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Sample records for affinity purified polyclonal

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

    PubMed

    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.

  2. Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

    PubMed Central

    Read, A.J.; Gauci, C.G.; Lightowlers, M.W.

    2009-01-01

    The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95. PMID:19349218

  3. A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

    PubMed

    Hermida, L; Rodríguez, R; Lazo, L; López, C; Márquez, G; Páez, R; Suárez, C; Espinosa, R; García, J; Guzmán, G; Guillén, G

    2002-03-28

    Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

  4. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    PubMed

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

  5. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  6. Antibody affinities and relative titers in polyclonal populations: surface plasmon resonance analysis of anti-DNA antibodies.

    PubMed

    Sem, D S; McNeeley, P A; Linnik, M D

    1999-12-01

    This paper presents the equations and methodology for the measurement and interpretation of apparent dissociation constants for polyclonal populations of antibodies, where antigen is kept trace relative to antibody concentration. Surface plasmon resonance is used to determine K(d)s for the binding of anti-DNA antibodies to trace amounts of DNA antigen on a chip. Since the approach taken relies on equilibrium measurements, kinetic mass transport artifacts are avoided. The apparent K(d) is a weighted average of all the K(d)s for the clonally related subpopulations within the polyclonal pool, where each weighting factor is the relative titer (fractional presence) of the subpopulation. Titration curves appear as if there is one monoclonal population with that titer-weighted-average K(d). Implications of changes in the antibody affinity distribution within the population are discussed. The equations described herein provide a better physical understanding of the apparent K(d) that is obtained when a heterogeneous population of receptors is titrated against a trace ligand.

  7. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA.

    PubMed

    Sousa, Fani; Prazeres, Duarte M F; Queiroz, João A

    2008-09-01

    The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.

  8. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  9. Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production.

    PubMed

    Nakkeeran, Ekambaram; Umesh-Kumar, Sukumaran; Subramanian, Rangaswamy

    2011-02-01

    Aspergillus carbonarius, when grown by submerged and solid-state fermentation, produces different molecular forms of polygalacturonase (PG; EC 3.2.1.15), among them a 42 kDa PG with a high specific activity of 7000 U/mg protein. When the enzymes were purified by integrated membrane process (IMP) and alginate affinity precipitation (AAP), the two processes concentrated different forms of the enzyme. The AAP process selectively purified and concentrated the high active PG whereas the IMP yielded different PGs and also amylase and protease. Evaluation of the AAP enzyme preparations for apple juice preparation under conditions usually employed commercially demonstrated that the high activity PG did not result in good juice clarity. With IMP processed enzymes, juice yields and clarity were similar to that obtained with commercial PG from A. niger.

  10. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  11. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

  12. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  13. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    PubMed

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.

  14. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  15. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  16. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  17. A novel affinity-based method for the isolation of highly purified extracellular vesicles

    PubMed Central

    Nakai, Wataru; Yoshida, Takeshi; Diez, Diego; Miyatake, Yuji; Nishibu, Takahiro; Imawaka, Naoko; Naruse, Ken; Sadamura, Yoshifusa; Hanayama, Rikinari

    2016-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies. PMID:27659060

  18. [Obtaining of the affinity purified antibodies against survivin for the structure functional study of the protein].

    PubMed

    Akhidova, E V; Volkova, T D; Koroev, D O; Yakupov, I Iu; Kalintseva, M V; Zavalishina, L E; Kaplun, A P; Zharskaia, O O; Zatsepina, O V; Vol'pina, O M

    2013-01-01

    Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.

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

    PubMed Central

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

    2015-01-01

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

  20. The antigenicity in guinea pigs and monkeys of three mycobacterial polysaccharides purified by affinity chromatography with concanavalin A.

    PubMed

    Daniel, T M

    1975-06-01

    The antigenicity of 3 polysaccharides purified from culture filtrates of Mycobacterim tuberculosis by affinity chromatography using a concanavalin A-agarose absorbent was studied. All 3 purified polysaccharides were found to be potent elicitors of delayed skin test reactions in sensitized guinea pigs and in a tuberculos monkey. This antigenicity could not be attributed to contaminating protein. Small dermal reactions were also observed in control guinea pigs. All 3 polysaccharides reacted with precipitating antibody in guinea pig sera, the antigenic specificity observed with the guinea pig sera differing from that demonstrated with reference goat antiserum. The 3 polysaccharides were also demonstrated to contain hemagglutination antigenic sites.

  1. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form.

  2. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  3. Rapid screening of textile dyes employed as affinity ligands to purify enzymes from yeast.

    PubMed

    Raya-Tonetti, G; Perotti, N

    1999-04-01

    A rapid method for screening potential dye ligands for use in affinity chromatography is described. Textile dyes were non-covalently coupled to a cross-linked polysaccharide Sepharose(R) matrix. Yeast alcohol dehydrogenase (ADH) was used as the model protein for evaluating the screening system. A homogenate from baker's yeast was used as the crude source of enzyme. Batchwise adsorption and elution were used to evaluate the individual dyes. The influence of pH and ionic strength in the binding and elution steps was evaluated. Batch isotherms were used to evaluate parameter characteristics. Experimental data obtained were fitted to Langmuir isotherms to determine the maximum binding capacity and the dissociation constant for each dye evaluated in this study. A dynamic binding capacity of 107.6 units of ADH/g of resin was determined for Procion Turquoise MXG dye by frontal analysis. Specific elution with NAD+ and non-specific elution with 50 mM Tris/HCl buffer, pH 8.5, were tested when Procion Turquoise MXG was used, giving purification factors of 53.5 and 4.4 respectively. This screening technique is inexpensive and can be performed in a few hours. It was possible to predict the performance of different reactive dyes in this way, and the influence of pH and salt on the binding behaviour was demonstrated.

  4. Affinity-purified respiratory syncytial virus antibodies from intravenous immunoglobulin exert potent antibody-dependent cellular cytotoxicity.

    PubMed

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F; Kazatchkine, Michel D; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

  5. Affinity-Purified Respiratory Syncytial Virus Antibodies from Intravenous Immunoglobulin Exert Potent Antibody-Dependent Cellular Cytotoxicity

    PubMed Central

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F.; Kazatchkine, Michel D.; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V.

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections. PMID:23894466

  6. Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

    PubMed Central

    Léguillette, Renaud; Zitouni, Nedjma B.; Govindaraju, Karuthapillai; Fong, Laura M.; Lauzon, Anne-Marie

    2008-01-01

    Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state. PMID:18614813

  7. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    PubMed

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  8. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    SciTech Connect

    Lazarovici, P.; Yavin, E.

    1986-11-04

    The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.

  9. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    PubMed

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Induction of in vitro heart block is not restricted to affinity purified anti-52 kDa Ro/SSA antibody from mothers of children with neonatal lupus.

    PubMed

    Viana, V S; Garcia, S; Nascimento, J H; Elkon, K B; Brot, N; Campos de Carvalho, A C; Bonfá, E

    1998-01-01

    The ability of affinity purified anti-52 kDa Ro/SSA antibody from patients without obstetric history of neonatal lupus to cause heart block using an experimental model was investigated. IgG-enriched fractions from sera of 20 systemic lupus erythematosus (SLE) and one Sjögren's syndrome (SS) all positives for anti-Ro/SSA antibodies as detected by CIE, were perfused on isolated whole rabbit hearts. Only six (29%) samples induced A-V block, five of them presenting low anti-Ro/SSA titre. All of them recognized the 52 kDa isoform on ELISA whereas only one had a concomitant binding to the 60 kDa protein. Moreover, affinity purified antibodies from two sera previously known to induce A-V block were obtained by affinity chromatography using a column containing the full-length 52 kDa Ro/SSA fusion protein. Paired eluate and effluent devoid of anti-52 kDa activity from the same patient were individually perfused in whole hearts. The ability to cause cardiac blockade was restricted to the affinity anti-52 kDa eluates. In addition, anti-52 kDa eluates from three IgG fractions that primarily failed to induce blockade remained ineffective. The present study has added to our knowledge that affinity anti-52 kDa Ro/SSA antibodies from mothers with healthy infants are capable of causing in vitro cardiac conduction disturbances. A prospective follow up of these patients will better delineate the clinical usefulness of this experimental model.

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

    PubMed

    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.

  12. Purification of polyclonal antibodies from Cohn fraction II + III, skim milk, and whey by affinity chromatography using a hexamer peptide ligand.

    PubMed

    Menegatti, Stefano; Naik, Amith D; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    HWRGWV, a peptide that binds specifically to the Fc fragment of human immunoglobulin G (IgG), was used for the purification of IgG from Cohn fraction II + III of human plasma and from bovine skim milk and whey. The concentration of sodium chloride and sodium caprylate in the binding buffer as well as the pH of the elution buffer were optimized to achieve high IgG yield and purity. Under optimized conditions, IgG was recovered from plasma fractions with yield and purity up to 84% and 95%, respectively. IgG was also purified from skim milk with 74% yield and 92% purity and from whey with 85% yield and 93% purity. Purification experiments were also performed with Protein A resin and the results were found to be similar to those obtained with the peptide adsorbent.

  13. Amino acid sequence and disulfide bridges of affinity purified Kunitz-type chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC).

    PubMed

    Kortt, A A; Burns, J E; Strike, P M

    1990-11-01

    The primary sequence of the affinity purified chymotrypsin inhibitor, WBCI, isolated from the albumin fraction of Psophocarpus tetragonolobus (L.) DC cv. UPS-122 seed was determined. The inhibitor consisted of a single polypeptide chain of 183 amino acids (Mr 20285) and the four half-cystine residues in the molecule formed two intramolecular disulfide bridges equivalent to those in other Kunitz-type seed inhibitors. The sequence of this chymotrypsin inhibitor was identical to that of chymotrypsin inhibitor-3 from cultivar UPS-31 and it showed about 50% sequence similarity to the winged bean acidic (WBTI-2, pI 5.1) and basic (WBTI-1, pI 8.9) trypsin inhibitors. Sequence similarities to other Kunitz-type seed inhibitors are discussed.

  14. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    PubMed

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

  15. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  16. LC-MS analysis of polyclonal human anti-Neu5Gc Xeno-autoantibodies IgG subclass and partial sequence using multi-step IVIG affinity purification and multi-enzymatic digestion

    PubMed Central

    Lu, Qiaozhen; Padler-Karavani, Vered; Yu, Hai; Chen, Xi; Wu, Shiaw-Lin; Varki, Ajit; Hancock, William S.

    2014-01-01

    Human polyclonal IgG antibodies directly against the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically-approved human IVIG (IgG). These results were used to select an appropriate sample for a multi-step affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multi-enzyme digestion procedure followed by nanoLC coupled to LTQ-FTMS. We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance. PMID:22390546

  17. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.

