal bleeding in a peritoneal dialysis patient

    Directory of Open Access Journals (Sweden)

    Eun-Mi Cho

    2013-07-01

    Full Text Available Polyclonal gammopathy represents the diffuse activation of B cells and is usually related to inflammation or immune-related diseases. However, the mechanisms leading to polyclonal gammopathy are essentially speculative. Generally, infectious, inflammatory, or various other reactive processes may be indicated by the presence of a broad-based peak or band in the gamma region on serum protein electrophoresis results. A 15-year-old girl, who had been receiving peritoneal dialysis, presented with polyclonal gammopathy and massive gross hematuria. Renal artery embolization was performed, after which the continuous bleeding subsided and albumin-globulin dissociation resolved. This is a rare case of polyclonal gammopathy related to renal bleeding.

  10. ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka

    2008-09-01

    Full Text Available The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5, pp.698-702, DOI:http://dx.doi.org/10.14429/dsj.58.1693

  11. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    DEFF Research Database (Denmark)

    Holm, B. E.; Bergmann, Ann Christina; Hansen, Paul Robert

    2015-01-01

    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme......-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...

  12. Preparation, Characterization, and Application of Antiharpinxoo Antibody

    Institute of Scientific and Technical Information of China (English)

    SHAO Min; LI Ming; PAN Xiao-mei; WANG Jin-sheng

    2006-01-01

    Polyclonal antiharpinxoo rabbit antibody has been prepared successfully using purified harpinxoo protein as an immunogen.The ELISA titer of the antiserum against harpinxoo was about 1:2 000. Western blot analysis showed that the antiserum could bind to the expression harpinxoo protein in particular. hrf1, encoding harpinxoo, is an expression in transgenic rice,detected by antiharpinxoo rabbit antibody. The rabbit antibody against harpinxoo can be used to study further about the biological function, harpinxoo localization, and hrf1 gene expression in other plants.

  13. Integrated Design of Antibodies for Systems Biology Using Ab Designer.

    Science.gov (United States)

    Pisitkun, Trairak; Dummer, Patrick; Somparn, Poorichaya; Hirankarn, Nattiya; Kopp, Jeffrey B; Knepper, Mark A

    2014-03-24

    In the current era of large-scale biology, systems biology has evolved as a powerful approach to identify complex interactions within biological systems. In addition to high throughput identification and quantification techniques, methods based on high-quality mono-specific antibodies remain an essential element of the approach. To assist the large-scale design and production of peptide-directed antibodies for systems biology studies, we developed a fully integrated online application, AbDesigner (http://helixweb.nih.gov/AbDesigner/), to help researchers select optimal peptide immunogens for antibody generation against relatively disordered regions of target proteins. Here we describe AbDesigner in terms of its features, comparing it to other software tools, and use it to design three antibodies against kidney disease-related proteins in human, viz. nephrin, podocin, and apolipoprotein L1.

  14. Antibody Engineering for Pursuing a Healthier Future

    Science.gov (United States)

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

  15. 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

    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...... 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...

  16. A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

    Science.gov (United States)

    Lee, Chingwei V; Koenig, Patrick; Fuh, Germaine

    2014-01-01

    A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

  17. Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues

    NARCIS (Netherlands)

    Coers, W; Timens, W; Kempinga, C; Klok, PA; Moshage, H

    1998-01-01

    Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several an

  18. Inhibition of Staphylococcus epidermidis biofilm formation by rabbit polyclonal antibodies against the SesC protein.

    NARCIS (Netherlands)

    Shahrooei, M.; Hira, V.; Stijlemans, B.; Merckx, R.; Hermans, P.W.M.; Eldere, J. van

    2009-01-01

    Several well-studied proteins with defined roles in Staphylococcus epidermidis biofilm formation are LPXTG motif-containing proteins. Here, we investigate the possible use of the LPXTG motif-containing protein SesC (S. epidermidis surface protein C; accession no. NP_765787) as a target for

  19. A novel combination approach of human polyclonal IVIG and antibiotics against multidrug-resistant Gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Sallam MM

    2016-12-01

    Full Text Available Mariam Madkour Sallam, Khaled Abou-Aisha, Mohamed El-Azizi Department of Microbiology, Immunology, and Biotechnology, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Cairo, Egypt Background: Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA and enterococci, have shown a remarkable ability to develop resistance to antimicrobial agents. Objective: We aimed to assess possible enhancement of the antimicrobial activity of vancomycin, amoxicillin, clarithromycin, and azithromycin by human polyclonal intravenous immunoglobulin G (IVIG against 34 multidrug-resistant (MDR bacterial isolates, including MRSA, Enterococcus faecium, and Enterococcus faecalis. Materials and methods: Double combinations of the antibiotics with the IVIG were assessed by checkerboard assay, where the interaction was evaluated with respect to the minimum inhibitory concentration (MIC of the antibiotics. The results of the checkerboard assay were verified in vitro using time-kill assay and in vivo using an invasive sepsis murine model. Results: The checkerboard assay showed that IVIG enhanced the antimicrobial activity of amoxicillin and clarithromycin against isolates from the three groups of bacteria, which were resistant to the same antibiotics when tested in the absence of IVIG. The efficacy of vancomycin against 15% of the tested isolates was enhanced when it was combined with the antibodies. Antagonism was demonstrated in 47% of the E. faecalis isolates when clarithromycin was combined with the IVIG. Synergism was proved in the time-kill assay when amoxicillin was combined with the antibodies; meanwhile, antagonism was not demonstrated in all tested combinations, even in combinations that showed such response in checkerboard assay. Conclusion: The suggested approach is promising and could be helpful to enhance the antimicrobial activity of not only effective antibiotics but also antibiotics that have

  20. Thyroid Antibodies

    Science.gov (United States)

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  1. Type 1 diabetes immunotherapy using polyclonal regulatory T cells.

    Science.gov (United States)

    Bluestone, Jeffrey A; Buckner, Jane H; Fitch, Mark; Gitelman, Stephen E; Gupta, Shipra; Hellerstein, Marc K; Herold, Kevan C; Lares, Angela; Lee, Michael R; Li, Kelvin; Liu, Weihong; Long, S Alice; Masiello, Lisa M; Nguyen, Vinh; Putnam, Amy L; Rieck, Mary; Sayre, Peter H; Tang, Qizhi

    2015-11-25

    Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing β cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo-expanded autologous CD4(+)CD127(lo/-)CD25(+) polyclonal Tregs (0.05 × 10(8) to 26 × 10(8) cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3(+)CD4(+)CD25(hi)CD127(lo) phenotype long-term. There were no infusion reactions or cell therapy-related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy. Copyright © 2015, American Association for the Advancement of Science.

  2. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

    DEFF Research Database (Denmark)

    Klausen, Joan; Andresen, Lars Ole; Barfod, Kristen

    2001-01-01

    A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6...

  3. 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

    . 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...

  4. Effects of anti-IgM suppression on polyclonally activated murine B cells: analysis of immunoglobulin mRNA, gene specific nuclear factors and cell cycle distribution.

    Science.gov (United States)

    Marcuzzi, A; Van Ness, B; Rouse, T; Lafrenz, D

    1989-01-01

    Polyclonal activation of murine B cells with bacterial lipopolysaccharide (LPS) and dextran sulfate (DxS) results in cell proliferation as well as increased immunoglobulin gene transcription and antibody secretion. When added to B cell cultures during mitogen activation, anti-mu antibody suppresses the rate of proliferation and immunoglobulin gene expression. Using this model system we show that co-cultures of B cells with LPS/DxS and anti-mu resulted in a decrease of both mu and kappa chain mRNA. Suppression did not prevent B cell entry into cycle nor a significant alteration in the distribution of cells in phases of cell cycle, although it did prolong the cycle transit time in a dose dependent fashion as determined by bromodeoxyuridine pulse labelling. Analysis of B cell specific nuclear binding factors, which previously have been shown to be important in regulating immunoglobulin gene transcription were examined. Results show that the kappa-specific enhancer binding activity of NF-kappa B was induced in activated as well as suppressed cultures. The lymphoid specific factor NF-A2, which recognizes the octamer sequence motif in the promoters of immunoglobulin genes, was induced by the polyclonal activation but was selectively lost in extracts from suppressed cells. Thus, specific regulation of the nuclear factor which plays a critical role in both heavy and light chain immunoglobulin gene expression may contribute to the transcriptional suppression observed in anti-mu treated B cells. Images PMID:2481271

  5. 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.