    PubMed

    Rudashevskaya, Elena L; Sacco, Roberto; Kratochwill, Klaus; Huber, Marie L; Gstaiger, Matthias; Superti-Furga, Giulio; Bennett, Keiryn L

    2013-01-01

    Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

  18. New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

    PubMed

    Lauer, I; Bonnewitz, B; Meunier, A; Beverini, M

    2000-01-14

    Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also

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

    PubMed

    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.

  20. Efficacy of intravenous immunoglobulin (IVIG) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris.

    PubMed

    Mimouni, D; Blank, M; Payne, A S; Anhalt, G J; Avivi, C; Barshack, I; David, M; Shoenfeld, Y

    2010-12-01

    Pemphigus vulgaris is a rare life-threatening autoimmune bullous disease caused by immunoglobulin G (IgG) autoantibodies directed against desmogleins 1 and 3. Previously, we showed that intravenous immunoglobulin (IVIG) ameliorates anti-desmoglein-induced experimental pemphigus vulgaris in newborn naive mice. The aim of this study was to examine the efficacy of anti-anti-desmoglein-specific IVIG in a similar model. Pemphigus-vulgaris-specific IVIG (PV-sIVIG) was affinity-purified from IVIG on a column of single-chain variable fragment (scFv) anti-desmogleins 1 and 3. The anti-idiotypic activity of PV-sIVIG was confirmed by enzyme-linked immunosorbent assay, inhibition assay. After induction of pemphigus by injection of anti-desmogleins 1 and 3 scFv to newborn mice, the animals were treated with PV-sIVIG, IVIG (low or high dose) or IgG from a healthy donor (n = 10 each). The skin was examined 24-48 h later, and samples of affected areas were analysed by histology and immunofluorescence. In vitro study showed that PV-sIVIG significantly inhibited anti-desmogleins 1 and 3 scFv binding to recombinant desmoglein-3 in a dose-dependent manner. Specificity was confirmed by inhibition assay. In vivo analysis revealed cutaneous lesions of pemphigus vulgaris in mice injected with normal IgG (nine of 10 mice) or low-dose IVIG (nine of 10 mice), but not in mice treated with PV-sIVIG (none of 10) or high-dose IVIG (none of 10). On immunopathological study, PV-sIVIG and regular IVIG prevented the formation of acantholysis and deposition of IgG in intercellular spaces. In conclusion, the PV-sIVIG preparation is more effective than native IVIG in inhibiting anti-desmoglein-induced pemphigus vulgaris in mice and might serve as a future therapy in patients with the clinical disease.

  1. Performance of two ELISAs for antifilaggrin autoantibodies, using either affinity purified or deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis

    PubMed Central

    Nogueira, L; Sebbag, M; Vincent, C; Arnaud, M; Fournie, B; Cantagrel, A; Jolivet, M; Serre, G

    2001-01-01

    OBJECTIVE—To develop a standardisable enzyme linked immunosorbent assay (ELISA), using human filaggrin, for detection of antifilaggrin autoantibodies in rheumatoid arthritis (RA). To compare the diagnostic performance of the ELISA with those of reference tests: "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin detected by immunoblotting (AhFA-IB).
METHODS—Two ELISAs were developed using either affinity purified neutral-acidic human epidermis filaggrin (AhFA-ELISA-pur) or a recombinant human filaggrin deiminated in vitro (AhFA-ELISA-rec) as immunosorbent. Antifilaggrin autoantibodies were assayed in 714 serum samples from patients with well characterised rheumatic diseases, including 241 RA and 473 other rheumatic diseases, using the two ELISAs. "AKA" and AhFA-IB tests were carried out in the same series of patients. The diagnostic performance of the four tests was compared and their relationships analysed.
RESULTS—The titres of "AKA", AhFA-IB, and the AhFA-ELISAs correlated strongly with each other. The diagnostic sensitivity of the AhFA-ELISA-rec, which was better than that of AhFA-ELISA-pur, was 0.52 for a specificity of 0.95. This performance was similar to those of "AKA" or AhFA-IB. However, combining AhFA-ELISA-rec with AhFA-IB led to a diagnostic sensitivity of 0.55 for a specificity of 0.99.
CONCLUSION—A simple and easily standardisable ELISA for detection of antifilaggrin autoantibodies was developed and validated on a large series of patients using a citrullinated recombinant human filaggrin. The diagnostic performance of the test was similar to that of the "AKA" and AhFA-IB. Nevertheless, combining the AhFA-ELISA-rec with one of the other tests clearly enhanced the performance.

 PMID:11502616

  2. Recombinant renewable polyclonal antibodies.

    PubMed

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

    PubMed

    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.

  4. Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals.

    PubMed

    Morenkov, O S; Fodor, N; Fodor, I

    1999-01-01

    Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.

  5. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

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

    PubMed Central

    Koolivand, Davoud; Bashir, Nemat Sokhandan; Behjatnia, Seyed Aliakbar; Joozani, Raziallah Jafari

    2016-01-01

    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. PMID:27721695

  7. Affinity-purified CCAAT-box-binding protein (YEBP) functionally regulates expression of a human class II major histocompatibility complex gene and the herpes simplex virus thymidine kinase gene

    SciTech Connect

    Zeleznik-Le, N.J.; Azizkhan, J.C.; Ting, J.P.Y. )

    1991-03-01

    Efficient major histocompatibility complex class II gene expression requires conseved protein-binding promoter elements, including X and Y elements. The authors affinity purified an HLA-DRA Y-element (CCAAT)-binding protein (YEBP) and used it to reconstitute Y-depleted HLA-DRA in vitro transcription. This directly demonstrates a positive functional role for YEBP in HLA-DRA transcription. The ability of YEBP to regulate divergent CCAAT elements was also assessed; YEBP was found to partially activate the thymidine kinase promoter. This functional analysis of YEBP shows that this protein plays an important role in the regulation of multiple genes.

  8. Polyclonal antibody to soman-tyrosine

    PubMed Central

    Li, Bin; Duysen, Ellen G.; Froment, Marie-Thérèse; Masson, Patrick; Nachon, Florian; Jiang, Wei; Schopfer, Lawrence M.; Thiele, Geoffrey M.; Klassen, Lynell W.; Cashman, John; Williams, Gareth R.; Lockridge, Oksana

    2013-01-01

    Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin, but not non-phosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10−11 M. The limit of detection on Western blots was 0.01 μg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity. PMID:23469927

  9. RPAP1, a Novel Human RNA Polymerase II-Associated Protein Affinity Purified with Recombinant Wild-Type and Mutated Polymerase Subunits

    PubMed Central

    Jeronimo, Célia; Langelier, Marie-France; Zeghouf, Mahel; Cojocaru, Marilena; Bergeron, Dominique; Baali, Dania; Forget, Diane; Mnaimneh, Sanie; Davierwala, Armaity P.; Pootoolal, Jeff; Chandy, Mark; Canadien, Veronica; Beattie, Bryan K.; Richards, Dawn P.; Workman, Jerry L.; Hughes, Timothy R.; Greenblatt, Jack; Coulombe, Benoit

    2004-01-01

    We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction. PMID:15282305

  10. Glycosylation is not necessary for recognition of the fusion glycoprotein domain of the human respiratory syncytial virus by a polyclonal antibody.

    PubMed

    Lim, S H E; Jahanshiri, F; Jalilian, F A; Rahim, R Abdul; Sekawi, Z; Yusoff, K

    2010-01-01

    Human respiratory syncytial virus (HRSV) is a leading pathogen causing lower respiratory tract infections in infants and young children worldwide. In line with the development of an effective vaccine against HRSV, a domain of the fusion (F) glycoprotein of HRSV was produced and its immunogenicity and antigenic properties, namely the effect of deficient glycosylation was examined. A His-tagged recombinant F (rF) protein was expressed in Escherichia coli, solubilized with 8 mol/l urea, purified by the Ni-NTA affinity chromatography and used for the raising of a polyclonal antibody in rabbits. The non-glycosylated rF protein proved to be a strong immunogen that induced a polyclonal antibody that was able to recognize also the glycosylated F1 subunit of native HRSV. The other way around, a polyclonal antibody prepared against the native HRSV was able to react with the rF protein. These results indicated that glycosylation was not necessary for the F domain aa 212-574 in order to be recognized by the specific polyclonal antibody.

  11. Purifying Nanomaterials

    NASA Technical Reports Server (NTRS)

    Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

    2014-01-01

    A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

  12. Methods for Purifying Enzymes for Mycoremediation

    NASA Technical Reports Server (NTRS)

    Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

    2014-01-01

    A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

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

    PubMed

    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.

  14. Validation of affinity reagents using antigen microarrays.

    PubMed

    Sjöberg, Ronald; Sundberg, Mårten; Gundberg, Anna; Sivertsson, Asa; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter

    2012-06-15

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  15. Water Purifier

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Floatron water purifier combines two space technologies - ionization for water purification and solar electric power generation. The water purification process involves introducing ionized minerals that kill microorganisms like algae and bacteria. The 12 inch unit floats in a pool while its solar panel collects sunlight that is converted to electricity. The resulting current energizes a specially alloyed mineral electrode below the waterline, causing release of metallic ions into the water. The electrode is the only part that needs replacing, and water purified by the system falls within EPA drinking water standards.

  16. [Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody].

    PubMed

    Qin, Yujie; Zhang, Tinghong; Ye, Xin

    2016-01-01

    Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.

  17. Water Purifiers

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Technology developed to purify the water aboard manned spacecraft has led to a number of spinoff applications. One of them is the Ambassador line of bacteriostatic water treatment systems, which employ high grade, high absorption media to inhibit bacteria growth and remove the medicinal taste and odor of chlorine. Company President, Ray Ward, originally became interested in the technology because of the "rusty" taste of his water supply.

  18. Expression profile of human immune-responsive gene 1 and generation and characterization of polyclonal antiserum.

    PubMed

    Xiao, Wei; Wang, Lan; Xiao, Ruijing; Wu, Mengjun; Tan, Jinquan; He, Yuling

    2011-07-01

    Murine immune-responsive gene 1 (IRG1) plays significant roles in embryonic implantation and neurodegeneration. The expression pattern of the human IRG1 gene, however, has not yet been established, and the predicted gene sequence has been revised several times according to computed expressed sequence tags (ESTs). To determine the human IRG1 gene expression profile, human fetal tissue samples, peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the human leukemia cell lines THP-1 and K-562 challenged with lipopolysaccharide (LPS) were subjected to RT-PCR using degenerate primers. The results indicated that the IRG1 gene is differentially expressed in human fetal PBMCs and LPS-stimulated adult PBMCs. The amplified gene fragment was cloned into the pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system. After affinity chromatography, human IRG1h fusion proteins were isolated by SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen to immunize rabbits. The titer and specificity of the purified rabbit antiserum were sufficient to measure human IRG1 gene expression in various tissues and cultures. This purified polyclonal antiserum will allow us to initiate studies to elucidate the biological roles of the human IRG1 gene.