  6. Size-dependence of tree growth response to drought for Norway spruce and European beech individuals in monospecific and mixed-species stands.

    Science.gov (United States)

    Ding, H; Pretzsch, H; Schütze, G; Rötzer, T

    2017-09-01

    Climate anomalies have resulted in changing forest productivity, increasing tree mortality in Central and Southern Europe. This has resulted in more severe and frequent ecological disturbances to forest stands. This study analysed the size-dependence of growth response to drought years based on 384 tree individuals of Norway spruce [Picea abies (L.) Karst.] and European beech [Fagus sylvatica ([L.)] in Bavaria, Germany. Samples were collected in both monospecific and mixed-species stands. To quantify the growth response to drought stress, indices for basal area increment, resistance, recovery and resilience were calculated from tree ring measurements of increment cores. Linear mixed models were developed to estimate the influence of drought periods. The results show that ageing-related growth decline is significant in drought years. Drought resilience and resistance decrease significantly with growth size among Norway spruce individuals. Evidence is also provided for robustness in the resilience capacity of European beech during drought stress. Spruce benefits from species mixing with deciduous beech, with over-yielding spruce in pure stands. The importance of the influence of size-dependence within tree growth studies during disturbances is highlighted and should be considered in future studies of disturbances, including drought. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  7. Biochemical compounds' dynamics during larval development of the carpet-shell clam Ruditapes decussatus (Linnaeus, 1758): effects of mono-specific diets and starvation

    Science.gov (United States)

    Matias, Domitília; Joaquim, Sandra; Ramos, Margarete; Sobral, Paula; Leitão, Alexandra

    2011-09-01

    Successful larval growth and development of bivalves depend on energy derived from internal (endotrophic phase) and external (exotrophic phase) sources. The present paper studies survival, growth and biochemical changes in the early developmental stages (from egg to pediveliger) of the clam Ruditapes decussatus in order to characterize the nutritional requirements and the transition from the endotrophic to the exotrophic phase. Three different feeding regimes were applied: starvation and two mono-specific microalgal diets ( Isochrysis aff galbana and Chaetoceros calcitrans). A comparison between fed and unfed larvae highlighted the importance of egg lipid reserves, especially neutral lipids, during a brief endotrophic phase of embryonic development (first 2 days after fertilization). Egg reserves, however, may energetically contribute to the maintenance of larvae beyond the embryonic development. In fed larvae, the endotrophic phase is followed by a mixotrophic phase extending to days 5-8 after fertilization and a subsequent exotrophic phase. Metamorphosis starts around day 20. The intense embryonic activities are supported by energy derived from lipids, mainly from neutral lipids, and the metamorphic activities are supported by energy derived essentially from proteins accumulated during the planktonic phase and depend on the nutritional value of diets. The diet of I. aff galbana proves to be more adequate to R. decussatus larval rearing. The results provide useful information for the successful production of R. decussatus aquaculture.

  8. Generation, use, and validation of receptor-selective antibodies.

    Science.gov (United States)

    Mackrill, John J

    2004-01-01

    Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties. In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described. Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage. Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described. Finally, methods are given for improvement of the titer and specificity of PAbs.

  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. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    Science.gov (United States)

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

  11. Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

    DEFF Research Database (Denmark)

    Knudsen, K; Madsen, AS; Mygind, P

    1999-01-01

    of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel...

  12. Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes.

    Science.gov (United States)

    Gunnarsson, Anders Patrik; Christensen, Rikke; Praetorius, Jeppe; Jensen, Uffe Birk

    2017-01-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive basal cells express higher levels of Itgβ1 the colony-forming efficiency is slightly lower than CSPG4-negative basal cells. Sorting the directly isolated keratinocytes based on Itgβ1 did not reveal differences in colony-forming efficiency between keratinocytes expressing high or low levels of Itgβ1. However, after the first passage Itgβ1 could be used to predict colony-forming efficiency whether the culture was established from CSPG4-positive or CSPG4-negative basal cell keratinocytes. Although we were unable to detect differences in the colony-forming assay, global gene expression profiling showed that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges the way we assess for stemness within the human epidermal basal layer.

  13. Effect of pH on antigen binding by clonotypic antibodies with different isoelectric points

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Y.; Miyai, K.; Hata, N.; Iijima, Y.

    1987-02-01

    Polyclonal rabbit antibodies to thyroxine, human myoglobin, human growth hormone, human thyrotropin, human alpha-fetoprotein, and human thyroglobulin were fractionated into clonotypic antibodies with different isoelectric points by agarose isoelectric focusing or chromatofocusing. The effect of pH on the binding of these antigens by their respective clonotypic antibodies was assessed by radioimmunoassay. The profiles of the pH effect differed both for different antigens and for different pI's of the antibodies used. The pH optima in the radioimmunoassays for protein antigens were found to be expressed as a function of pI and molecular weight of both antigen and antibody molecules.

  14. Microbial gut overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill.

    Science.gov (United States)

    van Saene, H K F; Taylor, N; Damjanovic, V; Sarginson, R E

    2008-05-01

    Polyclonality is defined as the occurrence of different genotypes of a bacterial species. We are of the opinion that these different clones originate within the patient. When infections and outbreaks occur, the terms of polyclonal infections and polyclonal outbreaks have been used, respectively. The origin of polyclonality has never been reported, although some authors suggest the acquisition of different clones from different animate and inanimate sources. We think that the gut of the critically ill patient with microbial overgrowth is the ideal site for the de-novo development of new clones, following increased spontaneous mutation.

  15. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    DEFF Research Database (Denmark)

    Holm, B.E.; Bergmann, A.C.; Hansen, Paul Robert

    2015-01-01

    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...... with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures...

  16. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  17. The Establishment of Immunochemistry Test Based on a Synthetic Peptide Antibody for the Detection of a Porcine Circovirus-Like Virus P1

    Institute of Scientific and Technical Information of China (English)

    Libin WEN; Aihua MAO; Junming ZHOU; Lixin LU; Jianping XIE; Fengzhi WANG; Kongwang HE; Yanxiu NI; Xuehan ZHANG; Rongli GUO; Bin LI; Xiaomin WANG; Zhengyu YU

    2014-01-01

    Recently, a novel porcine circovirus-like virus P1 with a circular DNA genome of 0.648 kb was identified. P1 antigen was detected both in vitro and in vivo by synthetic peptide-derived polyclonal antibody-based immunochemistry. The designed peptides were synthesized by solid-phase technique, purified by high per-formance liquid chromatography, coupled to Keyhole limpet hemocyanin, and injected into rabbits to prepare polyclonal antibody. The emergence of positive cells revealed that synthetic peptide could elicit antibodies against P1 and viral protein could be synthesized. The polyclonal peptide antibodies described here was successfully ap-plied to immunochemical staining and proved helpful in diagnosing P1.

  18. 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.

  19. Association between polyclonal and mixed mycobacterial Mycobacterium avium complex infection and environmental exposure.

    Science.gov (United States)

    Fujita, Kohei; Ito, Yutaka; Hirai, Toyohiro; Kubo, Takeshi; Maekawa, Koichi; Togashi, Kaori; Ichiyama, Satoshi; Mishima, Michiaki

    2014-01-01

    Polyclonal and mixed mycobacterial Mycobacterium avium complex (MAC) infection is observed in pulmonary MAC disease. Human living environments contain multiple species or genotypes of nontuberculous mycobacterial strains and are considered sources of infection. To investigate the association of environmental exposure with polyclonal and mixed mycobacterial infection in pulmonary MAC disease after adjustments for potential confounding diseases and conditions and radiographic findings. We collected two separate sputum samples from 102 patients and single sputum samples from 18 patients in whom the second MAC strain was not isolated in our prospective cohort of pulmonary MAC disease. MAC isolates from sputum samples and patients' residential soils were used for variable number of tandem repeats (VNTR) analyses. Polyclonal and mixed mycobacterial MAC infections were defined as having different VNTR genotypes and other mycobacterial species, respectively. Monoclonal MAC infection was defined as all isolates showing a single VNTR genotype. Associations of the type of infection with clinical and radiographic findings and environmental exposure were measured. Polyclonal and mixed mycobacterial MAC and monoclonal infections were observed in 42 and 78 patients, respectively. By stepwise regression analysis, patients with polyclonal and mixed mycobacterial MAC infections were associated with history of asthma (odds ratio [OR], 11.56; 95% confidence interval [CI], 1.41-255.77; P = 0.021), high soil exposure (≥2 h/wk; OR, 4.31; 95% CI, 1.72-11.45; P swimming in a pool (OR, 9.69; 95% CI, 1.21-206.92; P < 0.01). Environmental exposure was associated with polyclonal and mixed mycobacterial MAC infection in pulmonary MAC disease.

  20. 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 ...

  1. Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

    OpenAIRE

    Lück, P. C.; Helbig, J H; Günter, U; Assmann, M.; Blau, R; Koch, H.; Klepp, M.

    1994-01-01

    A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodie...

  2. Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

    OpenAIRE

    2006-01-01

    AIM: To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes.

  3. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  4. Constant domain-regulated antibody catalysis.

    Science.gov (United States)

    Sapparapu, Gopal; Planque, Stephanie; Mitsuda, Yukie; McLean, Gary; Nishiyama, Yasuhiro; Paul, Sudhir

    2012-10-19

    Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

  5. Competition for light and light use efficiency for Acacia mangium and Eucalyptus grandis trees in mono-specific and mixed-species plantations in Brazil

    Science.gov (United States)

    Le Maire, G.; Nouvellon, Y.; Gonçalves, J.; Bouillet, J.; Laclau, J.

    2010-12-01

    stemwood since half of the canopy (Acacias) are dominated, and the other half does not benefit much in terms of tree growth compared to absorbed light. The eventual benefit of the nitrogen-fixing species is not visible in the mixture with 50% of each species. More attention has to be paid to introducing acacias in an additive series with the same density of eucalyptus trees as in the monospecific stands.

  6. Long-term antibody synthesis in vitro- IV. Independent segregation of antibodies directed to different determinants of an antigen molecule in its native configuration.

    Science.gov (United States)

    Conway de Macario, E; Macario, A J

    1976-10-01

    Independent segregation of antibody populations directed to different portions of E. coli beta-d-galactosidase occurs during the immune response against the enzyme. Anti-enzyme antibodies able to interact and activate a naturally occurring ligand, the mutant-defective enzyme AMEF (Antibody Mediated Enzyme Factor), do not parallel anti-enzyme antibodies which are measured by a coprecipitation assay involving precipitation of the wild-type molecule. Dissociation of the two antibody populations is best achieved in microcultures sustaining long-lasting responses. Similarly, anti-NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) antibodies could be elicited without concomitant synthesis of anti-carrier antibodies by short-term challenge in vitro of ovalbumin-NIP-primed lymph nodes with a heterologous conjugate in which the hapten NIP was coupled to a carrier known to be non-immunogenic under the conditions of challenge. The potential applications of these findings are indicated, namely: large-scale production of monospecific antibodies in vitro; and the possibility of studying the regulatory role of antibodies directed towards on portion of the immunogenic molecule on the response to other regions of the same molecule.

  7. A novel combination approach of human polyclonal IVIG and antibiotics against multidrug-resistant Gram-positive bacteria

    Science.gov (United States)

    Sallam, Mariam Madkour; Abou-Aisha, Khaled; El-Azizi, Mohamed

    2016-01-01

    Background Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and enterococci, have shown a remarkable ability to develop resistance to antimicrobial agents. Objective We aimed to assess possible enhancement of the antimicrobial activity of vancomycin, amoxicillin, clarithromycin, and azithromycin by human polyclonal intravenous immunoglobulin G (IVIG) against 34 multidrug-resistant (MDR) bacterial isolates, including MRSA, Enterococcus faecium, and Enterococcus faecalis. Materials and methods Double combinations of the antibiotics with the IVIG were assessed by checkerboard assay, where the interaction was evaluated with respect to the minimum inhibitory concentration (MIC) of the antibiotics. The results of the checkerboard assay were verified in vitro using time-kill assay and in vivo using an invasive sepsis murine model. Results The checkerboard assay showed that IVIG enhanced the antimicrobial activity of amoxicillin and clarithromycin against isolates from the three groups of bacteria, which were resistant to the same antibiotics when tested in the absence of IVIG. The efficacy of vancomycin against 15% of the tested isolates was enhanced when it was combined with the antibodies. Antagonism was demonstrated in 47% of the E. faecalis isolates when clarithromycin was combined with the IVIG. Synergism was proved in the time-kill assay when amoxicillin was combined with the antibodies; meanwhile, antagonism was not demonstrated in all tested combinations, even in combinations that showed such response in checkerboard assay. Conclusion The suggested approach is promising and could be helpful to enhance the antimicrobial activity of not only effective antibiotics but also antibiotics that have been proven to be ineffective against MDR bacteria. To our knowledge, this combinatorial approach against MDR bacteria, such as MRSA and enterococci, has not been investigated before. PMID:27994476

  8. Changes in antibody profile after treatment of human onchocerciasis.

    Science.gov (United States)

    Lee, S J; Francis, H L; Awadzi, K; Ottesen, E A; Nutman, T B

    1990-08-01

    To define the changes in antibody response to Onchocerca volvulus antigens after treatment of patients with onchocerciasis, IgG and IgE antibodies were examined quantitatively and qualitatively in 21 patients and 3 control individuals before and sequentially for 14 days after treatment with diethylcarbamazine. The quantitative levels of IgE and IgG responses (both polyclonal and O. volvulus-specific) remained essentially unchanged for all patients, but 9 of the 21 patients showed intensified responses to one or more parasite-specific antigens, and 8 of 21 developed antibodies to previously undetected antigens. There was a significant correlation between the intensities of infection and the development of newly recognized anti-O. volvulus antibodies. These studies demonstrate that O. volvulus-specific IgE and IgG antibody responses are, at least transiently, enhanced by treatment with diethylcarbamazine and that after treatment, parasites possibly release antigens previously hidden from the host's immune response.

  9. 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...... defined, mostly due to the lack of appropriate working tools like antibodies and functional assays. Membrane bound molecules like immunoglobulins (Ig) serve as cell surface markers for specific cell subsets and the identification of cells relies upon the production of specific antibodies towards...... 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...

  10. Feasibility studies of using the Catfish Immune System to produce monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Poston, T.M.

    1987-03-01

    The objective of these studies was to determine the feasibility of using a teleost cell line to produce monoclonal antibodies. Studies were undertaken to demonstrate the production of a polyclonal response of channel catfish (Icatalurus punctatus) challenged with mycotoxins coupled to a protein carrier. Companion studies were also performed to induce a permanent cell line with catfish lymphocytes. Attempts to demonstrate a polyclonal response to haptenized mycotoxins were inconclusive. Tests to induce an immortal, permanent cell line with benzene and x-ray irradiated cells were also inconclusive. 3 refs., 13 tabs.

  11. Antibody-based resistance to plant pathogens.

    Science.gov (United States)

    Schillberg, S; Zimmermann, S; Zhang, M Y; Fischer, R

    2001-01-01

    Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

  12. 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.

  13. Estimativa do coeficiente Priestley-Taylor em floresta monodominante Cambarazal no Pantanal Estimation of the Priestley-Taylor coefficient in the monospecific forest in northern Pantanal, Brazil

    Directory of Open Access Journals (Sweden)

    Luciana Sanches

    2010-12-01

    Full Text Available O coeficiente Priestley-Taylor (α foi calculado baseado na estimativa da evapotranspiração pelo método de Bowen para floresta monodominante Vochysia divergens no Pantanal, Brasil. A área em estudo estava localizada no noroeste do Pantanal a aproximadamente 160 km de Cuiabá, Mato Grossso, Brasil (16º39'50''S; 56º47'50''O. Medições micrometeorológicas contínuas, em uma torre a uma altura de 32 m de altura, foram feitas de janeiro a dezembro de 2007. A evapotranspiração variou de 2,50 mm dia-1 (estação seca a 4,10 mm dia-1 (estação úmida. O coeficiente Pristley-Taylor (α variou durante o ano com valores máximos e mínimos nas estações seca e úmida, respectivamente, com média anual de 0,65 ± 0,18 de acordo com o padrão climático da área em estudo em função do aumento do conteúdo de água no solo/lâmina d'água de inundação e/ou diminuição na demanda evaporativa. Com a determinação empírica das dimensões de α, as estimativas da evapotranspiração podem ser melhoradas para florestas de Vochysia divergens na planície de inundação do Pantanal.The Priestley-Taylor coefficient (α was calculated based on the Bowen method evapotranspiration estimative for Vochysia divergens monospecific forests in Pantanal, Brazil. The study area was located at the northeastern Pantanal, approximately 160 km from Cuiabá city, Mato Grosso State, Brazil (16º39'50''S; 56º47'50''W. Continuous micrometeorological flux measurements at a 32 m tower height were made from January to December of 2007. The evapotranspiration ranged from 2.26 mm day-1 (dry season to 4.50 mm day-1 (wet season. The average estimated α value ranged along the year from a maximum and a minimum during the dry and wet season respectively, with an annual average of 0.65±0.18 according to the weather pattern over the study area due to the increase of water content in soil/water flooding depth and/or decrease in evaporative demand. Based on empirical α values

  14. Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein

    DEFF Research Database (Denmark)

    Keck, Zhenyong; Wang, Wenyan; Wang, Yong

    2013-01-01

    polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay......A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While...... between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions...