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

    SciTech Connect

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

    1990-09-25

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

  20. Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins

    PubMed Central

    Ferreira Júnior, Álvaro; Santiago, Fernanda M.; Silva, Murilo V.; Ferreira, Flávia B.; Macêdo Júnior, Arlindo G.; Mota, Caroline M.; Faria, Matheus S.; Filho, Hercílio H. Silva; Silva, Deise A. O.; Cunha-Júnior, Jair P.; Mineo, José R.; Mineo, Tiago W. P.

    2012-01-01

    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. PMID:22808150

  1. Immunoreactivity of polyclonal antibodies generated against the carboxy terminus of the predicted amino acid sequence of the Huntington disease gene

    SciTech Connect

    Alkatib, G.; Graham, R.; Pelmear-Telenius, A.

    1994-09-01

    A cDNA fragment spanning the 3{prime}-end of the Huntington disease gene (from 8052 to 9252) was cloned into a prokaryotic expression vector containing the E. Coli lac promoter and a portion of the coding sequence for {beta}-galactosidase. The truncated {beta}-galactosidase gene was cleaved with BamHl and fused in frame to the BamHl fragment of the Huntington disease gene 3{prime}-end. Expression analysis of proteins made in E. Coli revealed that 20-30% of the total cellular proteins was represented by the {beta}-galactosidase-huntingtin fusion protein. The identity of the Huntington disease protein amino acid sequences was confirmed by protein sequence analysis. Affinity chromatography was used to purify large quantities of the fusion protein from bacterial cell lysates. Affinity-purified proteins were used to immunize New Zealand white rabbits for antibody production. The generated polyclonal antibodies were used to immunoprecipitate the Huntington disease gene product expressed in a neuroblastoma cell line. In this cell line the antibodies precipitated two protein bands of apparent gel migrations of 200 and 150 kd which together, correspond to the calculated molecular weight of the Huntington disease gene product (350 kd). Immunoblotting experiments revealed the presence of a large precursor protein in the range of 350-750 kd which is in agreement with the predicted molecular weight of the protein without post-translational modifications. These results indicate that the huntingtin protein is cleaved into two subunits in this neuroblastoma cell line and implicate that cleavage of a large precursor protein may contribute to its biological activity. Experiments are ongoing to determine the precursor-product relationship and to examine the synthesis of the huntingtin protein in freshly isolated rat brains, and to determine cellular and subcellular distribution of the gene product.

  2. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  3. Production of Polyclonal Antibody to the HPV58 E7 Protein and Its Detection in Cervical Cancer

    PubMed Central

    Zheng, Qiaoli; Wang, Tuan; Jiang, Shaojie; Han, Rui; Jin, Na; Zhu, Jiang; Zhou, Qiang; Wang, Hui; Chen, Xianzhen; Cheng, Hao

    2016-01-01

    The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer worldwide, and HPV type 58 is the third most common HPV type in eastern Asia. The E7 oncoprotein is constitutively expressed in HPV58-associated cervical cancer cells and plays a key role during tumorigenesis. To study the biological function of HPV58 E7 and to characterize E7 protein-host cell interactions, we cloned the human HPV58 E7 gene and produced specific E7 antibodies. The HPV58 E7 gene was cloned into a prokaryotic expression vector, pGEX-4T2. The recombinant plasmid pGEX-4T2-(HPV58-E7) was transformed into Escherichia coli DH5α and expressed as a fusion protein containing a GST tag. After purification and removal of the GST affinity tag, the E7 protein was used as an antigen for the production of antiserum in rabbits. The specificity of the purified HPV58 E7 antibody was detected by western blotting, immunofluorescence and immunohistochemistry analysis. These methods demonstrated that the polyclonal antibody could specifically recognize the endogenous and the recombinant HPV58 E7 proteins. Immunohistochemistry analysis indicated that the E7 protein was localized in the nucleus of cervical cancer cells. PMID:28033368

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

    PubMed

    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.

  5. Production of metallothionein polyclonal antibodies using chickens as model.

    PubMed

    Ortiz-Bueno, Angélica María; León-Chávez, Bertha Alicia; Ruiz-Tagle, Alejandro; Lozano-Zarain, Patricia; Castro-Caballero, Leopoldo; Achanzar, William E; Brambila, Eduardo

    2009-09-01

    The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

  6. Gas stream purifier

    NASA Technical Reports Server (NTRS)

    Adam, Steven J.

    1994-01-01

    A gas stream purifier has been developed that is capable of removing corrosive acid, base, solvent, organic, inorganic, and water vapors as well as particulates from an inert mixed gas stream using only solid scrubbing agents. This small, lightweight purifier has demonstrated the ability to remove contaminants from an inert gas stream with a greater than 99 percent removal efficiency. The Gas Stream Purifier has outstanding market and sales potential in manufacturing, laboratory and science industries, medical, automotive, or any commercial industry where pollution, contamination, or gas stream purification is a concern. The purifier was developed under NASA contract NAS9-18200 Schedule A for use in the international Space Station. A patent application for the Gas Stream Purifier is currently on file with the United States Patent and Trademark Office.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota himalayan.

    PubMed

    Yang, Yan; Huang, Huang; Zhang, Zhenghua; Wang, Baoju; Tian, Yongjun; Lu, Mengji; Yang, Dongliang

    2007-08-01

    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.

  9. METHOD FOR PURIFYING URANIUM

    DOEpatents

    Knighton, J.B.; Feder, H.M.

    1960-04-26

    A process is given for purifying a uranium-base nuclear material. The nuclear material is dissolved in zinc or a zinc-magnesium alloy and the concentration of magnesium is increased until uranium precipitates.

  10. Pancreatic Cancer Metastases Harbor Evidence of Polyclonality

    PubMed Central

    Maddipati, Ravikanth; Stanger, Ben Z.

    2015-01-01

    Studies of the cancer genome have demonstrated that tumors are comprised of multiple sub-clones with varied genetic and phenotypic properties. However, little is known about how metastases arise and evolve from these sub-clones. To understand the cellular dynamics that drive metastasis, we used multi-color lineage tracing technology in an autochthonous mouse model of pancreatic cancer. Here, we report that precursor lesions exhibit significant clonal heterogeneity but that this diversity decreases during pre-malignant progression. Furthermore, we present evidence that a significant fraction of metastases are polyclonally seeded by distinct tumor sub-clones. Finally, we show that clonality during metastatic growth – leading to either monoclonal or polyclonal expansion – differs based on the site of metastatic invasion. These results provide an unprecedented window into the cellular dynamics of tumor evolution and suggest that heterotypic interactions between tumor subpopulations contribute to metastatic progression in native tumors. PMID:26209539

  11. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  12. Purified silicon production system

    DOEpatents

    Wang, Tihu; Ciszek, Theodore F.

    2004-03-30

    Method and apparatus for producing purified bulk silicon from highly impure metallurgical-grade silicon source material at atmospheric pressure. Method involves: (1) initially reacting iodine and metallurgical-grade silicon to create silicon tetraiodide and impurity iodide byproducts in a cold-wall reactor chamber; (2) isolating silicon tetraiodide from the impurity iodide byproducts and purifying it by distillation in a distillation chamber; and (3) transferring the purified silicon tetraiodide back to the cold-wall reactor chamber, reacting it with additional iodine and metallurgical-grade silicon to produce silicon diiodide and depositing the silicon diiodide onto a substrate within the cold-wall reactor chamber. The two chambers are at atmospheric pressure and the system is open to allow the introduction of additional source material and to remove and replace finished substrates.

  13. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L

    2011-03-15

    Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace

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

    PubMed

    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.

  15. Generation of metastatic melanoma specific antibodies by affinity purification

    PubMed Central

    Schütz, Birgit; Koppensteiner, Anita; Schörghofer, David; Kinslechner, Katharina; Timelthaler, Gerald; Eferl, Robert; Hengstschläger, Markus; Missbichler, Albert; Hundsberger, Harald; Mikula, Mario

    2016-01-01

    Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis. PMID:27853253

  16. Method of purifying isosaccharinate

    DOEpatents

    Rai, Dhanpat; Moore, Robert C.; Tucker, Mark D.

    2010-09-07

    A method of purifying isosaccharinate by mixing sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide with calcium isosaccharinate, removing the precipitated calcium carbonate and adjusting the pH to between approximately 4.5 to 5.0 thereby removing excess carbonate and hydroxide to provide an acidic solution containing isosaccharinate.

  17. Purifying Water by Imbibition

    NASA Technical Reports Server (NTRS)

    Lawton, E. A.

    1986-01-01

    Concept for purifying water uses absorbent material to remove organic substances. Entire bulk of material employed, not just surface. Proposed purification process uses inexpensive equipment and low energy. Material is methyl acrylate polymer. Material cheap and regenerated by rinsing with methanol or by allowing absorbed compounds to evaporate from it.

  18. Production of polyclonal antibodies to feline tumor necrosis factor.

    PubMed Central

    Otto, C M; Niagro, F; McGraw, R A; Rawlings, C A

    1997-01-01

    Two 13-amino-acid peptides were synthesized based on the putative feline tumor necrosis factor (FeTNF) sequence. The synthesized peptides were conjugated to keyhole limpet hemocyanin, emulsified in complete Freund's adjuvant, and injected into rabbits. The gene for FeTNF was cloned into the FLAG (International Biotechnologies Inc. [IBI], Kodak, New Haven, Conn.) fusion protein expression vector. The expressed fusion protein was purified by using the M-1 anti-FLAG octapeptide monoclonal antibody (IBI, Kodak). The fusion protein was emulsified in complete Freund's adjuvant and injected into chickens. The immune sera generated to the synthetic peptides and the fusion protein recognized the recombinant FeTNF fusion protein on Western or dot blot assay. The preimmune and immune sera were incubated with naturally occurring FeTNF (supernatants from lipopolysaccharide-stimulated cultured feline peritoneal exudate or peripheral mononuclear cells). The antibody raised to the recombinant FeTNF fusion protein and N-terminal synthetic peptide neutralized bioactivity of native FeTNF and recombinant human TNF. Preimmune sera did not have any neutralizing activity. The polyclonal antibodies were not specific for FeTNF, since both porcine and human recombinant TNF were neutralized by the fusion protein antibodies. The synthetic peptide antibodies recognized recombinant feline and equine TNF on a Western blot. PMID:9220170

  19. Generation and characterization of polyclonal antibodies against mouse T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain by DNA-based immunization.

    PubMed

    Gao, Y; Cui, J; He, W; Yue, J; Yu, D; Cai, L; Xu, H; Yang, C; Chen, Z K; Zhou, H

    2014-01-01

    Mouse T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) is a newly identified surface protein expressed in regulatory, memory, natural killer (NK), and activated T cells. Several studies indicate that mouse TIGIT is a vital immunomodulator that can control the activities of both NK and T cells and plays an important role in transplantation tolerance. In this study, we designed a vector, TIGIT-pcDNA3.1 (+), that encodes the complete coding sequence of mouse TIGIT. The vector was intramuscularly injected into rats, and then the specific antisera were harvested and purified using a protein A/G PLUS-agarose affinity column. Western blot and immunohistochemistry analyses revealed that the antibodies generated by DNA immunization can bind with the mouse TIGIT. Using these antibodies in immunoblots, TIGIT was detected in lysates of mouse organs, T cells from mouse lymph nodes, and recombinant mouse fusion protein of TIGIT and Fc fragment. Immunohistochemistry analysis of normal mouse kidney showed that immunoreactivity was located on endothelial cells of glomerular capillary loops and peritubular capillaries. Our results demonstrated that the DNA immunization of rats through intramuscular injection was a simple and easily available method of producing polyclonal antibodies that can be used to detect and analyze mouse TIGIT expression in mouse systems.

  20. Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

    PubMed Central

    Wine, Yariv; Boutz, Daniel R.; Lavinder, Jason J.; Miklos, Aleksandr E.; Hughes, Randall A.; Hoi, Kam Hon; Jung, Sang Taek; Horton, Andrew P.; Murrin, Ellen M.; Ellington, Andrew D.; Marcotte, Edward M.; Georgiou, George

    2013-01-01

    We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography–high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique VH CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody VH clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease. PMID:23382245

  1. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    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.

  2. Clostridium difficile infection: monoclonal or polyclonal genesis?

    PubMed

    Hell, M; Permoser, M; Chmelizek, G; Kern, J M; Maass, M; Huhulescu, S; Indra, A; Allerberger, F

    2011-10-01

    Clostridium difficile is considered to be a leading cause of hospital-acquired diarrhea. C. difficile (CDI) infection shows a high rate of recurrence. There would have to be a predominantly monoclonal mechanism of CDI within individual patients in order for molecular epidemiologic tools such as polymerase chain reaction (PCR) ribotyping to be useful in outbreak investigation or differentiation between infection relapse versus re-infection. It was the aim of our study to determine whether CDI is of monoclonal or of polyclonal genesis. Between December 2009 and June 2010, 11 patients with nosocomial CDI were chosen arbitrarily. Five individual colonies of C. difficile were picked from each of the primary culture plates. Of 55 isolates gained, 47 were available for PCR ribotyping (eight isolates failed attempts to re-culture). Among these 47 isolates, eight different PCR ribotypes were identified. Only one of the 11 patients had a stool sample that yielded more than one ribotype (PCR ribotypes 438 and 232); this 67-year-old female cancer patient was already suffering from recurring diarrhea prior to the fatal episode of colitis which was subsequently investigated. We conclude that polyclonal infections may occasionally occur in patients with CDI. Our findings of predominantly monoclonal origin of CDI within patients suggest that molecular epidemiologic investigations can be used reliably for outbreak investigations or discrimination between relapse and re-infection.

  3. PROCESS OF PURIFYING URANIUM

    DOEpatents

    Seaborg, G.T.; Orlemann, E.F.; Jensen, L.H.

    1958-12-23

    A method of obtaining substantially pure uranium from a uranium composition contaminated with light element impurities such as sodium, magnesium, beryllium, and the like is described. An acidic aqueous solution containing tetravalent uranium is treated with a soluble molybdate to form insoluble uranous molybdate which is removed. This material after washing is dissolved in concentrated nitric acid to obtaln a uranyl nitrate solution from which highly purified uranium is obtained by extraction with ether.

  4. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle

    PubMed Central

    Dye, John M.; Wu, Hua; Hooper, Jay W.; Khurana, Surender; Kuehne, Ana I.; Coyle, Elizabeth M.; Ortiz, Ramon A.; Fuentes, Sandra; Herbert, Andrew S.; Golding, Hana; Bakken, Russell A.; Brannan, Jennifer M.; Kwilas, Steve A.; Sullivan, Eddie J.; Luke, Thomas C.; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W.; Tripp, Ralph A.; Jiao, Jin-an

    2016-01-01

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities. PMID:27109916

  5. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle.

    PubMed

    Dye, John M; Wu, Hua; Hooper, Jay W; Khurana, Surender; Kuehne, Ana I; Coyle, Elizabeth M; Ortiz, Ramon A; Fuentes, Sandra; Herbert, Andrew S; Golding, Hana; Bakken, Russell A; Brannan, Jennifer M; Kwilas, Steve A; Sullivan, Eddie J; Luke, Thomas C; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W; Tripp, Ralph A; Jiao, Jin-An

    2016-04-25

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.

  6. T-cell regulation of polyclonal B-cell activation induced by extracts of oral bacteria associated with periodontal diseases.

    PubMed Central

    Carpenter, A B; Sully, E C; Ranney, R R; Bick, P H

    1984-01-01

    These studies were designed to examine the role of regulatory T cells in the polyclonal antibody response of human peripheral blood lymphocytes to extracts of bacterial isolates commonly associated with periodontal disease. Polyclonal antibody responses to the organisms tested were found to be T cell dependent, as are most of the B-cell activators in the human system. Functional T helper activity was resistant to 1,500 rads of irradiation. Optimal polyclonal antibody responses to the bacterial extracts occurred at a 3:1 T-cell-to-B-cell ratio, whereas pokeweed mitogen-induced responses peaked at a 1:1 ratio, suggesting a difference in T-cell regulatory influences in response to these activators. Purified populations of T helper and suppressor cells exerted potent regulatory control of the responses to the bacterial extracts. These findings support the conclusion that regulatory T lymphocytes exert a potent modulating influence over the polyclonal response to periodontally associated bacteria and may play an important role in regulating the lymphocyte response in the diseased site. PMID:6197378

  7. Natural Air Purifier

    NASA Technical Reports Server (NTRS)

    1993-01-01

    NASA environmental research has led to a plant-based air filtering system. Dr. B.C. Wolverton, a former NASA engineer who developed a biological filtering system for space life support, served as a consultant to Terra Firma Environmental. The company is marketing the BioFilter, a natural air purifier that combines activated carbon and other filter media with living plants and microorganisms. The filter material traps and holds indoor pollutants; plant roots and microorganisms then convert the pollutants into food for the plant. Most non-flowering house plants will work. After pollutants have been removed, the cleansed air is returned to the room through slits in the planter. Terra Firma is currently developing a filter that will also disinfect the air.

  8. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

  9. Development of polyclonal antibody based enzyme-linked immunosorbent assay for the analysis of the agricultural insecticide imidacloprid: food quality and safety.

    PubMed

    Li, Zhixiong; Liu, Yingli; Sun, Yuanming; Wu, Qing; Lei, Hongtao; Wang, Hong; Xiao, Zhili

    2007-01-01

    In order to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the insecticide imidacloprid, the anti-imidacloprid polyclonal antibody was produced and the affinity of three different coating antigens with the polyclonal antibody was compared. The antigenic determinant in antigen aiming at the polyclonal antibody was raised and analyzed. The standard curve for imidacloprid had been developed. The effects of organic solvents and buffer ionic strength on the ELISA for insecticide were studied and conditions of analysis were also optimized in this research. Experiment result showed that the coating antigen IMMP-OVA ,whose binding ratio was 14:1, had stronger affinity with the anti-imidacloprid polyclonal antibody than IMMB-OVA and IMEB-OVA ; the IC50 value in the standard curve was 995.4 ng.mL-1 and the limit of detection (LOD) was 30 ng.mL-1; the cross reaction ratios with nitenpyram and acetamiprid were 5.73% and 11.31% respectively.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    2012-01-01

    Purpose To extend the study of the camel milk proteins which have antiviral activity against HCV, camel naïve polyclonal IgGs, α-lactalbumin were purified from camel milk and their anti-HCV effect was examined using PBMCs and Huh7.5 cell-lines. They were compared with the activity of human polyclonal IgGs and camel lactoferrin and casein. Material and methods Three types of experiments were performed on PBMCs and HuH7.5 cell. HCV was directly incubated with the purified proteins and then mixed with both cell types, or the proteins were incubated with the cells and then exposed to HCV, or the HCV pre-infected cells were treated with the proteins to inhibit intracellular replication. The proteins were added to cells or virus at different concentrations and time intervals. Results Pretreated PBMCs and Huh7.5 cells with milk proteins were not protected when exposed to HCV infection. The direct interaction between HCV and camel IgGs and camel lactoferrin (cLf) led to a complete inhibition of HCV entry into cells, while casein, α-lactalbumin and human IgGs failed to inhibit HCV entry at any tested concentration. Camel IgGs showed ability to recognize HCV peptides with a significant titer (12 × 103) in comparison with human IgGs which failed to do it. Camel lactoferrin was capable of inhibiting the intracellular HCV replication at concentrations of 0.25-1.25 mg/ml. Conclusion Camel milk naïve polyclonal IgGs isolated from camel milk could inhibit the HCV infectivity and demonstrated strong signal against its synthetic peptides. Lactoferrin inhibit the HCV infectivity started from 0.25 mg/ml. However, α-lactalbumin, human IgGs and casein failed to demonstrate any activity against HCV infectivity. PMID:22978304

  13. A defined fragment of bacterial protein I (OmpF) is a polyclonal B-cell activator.

    PubMed Central

    Vordermeier, M; Stäb, K; Bessler, W G

    1986-01-01

    Protein I from the outer membrane of Escherichia coli and other members of the family Enterobacteriaceae is a potent mitogen and polyclonal B-lymphocyte activator. To determine the part of the polypeptide responsible for biological activity, we cleaved the molecule into defined polypeptide fragments of approximate molecular weights 24,000, 15,000, 9,000, 7,000, and 3,000 by using the cyanogen bromide method. The fragments were purified by gel permeation chromatography and by preparative polyacrylamide gel electrophoresis. They were investigated for mitogenicity and for the induction of immunoglobulin synthesis in lymphocyte cultures from several inbred mouse strains. The fragment of molecular weight 24,000 turned out to be a potent polyclonal B-lymphocyte activator comparable to native protein I. The low-molecular-weight fragments exhibited only marginal effects. Neither purified T lymphocytes nor thymocytes were activated. Our results show that a defined fragment of protein I is responsible for its lymphocyte-stimulating activity. Images PMID:3484458

  14. Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

    SciTech Connect

    Senogles, S.E.; Caron, M.G.

    1986-05-01

    The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..S binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.

  15. Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection.

    PubMed

    Yari, Sh; Hadizadeh Tasbiti, A; Fateh, A; Karimi, A; Yari, F; Sakhai, F; Ghazanfari, M; Bahrmand, A

    2011-11-01

    Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and cross-reactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection.

  16. Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

    PubMed

    Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

    2014-01-01

    Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

  17. In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

    PubMed

    Kumar, G Ravi; Saxena, Shikha; Saxena, Lovleen; Chaturvedi, Uttara; Santra, Lakshman; Kumar, Rajiv; Sahoo, Aditya Prasad; Rajmani; Kumar, Aswani; Desai, G S; Kumar, Sudesh; Tiwari, Ashok K

    2012-09-01

    The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

  18. Methods for purifying carbon materials

    DOEpatents

    Dailly, Anne; Ahn, Channing; Yazami, Rachid; Fultz, Brent T.