  15. Considerations in producing preferentially reduced half-antibody fragments.

    Science.gov (United States)

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

  16. Effect of polyclonal activators on cytokine production by blood cells and by malignant breast cancer cells.

    Science.gov (United States)

    Kunts, T A; Karpukhina, K V; Mikhaylova, E S; Marinkin, I O; Varaksin, N A; Autenshlyus, A I; Lyakhovich, V V

    2016-01-01

    The production of cytokines by peripheral blood cells and biopsy specimens of tumors stimulated by polyclonal activators (PAs) was evaluated in 34 patients with invasive ductal breast carcinoma using enzyme-linked immunosorbent assay (ELISA). Positive correlation between the stimulation index of polyclonal activators (SIPA) for IL-18 production by the tumor and the relative content of poorly differentiated cells was revealed. The latter, in turn, was positively correlated with the numbers of normal and pathologic mitoses and the degree of malignancy. Cancer cells can produce IL-18, which is involved in the process of angiogenesis, stimulates invasion and metastasis. Decrease in SIPA for the production of IL-6 and GCSF by peripheral blood cells could serve as an indicator of malignant progression in invasive ductal breast carcinoma.

  17. A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection

    Science.gov (United States)

    Xia, Yanyan; Shen, Han; Zhu, Yefei; Xu, Hongpan; Li, Zhiyang; Si, Jin

    2017-01-01

    Golgi protein 73 (GP73) is a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) that has been found to be abnormally elevated in liver disease. A latex particle-enhanced turbidimetric immunoassay (LTIA) was recently introduced and licensed for application in a variety of automated clinical chemistry analyzers. However, no studies have reported sufficient data on analytical performance of this method when using 3 monoclonal antibodies for GP73 measurement. The experimental conditions were firstly optimized and range of linearity, diagnostic potential, clinical relevance were compared with the LTIA based on polyclonal antibodies and ELISA. Dilution tests for the LTIA using 3 monoclonal antibodies produced a calibration curve from 10 to 350 ng/mL while the polyclonal antibodies produced the curve from 20 to 320 ng/mL. The detection limit was achieved at 1.82 ng/mL concentration. Within-run CV was obtained in the range of 1.5–2.9% and ROC curves indicated sensitivity and specificity of the LTIA based on 3 monoclonal antibodies were 96.7% and 93.3%, respectively, higher than for the polyclonal antibodies (94.6% and 72.4%) and ELISA (70.0% and 83.3%). Therefore, the LTIA assay based on 3 monoclonal antibodies is thus applicable in quantification of GP73 concentration in automated biochemistry analyzers. PMID:28054632

  18. Lipoproteins are Major Targets of the Polyclonal Human T-cell Response to M. tuberculosis1

    OpenAIRE

    2012-01-01

    Most vaccines and basic studies of T cell epitopes in M. tuberculosis emphasize water soluble proteins that are secreted into the extracellular space and presented in the context of MHC Class II. Much less is known about the role of antigens retained within the cell wall. We used polyclonal T cells from infected humans to probe for responses to immunodominant antigens in the M. tuberculosis cell wall. We found that the magnitude of response to secreted or cell wall intrinsic compounds was sim...

  19. Monoclonal antibody-based time-resolved fluorescence immunoassays for daidzein, genistein and equol in blood and urine

    DEFF Research Database (Denmark)

    Talbot, Duncan C.S.; Ogborne, Richard M.; Dadd, Tony

    2007-01-01

    of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genestein and equol erquired diethyl-ether extraction. Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA and gas chromatography-mass spectrometry...

  20. Balancing speed and accuracy of polyclonal T cell activation: a role for extracellular feedback

    Directory of Open Access Journals (Sweden)

    Savir Yonatan

    2012-08-01

    Full Text Available Abstract Background Extracellular feedback is an abundant module of intercellular communication networks, yet a detailed understanding of its role is still lacking. Here, we study interactions between polyclonal activated T cells that are mediated by IL-2 extracellular feedback as a model system. Results Using mathematical modeling we show that extracellular feedback can give rise to opposite outcomes: competition or cooperation between interacting T cells, depending on their relative levels of activation. Furthermore, the outcome of the interaction also depends on the relative timing of activation of the cells. A critical time window exists after which a cell that has been more strongly activated nevertheless cannot exclude an inferior competitor. Conclusions In a number of experimental studies of polyclonal T-cell systems, outcomes ranging from cooperation to competition as well as time dependent competition were observed. Our model suggests that extracellular feedback can contribute to these observed behaviors as it translates quantitative differences in T cells’ activation strength and in their relative activation time into qualitatively different outcomes. We propose extracellular feedback as a general mechanism that can balance speed and accuracy – choosing the most suitable responders out of a polyclonal population under the clock of an escalating threat.

  1. Association between polyclonal B cell activation and the presence of autoantibodies in mice infected with Yersinia enterocolitica O:3

    Directory of Open Access Journals (Sweden)

    B.M.M. Medeiros

    1997-03-01

    Full Text Available Eight-week old conventional female Swiss mice were inoculated intravenously with Yersinia enterocolitica O:3. A second group of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed for spleen cell collection on the 3rd, 5th, 7th, 10th, 14th and 21st day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. Total immunoglobulin levels were determined in mouse serum by single radial immunodiffusion and the presence of autoantibodies was determined by ELISA. We observed a marked increase in the total number of cells secreting immunoglobulins of all isotypes as early as on the 3rd day post-infection and the peak of secretion occurred on the 7th day. At the peak of the immunoglobulin response, the total number of secreting cells was 19 times higher than that of control mice and most immunoglobulin-secreting cells were of the IgG2a isotype. On the 10th day post-infection, total serum immunoglobulin values were 2 times higher in infected animals when compared to the control group, and continued at this level up to the 21st day post-infection. Serum absorption with viable Y. enterocolitica cells had little effect on antibody levels detected by single radial immunodiffusion. Analysis of serum autoantibody levels revealed that Y. enterocolitica infection induced an increase of anti-myosin and anti-myelin immunoglobulins. The sera did not react with collagen. The present study demonstrates that Y. enterocolitica O:3 infection induces polyclonal activation of murine B cells which is correlated with the activation of some autoreactive lymphocyte clones

  2. The specific detection of E.coli using commercial polyclonal serum for the development of a biosensor

    CSIR Research Space (South Africa)

    Kumar, S

    2013-11-01

    Full Text Available from waste water. Further cross reactivity to other bacteria from the family of Enterobacteriaceae is evident in figure 2. This highlights the lack of specificity of the polyclonal serum to E. coli. Fig 3A...

  3. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Science.gov (United States)

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  4. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Directory of Open Access Journals (Sweden)

    Tong Li

    2015-07-01

    Full Text Available The effects of somatic mutations that transform polyspecific germline (GL antibodies to affinity mature (AM antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM. We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab, and subsequently, the DCM was combined with molecular dynamics (MD simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  5. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  6. Purification and characterization of osteopontin from human milk

    DEFF Research Database (Denmark)

    Sørensen, Steen; Justesen, Steen Just; Johnsen, Anders H

    2003-01-01

    separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when...... investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies....

  7. Common epitope on urinary antigen derived from different Legionella pneumophila serogroup 1 strains recognized by a monoclonal antibody.

    Science.gov (United States)

    Helbig, J H; Lück, P C; Pilz, C; Witzleb, W

    1990-09-01

    Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

  8. Polyclonal hypergammaglobulinemia and high smooth-muscle autoantibody titers with specificity against filamentous actin: consider visceral leishmaniasis, not just autoimmune hepatitis.