    2009-05-26

    Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

  19. Purifying Aluminum by Vacuum Distillation

    NASA Technical Reports Server (NTRS)

    Du Fresne, E. R.

    1985-01-01

    Proposed method for purifying aluminum employs one-step vacuum distillation. Raw material for process impure aluminum produced in electrolysis of aluminum ore. Impure metal melted in vacuum. Since aluminum has much higher vapor pressure than other constituents, boils off and condenses on nearby cold surfaces in proportions much greater than those of other constituents.

  20. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

  1. Characterization and Epitope Mapping of the Polyclonal Antibody Repertoire Elicited by Ricin Holotoxin-Based Vaccination

    PubMed Central

    Cohen, Ofer; Mechaly, Adva; Sabo, Tamar; Alcalay, Ron; Aloni-Grinstein, Ronit; Seliger, Nehama; Kronman, Chanoch

    2014-01-01

    Ricin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine. PMID:25209559

  2. Characterization and epitope mapping of the polyclonal antibody repertoire elicited by ricin holotoxin-based vaccination.

    PubMed

    Cohen, Ofer; Mechaly, Adva; Sabo, Tamar; Alcalay, Ron; Aloni-Grinstein, Ronit; Seliger, Nehama; Kronman, Chanoch; Mazor, Ohad

    2014-11-01

    Ricin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine.

  3. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  4. Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data.

    SciTech Connect

    Stevens, F. J.; Bobrovnik, S. A.; Biosciences Division; Palladin Inst. Biochemistry

    2007-12-01

    Physiological responses of the adaptive immune system are polyclonal in nature whether induced by a naturally occurring infection, by vaccination to prevent infection or, in the case of animals, by challenge with antigen to generate reagents of research or commercial significance. The composition of the polyclonal responses is distinct to each individual or animal and changes over time. Differences exist in the affinities of the constituents and their relative proportion of the responsive population. In addition, some of the antibodies bind to different sites on the antigen, whereas other pairs of antibodies are sterically restricted from concurrent interaction with the antigen. Even if generation of a monoclonal antibody is the ultimate goal of a project, the quality of the resulting reagent is ultimately related to the characteristics of the initial immune response. It is probably impossible to quantitatively parse the composition of a polyclonal response to antigen. However, molecular regression allows further parameterization of a polyclonal antiserum in the context of certain simplifying assumptions. The antiserum is described as consisting of two competing populations of high- and low-affinity and unknown relative proportions. This simple model allows the quantitative determination of representative affinities and proportions. These parameters may be of use in evaluating responses to vaccines, to evaluating continuity of antibody production whether in vaccine recipients or animals used for the production of antisera, or in optimizing selection of donors for the production of monoclonal antibodies.

  5. Therapeutic Human Hyperimmune Polyclonal Antibodies Against Staphylococcal Enterotoxin B

    DTIC Science & Technology

    2008-05-01

    AD_________________ Award Number: W81XWH-08-C-0004 TITLE: Therapeutic Human Hyperimmune Polyclonal Antibodies against Staphylococcal...01-05-2008 2. REPORT TYPE Final, Phase I 3. DATES COVERED 19 OCT 2007 - 18 APR 2008 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic ...aerosol in a biowarfare scenario. The primary goal of Phase I was to demonstrate the feasibility of therapeutic intervention with immunoglobulin

  6. Process for purifying geothermal steam

    DOEpatents

    Li, Charles T.

    1980-01-01

    Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

  7. Process for purifying geothermal steam

    DOEpatents

    Li, C.T.

    Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

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

    PubMed

    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.

  9. Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins

    PubMed Central

    Requena, Mary; Bouhlal, Hicham; Nasreddine, Nadine; Saidi, Hela; Gody, Jean-Chrysostome; Aubry, Sylvie; Grésenguet, Gérard; Kazatchkine, Michel D; Sekaly, Rafick-Pierre; Bélec, Laurent; Hocini, Hakim

    2008-01-01

    The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342–amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 µg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1JRCSF, an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals. PMID:17999675

  10. Process for purifying zirconium sponge

    SciTech Connect

    Abodishish, H.A.M.; Kimball, L.S.

    1992-03-31

    This patent describes a Kroll reduction process wherein a zirconium sponge contaminated with unreacted magnesium and by-product magnesium chloride is produced as a regulus, a process for purifying the zirconium sponge. It comprises: distilling magnesium and magnesium chloride from: a regulus containing a zirconium sponge and magnesium and magnesium chloride at a temperature above about 800{degrees} C and at an absolute pressure less than about 10 mmHg in a distillation vessel to purify the zirconium sponge; condensing the magnesium and the magnesium chloride distilled from the zirconium sponge in a condenser; and then backfilling the vessel containing the zirconium sponge and the condenser containing the magnesium and the magnesium chloride with a gas; recirculating the gas between the vessel and the condenser to cool the zirconium sponge from above about 800{degrees} C to below about 300{degrees} C; and cooling the recirculating gas in the condenser containing the condensed magnesium and the condensed magnesium chloride as the gas cools the zirconium sponge to below about 300{degrees} C.

  11. Antigenic mimicry of Clostridium chauvoei flagella by polyclonal anti-idiotypic antibodies.

    PubMed

    Kijima-Tanaka, M; Tamura, Y; Suzuki, S; Nagamine, N; Nakamura, M

    1994-01-01

    Polyclonal rabbit anti-idiotypic (anti-Id) antibodies against two monoclonal antibodies (MAbs) specific for the flagella of Clostridium chauvoei were produced, purified and characterised. Lack of cross-reactivity with heterologous MAbs indicated that the anti-Id antibodies were highly specific. The surface-exposed epitopes of the flagellar filament recognised with protective MAb were further distinguished by the anti-Id antibodies. Moreover, each anti-Id antibody inhibited the binding of its related MAb to flagellar antigens in a competitive enzyme-linked immunosorbent assay, suggesting that the anti-Id antibodies bore an internal image of the flagellar antigens. The survival rate of mice was increased to nearly twice that of controls by immunisation with anti-Id 41, which had been produced with a protective MAb; in contrast, anti-Id 114, produced with a non-protective MAb, failed to immunise. The results suggest that an anti-Id antibody containing an internal image of C. chauvoei flagella might be used as a vaccine.

  12. A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

    PubMed

    Xiao, Bin; Kuang, Zhenzhan; Zhan, Yanli; Chen, Daxiang; Gao, Yang; Li, Ming; Luo, Shuhong; Hao, Wenbo

    2016-02-01

    The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na(+) and H(+) ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca(2+). In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

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

    PubMed

    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.

  14. Polyclonal outbreak of Burkholderia cepacia complex bacteraemia in haemodialysis patients.

    PubMed

    Magalhães, M; Doherty, C; Govan, J R W; Vandamme, P

    2003-06-01

    We report a polyclonal outbreak of bacteraemia involving 24 patients at a haemodialysis facility in Recife (Brazil). During the outbreak period (4 June to 11 July, 2001), three Burkholderia cepacia complex strains were isolated from human blood and from various water samples collected at different sites in the haemodialysis unit and from dialysate fluids. Out of 14 patients with positive blood cultures, six were infected by Burkholderia cepacia complex bacteria: three with Burkholderia cepacia genomovar III, two with a first strain of Burkholderia vietnamiensis, and one with the Burkholderia cepacia genomovar III strain and a second B. vietnamiensis strain.

  15. Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin.

    PubMed Central

    Abouzied, M M; Azcona-Olivera, J I; Yoshizawa, T; Pestka, J J

    1993-01-01

    The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis. PMID:8517728

  16. Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen).

    PubMed Central

    Sundaram, P; Xiao, W; Brandsma, J L

    1996-01-01

    Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta-galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants. PMID:8614644

  17. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-04-01

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

  20. Turbocharged engine with exhaust purifier

    SciTech Connect

    Tadokoro, T.; Matsuda, I.; Okimoto, H.

    1986-09-23

    The patent described a control system for an automobile engine having intake and exhaust systems for respectively conducting intake gases to and exhaust gases from the engine, which comprises, in combination: a turbocharger including a turbine disposed in the exhaust system and adapted to be driven by the flow of the exhaust gases therethrough and a blower disposed in the intake system and drivingly connected with the turbine for supercharging the intake gases; and exhaust purifying device disposed in the exhaust system downstream of the turbine with respect to the direction of flow of the exhaust gases; a regulating means for varying the effective cross-section of a portion of the exhaust system leading to the turbine; a control means for controlling the regulating means in dependence on an operating condition of the engine, the control means causing the regulating means to decrease the effective cross-section during a low speed operating condition, but to increase the effective cross-section during a high speed operating condition of the engine.

  1. Microstructure of Purified Rubber Particles.

    PubMed

    Wood; Cornish

    2000-05-01

    Purified rubber particles from Hevea brasiliensis (Brazilian rubber tree), Parthenium argentatum (guayule), Ficus elastica (Indian rubber tree), and Euphorbia lactiflua were examined and compared using conventional scanning electron microscopy (SEM), field-emission SEM, cryo-SEM, and transmission electron microscopy (TEM). Rubber particles of all four species were spherical; they varied in size and had a uniform homogeneous material, the rubber core, surrounded by a contiguous monolayer (half-unit) membrane. Frozen-hydrated and/or untreated particles from H. brasiliensis and P. argentatum deformed and fused readily, whereas those from F. elastica and E. lactiflua retained their spherical shapes. These results indicate that the surface components of the H. brasiliensis and P. argentatum particles are more fluid than those of F. elastica or E. lactiflua. When fixed in aldehyde, F. elastica particles retained their spherical exterior shapes but had hollow centers, whereas H. brasiliensis and P. argentatum particles completely collapsed. In aldehyde-osmium tetroxide-fixed material, the rubber core of F. elastica was poorly preserved in some particles in which only a small amount of the rubber core remained adhering to the monolayer membrane, leaving a hollow center. Euphorbia lactiflua particles were well preserved in terms of retaining the rubber core; however, the membrane was not as easily discernible as it was in the other three species. Both H. brasiliensis and P. argentatum were well preserved following fixation; their cores remained filled with rubber, and their monolayer membranes were defined. The addition of potassium permanganate to the fixation-staining regime resulted in higher-contrast micrographs and more well defined monolayer membranes.

  2. Back to the future: recombinant polyclonal antibody therapeutics

    PubMed Central

    Wang, Xian-zhe; Coljee, Vincent W.; Maynard, Jennifer A.

    2013-01-01

    Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed. PMID:24443710

  3. Polyclonal hyperviscosity syndrome in IgG4-related disease and associated conditions

    PubMed Central

    Chen, Luke YC; Wong, Patrick CW; Noda, Shinji; Collins, David R; Sreenivasan, Gayatri M; Coupland, Robert C

    2015-01-01

    Key Clinical Message Polyclonal hyperviscosity syndrome (HVS) is rare and has been reported in various disorders of immune dysregulation and lymphoid hyperplasia. IgG4-Related Disease (IgG4-RD) is an emerging disorder often associated with exuberant hypergammaglobulinemia, and this review of seven cases establishes IgG4-RD as an important cause of polyclonal HVS. PMID:25914812

  4. Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

    PubMed

    Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

    2013-09-01

    Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins.