    Science.gov (United States)

    Makaritsis, Konstantinos P; Gatselis, Nikolaos K; Ioannou, Maria; Petinaki, Efthimia; Dalekos, George N

    2009-07-01

    Visceral leishmaniasis (VL) remains a public health problem in most countries bordering the Mediterranean basin. Its diagnosis is challenging and often delayed, as the main clinical picture is often indistinguishable from that of other infectious and non-infectious diseases. Herein, we report two unusual cases of VL that presented with several characteristics of autoimmune hepatitis (AIH). Neither patient had a history of fever, only generalized symptoms accompanied by polyclonal hypergammaglobulinemia, cytopenias, signs of portal hypertension, elevated transaminases, and high titers of antinuclear and smooth-muscle autoantibodies (SMA) with reactivity against filamentous actin (F-actin), which has been recognized as specific to AIH. A clinical diagnosis of AIH was considered, but a bone marrow biopsy was performed before a liver biopsy to exclude a primary bone marrow disease. The biopsy led to the diagnosis of VL. The diagnosis was further confirmed by IgG antibodies against Leishmania spp. using ELISA and PCR-based assays. Treatment with amphotericin in the first case and pentamidine in the second (because of a severe reaction to amphotericin) was effective. From the clinical point of view, it should be emphasized that, in cases with high titers of anti-F-actin AIH-specific SMA accompanied by polyclonal hypergammaglobulinemia, the possibility of AIH should be cautiously differentiated from VL; this distinction is of paramount importance because initiation of immunosuppression for AIH treatment would be detrimental to a patient with underlying leishmaniasis. Therefore, in such cases and in areas where the disease is still present, it seems rational to exclude VL before starting any immunosuppressive therapy.

  9. Production of Polyclonal and Monoclonal Antibodies Against the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3Aa16

    OpenAIRE

    Ben Hamadou-Charfi, D.; Sauer, A.; Abdelkafi-Mesrati, L.; Jaoua, S.; Dietrich, S.

    2014-01-01

    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. ...

  10. PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST PELARGONIUM ZONATE SPOT VIRUS COAT PROTEIN EXPRESSED IN ESCHERICHIA COLI AND APPLICATION FOR IMMUNODIAGNOSIS

    Science.gov (United States)

    Pelargonium zonate spot virus (PZSV), a new emerging disease on tomato in the United States, has been classified as the first member of new proposed genus, Anulavirus, within the family Bromoviridae and characterized as having unstable virions with weakly immunogenic properties. To develop serologic...

  11. [Diagnosis of Legionella pneumonia by detection of antigenuria using an enzyme immunoassay with 6 antibody specificities].

    Science.gov (United States)

    Helbig, J H; Lück, P C; Witzleb, W

    1989-10-01

    In patients with microbiologically and clinically suspected Legionella caused pneumonia antigenuria was investigated by means of a direct two-site binding assay (ELISA) with polyclonal antibodies against Legionella (L.) pneumonia serogroup 1, 2, 3, 5 and 6 and L. micdadei. By application of antibodies only against L. pneumonia serogroup 1 antigenuria was found in 27 of 66 patients (= 41%). The expanding of the used specificities of antibodies in 47 out of this cases resulted in an increase of positive urinary antigen findings from 38% to 55%. Possibilities and limits of the detection of antigenuria with regard to efficient and rapid diagnostics of legionellosis are discussed.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Muthu Ramakrishnan

    Full Text Available BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: High sensitivity calorimetric determination of antibody binding to

  14. Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics.

    Science.gov (United States)

    Yakes, B J; Buijs, J; Elliott, C T; Campbell, K

    2016-08-15

    Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein.

  15. Lipoproteins are major targets of the polyclonal human T cell response to Mycobacterium tuberculosis.

    Science.gov (United States)

    Seshadri, Chetan; Turner, Marie T; Lewinsohn, David M; Moody, D Branch; Van Rhijn, Ildiko

    2013-01-01

    Most vaccines and basic studies of T cell epitopes in Mycobacterium tuberculosis emphasize water-soluble proteins that are secreted into the extracellular space and presented in the context of MHC class II. Much less is known about the role of Ags retained within the cell wall. We used polyclonal T cells from infected humans to probe for responses to immunodominant Ags in the M. tuberculosis cell wall. We found that the magnitude of response to secreted or cell wall intrinsic compounds was similar among healthy controls, patients with latent tuberculosis, and patients with active tuberculosis. Individual responses to secreted Ags and cell wall extract were strongly correlated (r(2) = 0.495, p = 0.001), suggesting that T cells responding to cell wall and secreted Ags are present at similar frequency. Surprisingly, T cell stimulatory factors intrinsic to the cell wall partition into organic solvents; however, these responses are not explained by CD1-mediated presentation of lipids. Instead, we find that molecules soluble in organic solvents are dependent upon MHC class II and recognized by IFN-γ-secreting CD4(+) T cells. We reasoned that MHC class II-dependent Ags extracting into lipid mixtures might be found among triacylated lipoproteins present in mycobacteria. We used M. tuberculosis lacking prolipoprotein signal peptidase A (lspA), an enzyme required for lipoprotein synthesis, to demonstrate loss of polyclonal T cell responses. Our results demonstrate the use of bacterial genetics to identify lipoproteins as an unexpected and immunodominant class of cell wall-associated Ags targeted by the polyclonal human T cell response to M. tuberculosis.

  16. 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.

  17. 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

    -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......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...

  18. 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

    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...... is only recognized (1) by a negative muscle tissue control that does not harbor the protein to be examined (fx. from knockout mouse) or (2) by use of a nonsense rabbit antibody that has been prepared in the same way as the antibody of interest. However, many muscle immuno(cyto/histo)chemical studies only...... 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...

  19. Antibodies recognizing different domains of the polymeric immunoglobulin receptor.

    Science.gov (United States)

    Solari, R; Kühn, L; Kraehenbuhl, J P

    1985-01-25

    The receptor responsible for the transepithelial transport of IgA dimer antibodies is a transmembrane glycoprotein known as membrane secretory component (SCm). During transport, the membrane anchoring domain is cleaved and the ectoplasmic domain of the receptor (SCs) remains tightly bound to the IgA dimer in exosecretions. We have produced monoclonal antibodies with distinct specificities against both cytoplasmic and ectoplasmic epitopes of rabbit SCm. One antibody (anti-SC303) reacted both with SCm and free SCs but not with SCs bound to IgA dimer (SIgA). Therefore, it recognized an epitope close to the IgA dimer binding site. The other monoclonal antibody (anti-SC166), which was unable to react with SCs, bound to the 15-kDa cytoplasmic extension of the membrane-spanning domain of the receptor. A polyclonal antibody (GaR-SC), raised in a goat against rabbit milk SCs, reacted with a subpopulation of SCs not recognized by the anti-SC303 monoclonal antibody and in addition also reacted with covalently bound sIgA. The three antibodies cross-reacted with rat SCm. We demonstrate the ability of the anti-SC166 monoclonal antibody to immunoadsorb subcellular organelles as a result of the cytoplasmic orientation of its epitope. Our data indicate that there are functional differences between the high- and low-molecular-weight families of SC in terms of IgA dimer binding.

  20. Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1.

    Science.gov (United States)

    Pedersen, Inger L; Klinck, Rasmus; Hecksher-Sorensen, Jacob; Zahn, Stefan; Madsen, Ole D; Serup, Palle; Jorgensen, Mette C

    2006-05-01

    We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine beta-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing beta-cells, making this transcription factor a unique beta-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/kappa subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the beta-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies.

  1. Human cysticercosis: antigens, antibodies and non-responders.

    Science.gov (United States)

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied. Images FIG. 1 FIG. 3 FIG. 6 PMID:7389197

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-05

    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 /sup 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.

  3. Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: implications for the production of polyspecific snake antivenoms.

    Science.gov (United States)

    Dos-Santos, Maria Cristina; Arroyo, Cynthia; Solano, Sergio; Herrera, María; Villalta, Mauren; Segura, Alvaro; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo

    2011-02-01

    Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. EBF recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies.