  5. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2010-01-01

    We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

  6. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  7. Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

    SciTech Connect

    Sandy, J.D.; Plaas, A.H.

    1989-06-01

    Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with (35S)sulfate, (3H)leucine, and (35S)cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with (35S)sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M.

  8. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schrödinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor.

  9. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  10. Hydrogen purifier module with membrane support

    DOEpatents

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

    2012-07-24

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

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

    PubMed

    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.

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

    PubMed Central

    Li, Jing; Wu, Ronghua; Chen, Haijiao; Zhou, Youlang; Li, Yan; Wang, Yongjun; Liu, Yan; Liu, Mei

    2013-01-01

    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. PMID:23615470

  13. Persistent polyclonal B-cell lymphocytosis with splenomegaly: histologic description of 2 cases.

    PubMed

    Martinez-Lopez, Azahara; Montes-Moreno, Santiago; Mazorra, Francisco; Miranda-Vallina, Cesar; Ulibarrena, Carlos; Martin, Juan Luis Alfonso; Bosch, Jose Miguel; Peri, Valeria; Burdaspal, Ana; Fernandez-Alvarez, Montse; Sanchez-Verde, Lidia; Piris, Miguel A

    2013-07-01

    Persistent polyclonal B-cell lymphocytosis is a rare, benign lymphoproliferative disorder characterized by a stable, polyclonal CD19-positive CD5-negative lymphocytosis, the presence of binucleated lymphocytes in peripheral blood, and a polyclonal increase in serum immunoglobulin-M that may occasionally be accompanied by splenomegaly. Histopathologic diagnosis of these splenectomy specimens is difficult because of the massive spleen infiltration and the rarity of the descriptions of this condition. We describe the histopathologic findings from 2 splenectomy specimens. These included a partially preserved architecture with infiltration of the red pulp by small lymphocytes and partial replacement of the white pulp. Suggestions for identifying the disorder are made.

  14. Production of polyclonal anti-dUCH (Drosophila ubiquitin carboxyl-terminal hydrolase) antibodies.

    PubMed

    Tram, Nguyen Thi Quynh; Trang, Nguyen Thi Thu; Thao, Dang Thi Phuong; Thuoc, Tran Linh

    2013-04-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), which is a member of the ubiquitin carboxyl-terminal hydrolase (UCH) family, is highly expressed in neurons. In vitro, UCH- L1 exhibits both ubiquitin hydrolase and ligase activity. Many studies have suggested that UCH-L1 is involved in the pathogenesis of Parkinson's disease and some different human cancer diseases, but its role in a living system is still unclear. Recently, Drosophila melanogaster has been shown to be a compatible model for studying human diseases. To investigate the role of UCH-L1 in a living system, the UCH-L1 homologous protein in Drosophila melanogaster (dUCH) is used for analyzing the role of the protein's function in transgenic Drosophila. Here, we used DNA molecular techniques to clone, express, and purify dUCH protein from Escherichia coli. The purified dUCH protein was injected into a rabbit to produce an anti-dUCH antibody, which was shown to have high specificity and sensitivity to the dUCH protein. The affinity of the antibody is 1:320,000 at 7.81 ng/μL antigen concentration. The 1:40,000 dilution-produced antibodies can detect antigen at a low concentration of 0.98 ng/μL. Success in producing this antibody provides good material for further experiments in the study of the role of UCH-L1 by a Drosophila model.

  15. Effects of purified alginate sponge on the regeneration of chondrocytes: in vitro and in vivo.

    PubMed

    Song, Jeong Eun; Kim, A Ram; Lee, Cheon Jung; Tripathy, Nirmalya; Yoon, Kun Ho; Lee, Dongwon; Khang, Gilson

    2015-01-01

    Regeneration science has been studied using tissue engineering techniques due to the self-renewal difficulties of damaged or degenerated cartilage. A scaffold with biodegradability and biocompatibility features plays a key role in developing cartilage tissue similar to human biological materials. Herein, we have fabricated three-dimensional sponge using purified alginate for the regeneration of chondrocytes cells and formation of cartilage. We demonstrated that the alginate purification can effectively minimize inflammatory reaction through reducing the content of mannuronic acid causing immune rejection. Cartilage regeneration research was performed using three-dimensional non-purified and purified alginate sponges synthesized by modified Korbutt method. In vitro cell viability and specific gene expression in the cartilage cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and reverse transcriptase-polymerase chain reaction (RT-PCR) after seeding chondrocytes on the as-fabricated sponges. Specific extracellular matrix (ECM) of chondrocytes, sGAG, and the content of collagen were also measured. Histological staining was carried out after purified alginate sponge seeded with chondrocytes and was implanted in subcutaneous nude mouse followed by extraction. Compared to the non-purified ones, the purified alginate sponges showed positive effects on maintaining affinities and phenotype of chondrocytes. From these results, it can be suggested that the purified alginate sponges provide a promising platform for cartilage regeneration.

  16. Single-batch production of recombinant human polyclonal antibodies.

    PubMed

    Nielsen, Lars S; Baer, Alexandra; Müller, Christian; Gregersen, Kristian; Mønster, Nina T; Rasmussen, Søren K; Weilguny, Dietmar; Tolstrup, Anne B

    2010-07-01

    We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress I technology. The Sympress I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

  17. Type 1 diabetes immunotherapy using polyclonal regulatory T cells

    PubMed Central

    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, Kevin; Liu, Weihong; Long, S. Alice; Masiello, Lisa M.; Nguyen, Vinh; Putnam, Amy L.; Rieck, Mary; Sayre, Peter; Tang, Qizhi

    2016-01-01

    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+CD127lo/−CD25+ polyclonal Tregs (0.05 × 108 to 26 × 108 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+CD25hiCD127lo 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. PMID:26606968

  18. [An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].

    PubMed

    Liu, Rong; Zhong, Qin-Ping; Jiang, Ming-Sen; Dong, Hui-Fen

    2011-10-01

    To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

  19. Binding of rabies virus to purified Torpedo acetylcholine receptor.

    PubMed

    Lentz, T L; Benson, R J; Klimowicz, D; Wilson, P T; Hawrot, E

    1986-12-01

    The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

  20. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

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

    DTIC Science & Technology

    1982-08-01

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

  2. Production of a Purified Marine Neurotoxin and Demonstration of its Binding Affinity to Ion Channel Receptors

    DTIC Science & Technology

    1989-06-10

    the ciguatera Implicated toxins, maitotoxin, does not displace brevetoxin from its unique receptor and therefore must produce its toxic 49octs with a...James R. Balthrop, John A. Babinchak, Penny B. Travis, Teresa L. Herring and Pam Y. Brown-Eyo Ciguatera is a tropical fish-borne disease in which both a...synaptosome bound toxin from free toxin following in vitro bindina. we have demonstruted that one of the ciguatera implicated toxins, maitotoxin

  3. Method for purifying bidentate organophosphorus compounds

    DOEpatents

    Schulz, Wallace W.

    1977-01-01

    Bidentate organophosphorus compounds useful for extracting actinide elements from acidic nuclear waste solutions are purified of undesirable acidic impurities by contacting the compounds with ethylene glycol which preferentially extracts the impurities found in technical grade bidentate compounds.

  4. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    PubMed Central

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  5. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera.

    PubMed

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species.

  6. Affine Sphere Relativity

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-03-01

    We investigate spacetimes whose light cones could be anisotropic. We prove the equivalence of the structures: (a) Lorentz-Finsler manifold for which the mean Cartan torsion vanishes, (b) Lorentz-Finsler manifold for which the indicatrix (observer space) at each point is a convex hyperbolic affine sphere centered on the zero section, and (c) pair given by a spacetime volume and a sharp convex cone distribution. The equivalence suggests to describe (affine sphere) spacetimes with this structure, so that no algebraic-metrical concept enters the definition. As a result, this work shows how the metric features of spacetime emerge from elementary concepts such as measure and order. Non-relativistic spacetimes are obtained replacing proper spheres with improper spheres, so the distinction does not call for group theoretical elements. In physical terms, in affine sphere spacetimes the light cone distribution and the spacetime measure determine the motion of massive and massless particles (hence the dispersion relation). Furthermore, it is shown that, more generally, for Lorentz-Finsler theories non-differentiable at the cone, the lightlike geodesics and the transport of the particle momentum over them are well defined, though the curve parametrization could be undefined. Causality theory is also well behaved. Several results for affine sphere spacetimes are presented. Some results in Finsler geometry, for instance in the characterization of Randers spaces, are also included.

  7. 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.

    PubMed

    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.

  8. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  9. Purified plasminogen activating factor produced by malignant lymphoid cells abrogates lymphocyte cytotoxicity.

    PubMed Central

    Sundar, S K; Bergeron, J; Menezes, J

    1984-01-01

    Immunosuppression is a generally observed phenomenon in patients with malignancies. Here we report that plasminogen activating factor (PAF) produced by human (P3HR-1) and simian (B95-8) lymphoid cells of malignant origin abrogates lymphocyte cytotoxicity. PAF has been purified from Epstein-Barr (EB) virus genome carrying lymphocyte cytotoxicity. PAF has been purified from Epstein-Barr (EB) virus genome carrying lymphoid lines by affinity chromatography using lysine-Sepharose columns. Purified PAF consistently inhibited Killer cell activity against the following targets: K-562, EB virus superinfected Raji cells and in vitro EB virus transformed autologous B lymphocytes. Furthermore PAF also inhibited the antibody-dependent cellular cytotoxicity. The results presented also indicate that PAF affects the effector lymphocytes and not the target cells. Taken together, these observations emphasize the importance of factors such as PAF, released by malignant cells, as inhibitors/modulators of immune mechanisms effective against tumour cells. PMID:6430612

  10. Immunogenicity of a purified fragment of 17D yellow fever envelope protein.

    PubMed

    Brandriss, M W; Schlesinger, J J; Walsh, E E

    1990-06-01

    Information on the immunogenic properties of purified flavivirus proteins may be useful in the development of recombinant or synthetic peptide vaccines. Using a monoclonal antibody, an attempt was made to purify the envelope (E) protein of 17D yellow fever virus (17D YF) by affinity chromatography. The purified material could not be identified as intact E protein but it did bear antigenic determinants of E as determined by selective reactivity with anti-E monoclonal antibodies. Rabbits immunized with this material produced antibodies that neutralized 17D YF and dengue-2 viruses in comparable titers, indicating that cross-reactive antigenic determinants were preserved. Immunization of mice resulted in protection against intracerebral challenge with 17D YF.

  11. Production of polyclonal antibody to a recombinant non-structural protein Nsp1a of human astrovirus.

    PubMed

    Liu, Chang; Liu, Wen-Hui; Kan, Li-Li; Li, Xin; Li, Yong-gang; Zhao, Wei

    2014-12-01

    Human astrovirus (HAstV) are important pathogens that cause acute viral diarrhea in infants. Little is known about the mechanisms of astrovirus-induced diarrhea. Previous studies have suggested that an apoptosis inducer may be encoded in the non-structural protein (nsP1a) of astrovirus and contribute to virus-induced diarrhea. To study the biological function of nsP1a and to gain further insight into nsP1a protein-host cell interactions, good quality antibodies must be produced. The nsP1agene of HAstV-1 was cloned into a bacterial expression vector Pgex-6P-1. The recombinant plasmid Pgex-6P-nsP1a was transformed into Escherichia coli BL21 (DE3) and expressed as a fusion protein that contains N-terminal GST tags. The expressed recombinant protein was purified and used as an antigen to produce an nsP1a antiserum in rabbits. ELISA was used to detect the titer of specific antibodies. Specificity activity was detected by Western blot and immunofluorescence analysis. The titer of specific antibodies was up to 1:30,000. Western blotting and immunofluorescence analysis indicated that the polyclonal antibody could recognize specifically the HAstV-1 nsP1a protein.

  12. Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata).

    PubMed

    Alonso, W R; Crock, J E; Croteau, R

    1993-02-15

    Limonene synthase, a monoterpene cyclase from the oil glands of spearmint (Mentha spicata) leaves that catalyzes the conversion of geranyl pyrophosphate to (-)-4S-limonene, was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. Immunoblotting analysis revealed that the antibodies were very specific for denatured limonene synthase from all Mentha species tested. However, no immunological cross-reactivity was observed with denatured limonene synthases from Valencia oranges (Citrus sinensis, Rutaceae) or wormseed (Chenopodium ambrosioides, Chenopodiaceae). Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiaceae, Asteraceae, and Umbellifereae, or from conifer species, and no cross-reactivity was demonstrated toward several sesquiterpene cyclases of higher plant and fungal origin. Although the antibody preparation was highly selective for denatured limonene cyclase from Mentha, the antibodies did not recognize the native protein in several different types of experiments. Nevertheless, specificity for the target enzyme was unambiguously demonstrated when the antibody preparation was shown to cross-react with the cyclase protein expressed in Escherichia coli that harbored the corresponding limonene synthase cDNA gene from M. spicata.

  13. Identification and functional characterization of platelet-activating factor receptors in human leukocyte populations using polyclonal anti-peptide antibody.

    PubMed Central

    Müller, E; Dagenais, P; Alami, N; Rola-Pleszczynski, M

    1993-01-01

    Recently, the successful cloning of a receptor for platelet-activating factor (PAF), a lipid mediator of inflammation, was reported. Here we investigated the distribution and potential diversity of human PAF receptors (hPAF-Rs) among individual leukocyte populations by (i) hPAF-R mRNA transcription studies and (ii) analysis of cell surface expression of hPAF-R protein using a polyclonal anti-peptide antibody (anti-hPAF-R164-173). Northern blot analysis, flow cytometry, and immunoblotting with anti-hPAF-R antibody indicated that monocytic, neutrophilic, and B-lymphocytic cell lines all shared a similar hPAF-R species, whereas resting T-cell and natural killer cell lines failed to express detectable levels of either hPAF-R protein or mRNA. Peripheral blood leukocyte populations showed a distribution of hPAF-R cell surface expression similar to that of the corresponding cell lines. Furthermore, binding of anti-hPAF-R164-173 antiserum, purified IgG, or Fab and F(ab')2 fragments to the receptor of all investigated PAF-R-positive cell lines induced an increase in intracellular free calcium concentration. The characterization of the expression of a lipid ligand receptor using antibodies against an intrinsic portion of the receptor protein has, to our knowledge, never been reported previously. Images Fig. 1 Fig. 5 Fig. 7 PMID:8390683

  14. Mullerian inhibiting substance fractionation by dye affinity chromatography.

    PubMed

    Budzik, G P; Powell, S M; Kamagata, S; Donahoe, P K

    1983-08-01

    Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.

  15. Method for purifying bidentate organophosphorous compounds

    DOEpatents

    McIsaac, Lyle D.; Krupa, Joseph F.; Schroeder, Norman C.

    1981-01-01

    Bidentate organophosphorous compounds are purified of undesirable impurities by contacting a solution of the compounds with a mercuric nitrate solution to form an insoluble mercuric bidentate compound which precipitates while the impurities remain in solution. The precipitate is washed and then contacted with a mixture of an aqueous solution of a strong mercuric ion complexing agent and an organic solvent to complex the mercuric ion away from the bidentate compound which then dissolves in the solvent. The purified bidentate compounds are useful for extracting the actinide elements from aqueous acidic nuclear waste solutions.

  16. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    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.

    2016-01-01

    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. PMID:26908053

  17. Establishment and characterization of monoclonal and polyclonal antibodies against human intestinal fatty acid-binding protein (I-FABP) using synthetic regional peptides and recombinant I-FABP.

    PubMed

    Kajiura, Satoshi; Yashiki, Tetsuya; Funaoka, Hiroyuki; Ohkaru, Yasuhiko; Nishikura, Ken; Kanda, Tatsuo; Ajioka, Yoichi; Igarashi, Michihiro; Hatakeyama, Katsuyoshi; Fujii, Hiroshi

    2008-01-01

    We have succeeded in raising highly specific anti-human intestinal fatty acid-binding protein (I-FABP) monoclonal antibodies by immunizing animals with three synthetic regional peptides, i.e., the amino terminal (RP-1: N-acetylated 1-19-cysteine), middle portion (RP-2: cysteinyl-91-107) and carboxylic terminal (RP-3: cysteinyl-121-131) regions of human I-FABP, and the whole I-FABP molecule as antigens. We also raised a polyclonal antibody by immunizing with a recombinant (r) I-FABP. To ascertain the specificity of these antibodies for human I-FABP, the immunological reactivity of each was examined by a binding assay using rI-FABP, partially purified native I-FABP and related proteins such as liver-type (L)-FABP, heart-type (H)-FABP, as well as the regional peptides as reactants, and by Western blot analysis. In addition, the expression and distribution of I-FABP in the human gastrointestinal tract were investigated by an immunohistochemical technique using a carboxylic terminal region-specific monoclonal antibody, 8F9, and a polyclonal antibody, DN-R2. Our results indicated that both the monoclonal and polyclonal antibodies established in this study were highly specific for I-FABP, but not for L-FABP and H-FABP. Especially, the monoclonal antibodies raised against the regional peptides, showed regional specificity for the I-FABP molecule. Immunoreactivity of I-FABP was demonstrated in the mucosal epithelium of the jejunum and ileum by immunohistochemical staining, and the immunoreactivity was based on the presence of the whole I-FABP molecule but not the presence of any precursors or degradation products containing a carboxylic terminal fragment. It is concluded that some of these monoclonal and polyclonal antibodies, such as 8F9, 4205, and DN-R2, will be suitable for use in research on the immunochemistry and clinical chemistry of I-FABP because those antibodies can recognize both types of native and denatured I-FABP. In order to detect I-FABP in blood samples, it

  18. Dermatitis from purified sea algae toxin (debromoaplysiatoxin).

    PubMed

    Solomon, A E; Stoughton, R B

    1978-09-01

    Cutaneous inflammation was induced by debromoaplysiatoxin, a purified toxin extracted from Lyngbya majuscula Gomont. This alga causes a seaweed dermatitis that occurs in persons who have swum off the coast of Oahu in Hawaii. By topical application, the toxin was found to produce an irritant pustular folliculitis in humans and to cause a severe cutaneous inflammatory reaction in the rabbit and in hairless mice.

  19. Improvement of Linde Kryotechnik's internal purifier

    NASA Astrophysics Data System (ADS)

    Decker, Lutz; Meier, Albert; Wilhelm, Hanspeter

    2014-01-01

    With the recent shortage in supply of helium, recovery solutions have experienced a new focus with a tendency to recover streams with higher impurity content. This development calls for purifier systems operating efficiently and with low impact on liquefaction capacity for helium streams with impurity levels in the percentage range. Linde Kryotechnik has answered this demand by improving the performance of its purifier technology. Since 1983, its standardized helium liquefiers of the L- and former TCF-series type contain an internal purifier which already allows efficient impurity removal with minimized space demand. Along with a line dryer to absorb humidity, it is designed to remove air impurities up to 5 mol%. However, with increasing impurity level, liquefaction capacity reduced significantly being furthermore restricted to an upper level of approx. 180 l/h and continuous purification became limited in time. With the current redesign of this purifier, the impact on liquefaction capacity is now minimized without any limitation within the capacity range of the L-series plants. Continuous purification is hence ensured beyond previous maximum impurity content. This paper provides the key design changes and the achievable performance, which has been verified in the recent L-series plants delivered to customers.

  20. Method of purifying neutral organophosphorus extractants

    DOEpatents

    Horwitz, E. Philip; Gatrone, Ralph C.; Chiarizia, Renato

    1988-01-01

    A method for removing acidic contaminants from neutral mono and bifunctional organophosphorous extractants by contacting the extractant with a macroporous cation exchange resin in the H.sup.+ state followed by contact with a macroporous anion exchange resin in the OH.sup.- state, whereupon the resins take up the acidic contaminants from the extractant, purifying the extractant and improving its extraction capability.

  1. Home Air Purifiers Eradicate Harmful Pathogens

    NASA Technical Reports Server (NTRS)

    2014-01-01

    Marshall Space Flight Center funded the University of Madison-Wisconsin to develop ethylene scrubbers to keep produce fresh in space. Akida Holdings of Jacksonville, Florida, licensed the technology and developed Airocide, an air purifier that can kill airborne pathogens. Previously designed for industrial spaces, there is now a specially designed unit for home use.

  2. Two systems developed for purifying inert atmospheres

    NASA Technical Reports Server (NTRS)

    Foster, M. S.; Johnson, C. E.; Kyle, M. L.

    1969-01-01

    Two systems, one for helium and one for argon, are used for purifying inert atmospheres. The helium system uses an activated charcoal bed at liquid nitrogen temperature to remove oxygen and nitrogen. The argon system uses heated titanium sponge to remove nitrogen and copper wool beds to remove oxygen. Both use molecular sieves to remove water vapor.

  3. Electrophoretic separator for purifying biologicals, part 1

    NASA Technical Reports Server (NTRS)

    Mccreight, L. R.

    1978-01-01

    A program to develop an engineering model of an electrophoretic separator for purifying biologicals is summarized. An extensive mathematical modeling study and numerous ground based tests were included. Focus was placed on developing an actual electrophoretic separator of the continuous flow type, configured and suitable for flight testing as a space processing applications rocket payload.

  4. Microbial gut overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill.

    PubMed

    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.

  5. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

    SciTech Connect

    Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

    1988-01-12

    Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor ..cap alpha.. subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-(..gamma..-/sup 32/P)triphosphate, a protein of 95 kilodaltons representing the insulin receptor ..beta.. subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.