    Science.gov (United States)

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

    2014-05-01

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

  5. Development and characterization of an enzyme-immunoassay with polyclonal antisera for benzene, toluene, and xylenes (BTX); Entwicklung und Charakterisierung eines Enzym-Immunoassays mit polyklonalen Antikoerpern fuer Benzol, Toluol und Xylole (BTX)

    Energy Technology Data Exchange (ETDEWEB)

    Beyer, K.; Knopp, D.; Niessner, R. [Technische Univ. Muenchen (Germany). Lehrstuhl fuer Hydrogeologie, Hydrochemie und Umweltanalytik

    1997-11-01

    An indirect competitive enzyme immunoassay for the detection of the volatile organic compounds benzene, toluene, and xylenes has been developed with polyclonal antibodies. The limit of determination for the equal amount of the five analytes in water was 210 {mu}g/l with a center point value of 1.7 mg/l. The addition of 10% of dimethyl sulfoxide (DMSO) to the sample decreased the limit of determination of 80 {mu}g/l and the center point value to 540 {mu}g/l. The specificity of the polyclonal antibodies was investigated based on its cross-reactivity. The influence of increasing concentrations of organic solvents and humic acid on the sensitivity of the antibodies was studied. Water samples were analysed both with GC-FID and by an immunochemical method in order to evaluate the suitability of the assay for environmental analysis. (orig.) [Deutsch] Zur Bestimmung von Benzol, Toluol und den drei Xylol-Isomeren (BTX) wird ein indirekter kompetitiver Enzym-Immunoassay auf der Basis von polyklonalen Antikoerpern beschrieben. Die Bestimmungsgrenze in Wasser liegt bei 210 {mu}g/l und der Testmittelpunkt der sigmoiden Kalibrierkurve bei 1,7 mg/l fuer ein aequivalentes Volumengemisch der fuenf Einzelverbindungen. Durch Loesemittelzusatz, zum Beispiel 10% Dimethylsulfoxid (DMSO), laesst sich die Bestimmungsgrenze auf 80 {mu}g/l und der Testmittelpunkt auf 540 {mu}g/l verringern. Die Spezifitaet der Antikoerper wurde durch Bestimmung von Kreuzreaktionen ermittelt. Weiterhin wurde der Einfluss unterschiedlicher Loesemittelgehalte und von Huminsaeure auf die Affinitaet der Antikoerper im Hinblick auf die Anwendung fuer Realproben untersucht. Um die Eignung des Tests fuer die Routineanalytik zu pruefen, wurde parallel zur immunochemischen Methode eine gaschromatographische Bestimmung BTX in Wasserproben durchgefuehrt. (orig.)

  6. Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

    OpenAIRE

    Sevigny, Leila M; Booth, Brian J.; Rowley, Kirk J.; Leav, Brett A.; Cheslock, Peter S.; Kerry A Garrity; Sloan, Susan E.; Thomas, William; Babcock, Gregory J.; Wang, Yang

    2013-01-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available ...

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

    Directory of Open Access Journals (Sweden)

    Bazargan SA

    1996-07-01

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

  8. [Immunohistochemical study of human breast tumors using monoclonal antibodies to intermediate filament proteins (nonproliferating epithelial structures in breast dysplasia)].

    Science.gov (United States)

    Gel'shteĭn, V I; Chipysheva, T A; Litvinova, L V; Ermilova, V D; Bannikov, G A

    1985-01-01

    An immunohistochemical analysis of nonproliferating epithelial structures was carried out in 10 samples of human breast dysplasia and in 4 samples of tissue surrounding mammary gland carcinoma. Monoclonal mouse antibodies against individual prekeratins of rat monolayer epithelial antibodies of clone C12 against rat prekeratin with the molecular mass 49 kilodalton and antibodies of clone E3 against rat prekeratin with the molecular mass 40 kilodalton-monoclonal antibodies against vimentin (clone 30), as well as polyclonal antibodies against smooth muscle myosin and against the basement membrane glycoprotein laminin were used. The lining epithelium of all glandular structures reacted only with C12 antibodies. Two variants of myoepithelial cells containing myosin were detected. Variant I contains myosin and vimentin and is localized in intralobular ducts. Variant 2 contains myosin and prekeratin, recognized by E3 antibodies and is found in extralobular ducts.

  9. Antibody to collapsin response mediator protein 1 promotes neurite outgrowth from rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Hongsheng Lin; Jing Chen; Wenbin Zhang; Xiaobing Gong; Biao Chen; Guoqing Guo

    2011-01-01

    This study examined the role of collapsin response mediator protein 1 (CRMP-1) on neurite outgrowth from rat hippocampal neurons by blocking its function using an antibody. Hippocampal neurons, cultured in vitro, were treated (blocked) using a polyclonal antibody to CRMP-1, and neurite outgrowth and cytoskeletal changes w ere captured using atomic force microscopy and laser confocal microscopy. Control cells, treated with normal rabbit IgG, established their characteristic morphology and had a large number of processes emerging from the soma, including numerous branches. Microtubules were clearly visible in the soma, formed an elaborate network, and were aligned in parallel arrays to form bundles which projected into neurites. After blocking with CRMP-1 antibody, the number of branches emerging from axons and dendrites significantly increased and were substantially longer, compared with control cells. However, the microtubule network nearly disappeared and only a few remnants were visible. When CRMP-1 antibody-blocked neurons were treated with the Rho inhibitor, Y27632, numerous neurites emerged from the soma, and branches were more abundant than in control neurons. Although the microtubules were not as clearly visible compared with neurons cultured in control medium, the microtubule network recovered in cells treated with Y27632, when compared with cells that were blocked by CRMP-1 antibody (but not treated with Y27632). These results demonstrate that neurite outgrowth from hippocampal neurons can be promoted by blocking CRMP-1 with a polyclonal antibody.

  10. 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.

  11. Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

    Science.gov (United States)

    Cedergren, A M; Miklasz, S D; Voss, E W

    1996-01-01

    Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

  12. Middle-Down 193-nm Ultraviolet Photodissociation for Unambiguous Antibody Identification and its Implications for Immunoproteomic Analysis.

    Science.gov (United States)

    Cotham, Victoria C; Horton, Andrew P; Lee, Jiwon; Georgiou, George; Brodbelt, Jennifer S

    2017-06-20

    Mass spectrometry (MS) has emerged as a powerful tool within the growing field of immunoproteomics, which aims to understand antibody-mediated immunity at the molecular-level based on the direct determination of serological antibody repertoire. To date, these methods have relied on the use of high-resolution bottom-up proteomic strategies that require effective sampling and characterization of low abundance peptides derived from the antigen-binding domains of polyclonal antibody mixtures. Herein, we describe a method that uses restricted Lys-C enzymatic digestion to increase the average mass of proteolytic IgG peptides (≥4.5 kDa) and produce peptides which uniquely derive from single antibody species. This enhances the capacity to discriminate between very similar antibodies present within polyclonal mixtures. Furthermore, our use of 193-nm ultraviolet photodissociation (UVPD) improves spectral coverage of the antibody sequence relative to conventional collision- and electron-based fragmentation methods. We apply these methods to both a monoclonal and an antibody mixture. By identifying from a database search of approximately 15 000 antibody sequences those which compose the mixture, we demonstrate the analytical potential of middle-down UVPD for MS-based serological repertoire analysis.

  13. 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.

  14. 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; ...

  15. 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...

  16. 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.

  17. Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin

    Science.gov (United States)

    Buszko, Maja; Cardini, Benno; Oberhuber, Rupert; Oberhuber, Lukas; Jakic, Bojana; Beierfuss, Anja; Wick, Georg; Cappellano, Giuseppe

    2017-01-01

    Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and (c) demonstrated the immunomodulatory effect of ATG under these experimental circumstances by prolongation of heart allograft survival. PMID:28257450

  18. Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of bio-therapeutic Fab fragments.

    Science.gov (United States)

    Cresswell, Chrissie; Newcombe, Anthony R; Davies, Susannah; Macpherson, Ian; Nelson, Paul; O'Donovan, Kieran; Francis, Richard

    2005-10-01

    In the present paper, we describe a rapid method for the determination of optimum conditions for papain digestion of polyclonal ovine IgG (purified by Na(2)SO(4) precipitation) for the production of bio-therapeutic Fabs (antigen-binding fragments). To determine the optimum conditions for digestion, a factorial approach to the design of experiments was undertaken. The resulting experimental data were used to construct the mathematical models using Design Expert 6.06(R) (Stat-Ease, Minneapolis, MN, U.S.A.) to predict the optimum conditions for a robust IgG digestion step. Optimum conditions were evaluated experimentally, and the applicability of the conditions for large-scale manufacture of bio-therapeutic Fab fragments was assessed. The results and methods described in the present paper suggest that, provided the time and temperature are maintained at the high settings evaluated (24 h, 40 degrees C), the modelled data predict IgG digestion close to 100% for all the papain concentrations used. Provided papain is used at >2.5% (w/w), either time and/or temperature may be reduced. The results and methods described in the present paper may also be applicable to the generation of therapeutic Fab fragments from other immunoglobulins, including monoclonal antibodies purified from mammalian cell culture.

  19. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen

    2002-01-01

    in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection......The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen...

  20. Anti-epitope antibody,a novel site-directed antibody against human acetylcholinesterase

    Institute of Scientific and Technical Information of China (English)

    Xing-mei ZHANG; Gang LIU; Man-ji SUN

    2004-01-01

    AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induction and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody.METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChE was chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE)polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

  1. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

    Science.gov (United States)

    He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E; Lee, Amanda J; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C; Ashkar, Ali A; Palese, Peter; Miller, Matthew S

    2016-10-18

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

  2. The immune response to Trypanoplasma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation

    NARCIS (Netherlands)

    Saeij, J.P.J.; Vries, de B.J.; Wiegertjes, G.F.

    2003-01-01

    Although Trypanoplasma borreli induces the production of non-specific antibodies, survival of infection is associated with the production of T. borreli specific antibodies, able to lyse this parasite in the presence of complement. During the lag phase of this acquired immune response, innate immune

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

    Science.gov (United States)

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

    2016-07-01

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

  4. Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella.

    Directory of Open Access Journals (Sweden)

    Girish Ramachandran

    Full Text Available Non-typhoidal Salmonella (NTS serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.

  5. Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella.

    Science.gov (United States)

    Ramachandran, Girish; Tennant, Sharon M; Boyd, Mary A; Wang, Jin Y; Tulapurkar, Mohan E; Pasetti, Marcela F; Levine, Myron M; Simon, Raphael

    2016-01-01

    Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.

  6. 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.

  7. Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition.

    Science.gov (United States)

    Croasdale, Rebecca; Wartha, Katharina; Schanzer, Juergen M; Kuenkele, Klaus-Peter; Ries, Carola; Mayer, Klaus; Gassner, Christian; Wagner, Martina; Dimoudis, Nikolaos; Herter, Sylvia; Jaeger, Christiane; Ferrara, Claudia; Hoffmann, Eike; Kling, Lothar; Lau, Wilma; Staack, Roland F; Heinrich, Julia; Scheuer, Werner; Stracke, Jan; Gerdes, Christian; Brinkmann, Ulrich; Umana, Pablo; Klein, Christian

    2012-10-15

    In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

  8. Evaluation of rabbit antibody response against 8 and 16 kDa recombinant subunits of antigen B fromEchinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Jahangir Abdi; Bahram Kazemi; Mohammad Hasan Karimfar; Mohammad Bagher Rokni

    2012-01-01

    ABSTRACT Objective:To immunize rabbits with12 and16 kDa recombinant subunits of antigenB from Echinococcus granulosus (E. granulosus) and measuring polyclonal antibody and humoral immune response usingELISA and gel diffusion.Methods:Two mentioned antigens were cloned and expressed in expression vector and purified by affinity chromatography.Four young rabbits were selected and challenged intradermally with yielded recombinant antigens.Rabbits’ sera were collected post infection and were tested usingELISA and gel diffusion for polyclonal antibody detection10 days after last injection.Results:The specific antibody against the recombinant peptides was efficiently produced within4 weeks post infection.Conclusions:Produced recombinants proteins could induce the immune response of the rabbits successfully. This process might improve the clarification of diagnosis and vaccination as regards hydatidosis.

  9. Protein expression and preparation of polydonal antibody of AD-004 and study on its expression in the adrenal and testis

    Institute of Scientific and Technical Information of China (English)

    乔洁

    2006-01-01

    Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni2+ -NTA column. The purified protein was used as an immunogene to prepare polyclonal

  10. In vitro infection with HIV enables human CD4+ T cell clones to induce noncognate contact-dependent polyclonal B cell activation.

    Science.gov (United States)

    Macchia, D; Parronchi, P; Piccinni, M P; Simonelli, C; Mazzetti, M; Ravina, A; Milo, D; Maggi, E; Romagnani, S

    1991-05-15

    Eleven (nine CD4+ and two CD8+) protein purified derivative-specific and eight tetanus toxoid-specific T cell clones (TCC), established from the peripheral blood of healthy persons, were cocultured in vitro with irradiated mononuclear cells from patients infected by HIV in the presence of PHA and polybrene. Two weeks post-HIV exposure, all 17 CD4+, but neither of the two CD8+, TCC exhibited integration of HIV in their genoma, as detected by polymerase chain reaction analysis, and released HIV into their supernatants, as detected by measuring both reverse transcriptase activity and p24 Ag. When co-cultured with either autologous or allogeneic B cells, all CD4+ HIV-infected TCC induced the synthesis of extraordinarily high amounts of IgM, IgG, and IgA. In contrast, their noninfected counterparts could provide helper function for Ig synthesis by autologous B cells only in the presence of the specific Ag (or anti-CD3 antibody), and induced allogeneic B cells to synthesize Ig only upon stimulation with anti-CD3 antibody. The supernatants of HIV-infected TCC failed to stimulate Ig synthesis in B cells. More importantly, when HIV-infected clonal T blasts and B cells were cultured in different chambers separated by a millipore membrane, permeable to molecules but not to cells, Ig synthesis did not occur. The Ig synthesis induced by HIV-infected TCC was also markedly inhibited by the addition in culture of either anti-CD4 or anti-LFA-1 antibody. In contrast, HIV-infected TCC maintained their ability to provide helper function for Ig synthesis in the absence of any stimulus, even after fixation with p-formaldehyde. These data demonstrate that in vitro infection with HIV enables human T cells to stimulate Ig synthesis by B cells by an Ag-nonspecific, MHC-unrestricted, contact-dependent mechanism. This may explain, at least in part, the hypergammaglobulinemia and other phenomena related to polyclonal B cell activation frequently seen in HIV-infected persons.

  11. The use of receptor-specific antibodies to study G-protein-coupled receptors.

    Science.gov (United States)

    Gupta, Achla; Devi, Lakshmi A

    2006-07-01

    The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs.

  12. Synthesis and Characterization of Hapten-Protein Conjugates for Antibody Production against Cyanogenic Glycosides.

    Science.gov (United States)

    Bolarinwa, Islamiyat Folashade

    2015-07-01

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

  13. Persistent Polyclonal B Cell Lymphocytosis B Cells Can Be Activated through CD40-CD154 Interaction

    Directory of Open Access Journals (Sweden)

    Emmanuelle Dugas-Bourdages

    2014-01-01

    Full Text Available Persistent polyclonal B cell lymphocytosis (PPBL is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19+CD27+IgM+ memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.

  14. 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.

  15. 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.

  16. Human secondary lymphoid organs typically contain polyclonally-activated proliferating regulatory T cells.

    Science.gov (United States)

    Peters, Jorieke H; Koenen, Hans J P M; Fasse, Esther; Tijssen, Henk J; Ijzermans, Jan N M; Groenen, Patricia J T A; Schaap, Nicolaas P M; Kwekkeboom, Jaap; Joosten, Irma

    2013-09-26

    Immunomodulating regulatory T-cell (Treg) therapy is a promising strategy in autoimmunity and transplantation. However, to achieve full clinical efficacy, better understanding of in vivo human Treg biology is warranted. Here, we demonstrate that in contrast to blood and bone marrow Tregs, which showed a resting phenotype, the majority of CD4(pos)CD25(pos)CD127(neg)FoxP3(pos) Tregs in secondary lymphoid organs were proliferating activated CD69(pos)CD45RA(neg) cells with a hyperdemethylated FOXP3 gene and a broad T-cell receptor-Vβ repertoire, implying polyclonal activation. Activated CD69(pos) Tregs were distributed over both T-cell and B-cell areas, distant from Aire(pos) and CD11c(pos) cells. In contrast to the anergic peripheral blood Tregs, lymphoid organ Tregs had significant ex vivo proliferative capacity and produced cytokines like interleukin-2, while revealing similar suppressive potential. Also, next to Treg-expressing chemokine receptors important for a prolonged stay in lymphoid organs, a significant part of the cells expressed peripheral tissue-associated, functional homing markers. In conclusion, our data suggest that human secondary lymphoid organs aid in the maintenance and regulation of Treg function and homeostasis. This knowledge may be exploited for further optimization of Treg immunotherapy, for example, by ex vivo selection of Tregs with capacity to migrate to lymphoid organs providing an in vivo platform for further Treg expansion.

  17. Polyclonal Expansion of NKG2C+ NK Cells in TAP-deficient Patients

    Directory of Open Access Journals (Sweden)

    vivien eBeziat

    2015-10-01

    Full Text Available Adaptive natural killer (NK cell responses to human cytomegalovirus (CMV infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs. Here, we set out to study the HLA class I-dependency of such NKG2C+ NK cell expansions. We demonstrate expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP-deficiency, whom express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR-profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR-repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. Thus, the emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to anti-viral immunity and potentially explain these patients’ low incidence of severe viral infections.

  18. Polyclonal Expansion of NKG2C+ NK Cells in TAP-Deficient Patients

    Science.gov (United States)

    Béziat, Vivien; Sleiman, Marwan; Goodridge, Jodie P.; Kaarbø, Mari; Liu, Lisa L.; Rollag, Halvor; Ljunggren, Hans-Gustaf; Zimmer, Jacques; Malmberg, Karl-Johan

    2015-01-01

    Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections. PMID:26500647

  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. Facile Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy.

    Science.gov (United States)

    Geng, Steven B; Wu, Jiemin; Alam, Magfur E; Schultz, Jason S; Dickinson, Craig D; Seminer, Carly R; Tessier, Peter M

    2016-10-19

    Protein-nanoparticle conjugates are widely used for conventional applications such as immunohistochemistry and biomolecular detection as well as emerging applications such as therapeutics and advanced materials. Nevertheless, it remains challenging to reproducibly prepare stable protein-nanoparticle conjugates with highly similar optical properties. Here we report an improved physisorption method for reproducibly preparing stable antibody-gold conjugates at acidic pH using polyclonal antibodies from a wide range of species (human, goat, rabbit, mouse, and rat). We find that gold particles synthesized using citrate alone or in combination with tannic acid are similar in size but display variable colloidal stability when conjugated to polyclonal antibodies. The variability in conjugate stability is due to differences in the pH and composition of the original gold colloid, which prevents reproducible preparation of stable antibody conjugates without additional purification of the particles prior to conjugation. Sedimentation-based purification of gold particles synthesized using different methods enabled reproducible generation of antibody-gold conjugates with high stability and similar plasmon wavelengths. We also find that antibody conjugates prepared using our improved procedure display excellent performance when applied to a high-throughput immunogold assay (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) for identifying monoclonal antibodies with low self-association, high solubility, and low viscosity. The stable antibody conjugates prepared with various types of gold colloid result in robust and reproducible AC-SINS measurements of antibody self-association using extremely dilute (microgram per mL) and unpurified antibody solutions. We expect that this improved methodology will be useful for reproducibly preparing stable antibody-gold conjugates for diverse applications.

  1. High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Koch, Claus; Jensen, Charlotte H

    2004-01-01

    A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies ...... of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.......A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies...... were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging...

  2. GD1b-specific antibodies may bind to complex of GQ1b and GM1, causing ataxia.

    Science.gov (United States)

    Yuki, Nobuhiro; Fukami, Yuki; Yanaka, Chiaki; Koike, Saiko; Hirata, Koichi

    2014-08-01

    Monospecific IgG antibodies to GD1b ganglioside (GD1b-specific antibodies) have been found in patients with acute ataxic neuropathy and Guillain-Barré syndrome, but the association of the GD1b-specific antibodies with specific neurological conditions has yet to be established. We tested sera from more than 10,000 patients with various neurological disorders, and found six sera, which contained IgG antibodies to GD1b, but not to LM1, GM1, GM1b, GD1a, GalNAc-GD1a, GT1a, GT1b and GQ1b. All six patients who carried GD1b-specific antibodies presented with acute onset of ataxia and monophasic course of the illness, of whom five demonstrated cerebellar-like ataxia. Four patients had antecedent symptoms of upper respiratory tract infection. The six patients demonstrated areflexia, and four complained of distal numbness. All the six patients who had the GD1b-specific antibodies carried IgG antibodies to complex of GQ1b/GM1 and GT1a/GM1. GD1b-specific antibodies were significantly absorbed by GQ1b/GM1 and GT1a/GM1 and anti-GQ1b/GM1 and -GT1a/GM1 antibodies were absorbed by GD1b. In conclusion, the GD1b-specific antibodies, which recognizes GQ1b/GM1 or GT1a/GM1 complex, are associated with acute ataxia.

  3. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  4. Molecular epidemiology of HIV-associated tuberculosis in Dar es Salaam, Tanzania: strain predominance, clustering, and polyclonal disease.

    Science.gov (United States)

    Adams, Lisa V; Kreiswirth, Barry N; Arbeit, Robert D; Soini, Hanna; Mtei, Lillian; Matee, Mecky; Bakari, Muhammad; Lahey, Timothy; Wieland-Alter, Wendy; Shashkina, Elena; Kurepina, Natalia; Driscoll, Jeffrey R; Pallangyo, Kisali; Horsburgh, C Robert; von Reyn, C Fordham

    2012-08-01

    Molecular typing of Mycobacterium tuberculosis can be used to elucidate the epidemiology of tuberculosis, including the rates of clustering, the frequency of polyclonal disease, and the distribution of genotypic families. We performed IS6110 typing and spoligotyping on M. tuberculosis strains isolated from HIV-infected subjects at baseline or during follow-up in the DarDar Trial in Tanzania and on selected community isolates. Clustering occurred in 203 (74%) of 275 subjects: 124 (80%) of 155 HIV-infected subjects with baseline isolates, 56 (69%) of 81 HIV-infected subjects with endpoint isolates, and 23 (59%) of 39 community controls. Overall, 113 (41%) subjects had an isolate representing the East Indian "GD" family. The rate of clustering was similar among vaccine and placebo recipients and among subjects with or without cellular immune responses to mycobacterial antigens. Polyclonal disease was detected in 6 (43%) of 14 patients with multiple specimens typed. Most cases of HIV-associated tuberculosis among subjects from this study in Dar es Salaam resulted from recently acquired infection. Polyclonal infection was detected and isolates representing the East Indian GD strain family were the most common.

  5. Characterization of Antibodies for Grain-Specific Gluten Detection.

    Science.gov (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

    2016-03-01

    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

  6. Antibodies Against Glycolipids Enhance Antifungal Activity of Macrophages and Reduce Fungal Burden After Infection with Paracoccidioides brasiliensis.

    Science.gov (United States)

    Bueno, Renata A; Thomaz, Luciana; Muñoz, Julian E; da Silva, Cássia J; Nosanchuk, Joshua D; Pinto, Márcia R; Travassos, Luiz R; Taborda, Carlos P

    2016-01-01

    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.

  7. Antibody-Conjugated Rubpy Dye-Doped Silica Nanoparticles as Signal Amplification for Microscopic Detection of Vibrio cholerae O1

    Directory of Open Access Journals (Sweden)

    Nualrahong Thepwiwatjit

    2013-01-01

    Full Text Available This study demonstrated the potential application of antibody-conjugated Rubpy dye-doped silica nanoparticles for immunofluorescence microscopic detection of Vibrio cholerae O1. The particle synthesis of 20X of the original ratio was accomplished yielding spherical nanoparticles with an average size of 45±3 nm. The nanoparticles were carboxyl functionalized and then conjugated with either monoclonal antibody or polyclonal antibody against V. cholerae O1. The antibody-conjugated nanoparticles were tested with two target bacteria and three challenge strains. The result showed that monoclonal antibody-conjugated Rubpy dye-doped silica nanoparticles could be effectively used as signal amplification to detect V. cholerae O1 under a fluorescence microscope. Their extremely strong fluorescence signal also enables the detection of a single cell bacterium.

  8. 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.

  9. 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

  10. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  11. Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

    Directory of Open Access Journals (Sweden)

    D.M. Grosso

    2008-12-01

    Full Text Available In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(- on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/- mice (6 per group were immunized with the following antibodies (100 µg each: mouse anti-LDL(- monoclonal IgG2b, rabbit anti-LDL(- polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol and then every week for 21 days. The passive immunization with anti-LDL(- monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD, respectively compared to control (124.9 ± 13.2 µm². Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the i