  6. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  7. Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

    SciTech Connect

    Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

    1987-09-01

    The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining (/sup 3/H)nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure (/sup 3/H)nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-(/sup 3/H)nicotine-binding characteristics.

  8. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  9. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  10. Functionalized multi-walled carbon nanotubes as affinity ligands

    NASA Astrophysics Data System (ADS)

    Yu, L.; Li, C. M.; Zhou, Q.; Gan, Y.; Bao, Q. L.

    2007-03-01

    Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of ~361 µg IgG/mg MWCNT (wet basis). About 75% of bound IgG was eluted from affinity MWCNTs (ANT-I and ANT-II) and ELISA confirmed that the biological activity of IgG was well preserved during the course of affinity separation. The functionalized MWCNTs could be potentially used in affinity chromatography.

  11. Preparation of polyclonal antibodies against pig trachea apomucin

    SciTech Connect

    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.

  12. Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies

    PubMed Central

    Deniger, Drew C.; Maiti, Sourindra N.; Mi, Tiejuan; Switzer, Kirsten C.; Ramachandran, Vijaya; Hurton, Lenka V.; Ang, Sonny; Olivares, Simon; Rabinovich, Brian A.; Huls, Helen; Lee, Dean A.; Bast, Robert C.; Champlin, Richard E.; Cooper, Laurence J.N.

    2014-01-01

    Purpose To activate and propagate populations of γδT cells expressing polyclonal repertoire of γ and δ TCR chains for adoptive immunotherapy for cancer, which has yet to be achieved. Experimental Design Clinical-grade artificial antigen presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδT cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. Results γδT cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL-2 and IL-21. Propagated γδT cells were polyclonal as they expressed Vδ1, Vδ2, Vδ3, Vδ5, Vδ7, and Vδ8 with Vγ2, Vγ3, Vγ7, Vγ8, Vγ9, Vγ10, and Vγ11 TCR chains. Interferon-γ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδantibody when added to co-cultures of polyclonal γδT cells and tumor cell lines. Polyclonal γδT cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδT cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδT cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts. Conclusions Polyclonal γδT cells can be activated and propagated with clinical-grade aAPC and demonstrate broad anti-tumor activities, which will facilitate the implementation of γδT cell cancer immunotherapies in humans. PMID:24833662

  13. Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

    NASA Technical Reports Server (NTRS)

    Knisley, Keith A.; Rodkey, L. Scott

    1986-01-01

    A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

  14. Lower Affinity T Cells are Critical Components and Active Participants of the Immune Response

    PubMed Central

    Martinez, Ryan J.; Evavold, Brian D.

    2015-01-01

    Kinetic and biophysical parameters of T cell receptor (TCR) and peptide:MHC (pMHC) interaction define intrinsic factors required for T cell activation and differentiation. Although receptor ligand kinetics are somewhat cumbersome to assess experimentally, TCR:pMHC affinity has been shown to predict peripheral T cell functionality and potential for forming memory. Multimeric forms of pMHC monomers have often been used to provide an indirect readout of higher affinity T cells due to their availability and ease of use while allowing simultaneous definition of other functional and phenotypic characteristics. However, multimeric pMHC reagents have introduced a bias that underestimates the lower affinity components contained in the highly diverse TCR repertoires of all polyclonal T cell responses. Advances in the identification of lower affinity cells have led to the examination of these cells and their contribution to the immune response. In this review, we discuss the identification of high- vs. low-affinity T cells as well as their attributed signaling and functional differences. Lastly, mechanisms are discussed that maintain a diverse range of low- and high-affinity T cells. PMID:26441973

  15. Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

    PubMed

    Hou, Shuai; Shi, Lei; Lei, Haixin

    2016-01-01

    RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

  16. Steroidogenesis in amlodipine treated purified Leydig cells

    SciTech Connect

    Latif, Rabia; Lodhi, Ghulam Mustafa; Hameed, Waqas; Aslam, Muhammad

    2012-01-01

    Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

  17. Utilization of purified cellulose in fiber studies.

    PubMed

    Penner, M H; Liaw, E T

    1990-01-01

    Purified cellulose-type fiber products are widely used in experimental nutrition. Their use in a broad spectrum of studies may potentially lead to the acceptance of the misconception that the various commercially available cellulose products are equivalent. In this paper we have attempted to show that this is not the case. The comparative structural data of Table 2 and the compositional data of Olsen et al provide examples which indicate that purified cellulose preparations should not necessarily be considered equivalent. Unfortunately, our current lack of understanding of how fibers are metabolized and how they may affect specific physiological parameters makes it difficult to determine which, if any, of the measurable structural and chemical properties will be of relevance for a given in vivo study. At present, it appears that researchers utilizing/evaluating the consequences of consuming a purified cellulose-type fiber would be prudent to provide at least a limited amount of data on the properties of the cellulose preparation used in their studies. The characterization of the cellulose product may be done by a variety of methods depending on the expertise of the laboratory. The methods and results discussed in this paper provide an example of the type of information which may be obtained from an in vitro characterization of cellulose products.

  18. Kernel Affine Projection Algorithms

    NASA Astrophysics Data System (ADS)

    Liu, Weifeng; Príncipe, José C.

    2008-12-01

    The combination of the famed kernel trick and affine projection algorithms (APAs) yields powerful nonlinear extensions, named collectively here, KAPA. This paper is a follow-up study of the recently introduced kernel least-mean-square algorithm (KLMS). KAPA inherits the simplicity and online nature of KLMS while reducing its gradient noise, boosting performance. More interestingly, it provides a unifying model for several neural network techniques, including kernel least-mean-square algorithms, kernel adaline, sliding-window kernel recursive-least squares (KRLS), and regularization networks. Therefore, many insights can be gained into the basic relations among them and the tradeoff between computation complexity and performance. Several simulations illustrate its wide applicability.

  19. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  20. Permeation of Calcium through Purified Connexin 26 Hemichannels*

    PubMed Central

    Fiori, Mariana C.; Figueroa, Vania; Zoghbi, Maria E.; Saéz, Juan C.; Reuss, Luis; Altenberg, Guillermo A.

    2012-01-01

    Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to Mr 1,000, including second messengers and metabolites. Intercellular Ca2+ signaling can occur by movement of a number of second messengers, including Ca2+, through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca2+ permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca2+ influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca2+ permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca2+ by measuring Ca2+ transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca2+-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca2+] in response to an imposed [Ca2+] gradient. We show that Ca2+ does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca2+ is high, similar to that for Na+. We suggest that hemichannels can be a significant pathway for Ca2+ influx into cells under conditions such as ischemia. PMID:23048025

  1. PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

    PubMed

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z; Gittes, George K

    2016-02-17

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

  2. PNA lectin for purifying mouse acinar cells from the inflamed pancreas

    PubMed Central

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z.; Gittes, George K.

    2016-01-01

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer. PMID:26884345

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

    DTIC Science & Technology

    2016-07-01

    recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV)-Makona isolate. Serum collected from these hyperimmunized Tc...countermeasures for EBOV infections in humans. Effective countermeasures that include vaccines , antivirals, and other prophylactic and therapeutic...traditional animal systems used to produce polyclonal antibodies, Tc bovines can be hyperimmunized over a long period of time with vaccines containing strong

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

    PubMed Central

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

    2016-01-01

    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. PMID:27380890

  5. Biological cost of fluoroquinolone resistance in Escherichia coli implicated in polyclonal infection.

    PubMed

    Bémer, P; Corvec, S; Guitton, C; Giraudeau, C; Le Gargasson, G; Espaze, E; Drugeon, H

    2007-07-01

    Polyclonal Escherichia coli strains were isolated in a transplanted patient who experienced successive septic shocks. Fluoroquinolone susceptible and resistant strains were corresponding to different PFGE fragment profiles. The gyrA S83L mutation was associated with a reduction in biological fitness. Resistant strain was selected by a long-term single use of ofloxacin.

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

    PubMed

    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.

  7. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B-cell stimulatory factor 1.

    PubMed Central

    Finkelman, F D; Katona, I M; Urban, J F; Snapper, C M; Ohara, J; Paul, W E

    1986-01-01

    The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses. PMID:3491987

  8. Air Purifiers Eliminate Pathogens, Preserve Food

    NASA Technical Reports Server (NTRS)

    2009-01-01

    NASA-funded researchers produced an ethylene reduction device for a plant growth unit. KES Science & Technology Inc., a Kennesaw, Georgia-based company specializing in sustaining perishable foods, licensed the ethylene scrubbing technology. KES partnered with Akida Holdings, of Jacksonville, Florida, which now markets the NASA-developed technology as AiroCide. According to the company, it is the only air purifier that completely destroys airborne bacteria, mold, fungi, mycotoxins, viruses, volatile organic compounds (like ethylene), and odors. What?s more, the devices have no filters that need changing and produce no harmful byproducts, such as the ozone created by some filtration systems.

  9. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

    Sulton, Deley L.; Boothe, Michael; Ball, David W.; Morales, Wilfredo

    1997-01-01

    Previous work indicated that polyphenyl thioethers possessed chemical properties, related to their electron affinities, which could allow them to function as vapor phase lubricants (VPL). Indeed, preliminary tribological tests revealed that the thioethers could function as vapor phase lubricants but not over a wide temperature and hertzian pressure range. Increasing the electron affinity of the thioethers may improve their VPL properties over this range. Adding a substituent group to the thioether will alter its electron affinity in many cases. Molecular orbital calculations were undertaken to determine the effect of five different substituent groups on the electron affinity of polyphenyl thioethers. It was found that the NO2, F, and I groups increased the thioethers electron affinity by the greatest amount. Future work will involve the addition of these groups to the thioethers followed by tribological testing to assess their VPL properties.

  10. Immunoprotection in sheep against Haemonchus contortus using its thiol-purified excretory/secretory proteins.

    PubMed

    Arunkumar, Selvarayar

    2012-01-01

    Excretory/Secretory antigen was prepared by culturing live adult worms of Haemonchus contortus in RPMI 1640 medium at a concentration of 50 worms per mL in a culture-flask at 37 ˚C for 24 hr and the culture supernatant was used as antigen. The E/S antigen was purified by thiol-sepharose affinity chromatography. On western blot analysis, it was demonstrated that thiol-purified antigen showed a single reactive band at 66 kDa. In immunization trial, sheep were administered intramuscularly with 500 µg of thiol-purified excretory/secretory antigen along with montanide as adjuvant on day 0, 30 and 60. On ELISA, it was observed that the mean absorbance values were significantly (p ≤ 0.01) higher up to 20 weeks post immunization in Group-I (purified antigen) compared to Group- II (unimmunized control). Further, the mean EPG values was lower in Group I (200.00 ± 40.82 to 400.00 ± 91.29) than Group II (2200.00 ± 108.01 to 5100.00 ± 169.56) and the percentage reduction in mean fecal egg counts was 88.50%. Similarly, the mean abomasal worm counts was lower in Group I (808.33 ± 78.29) than Group II (3280.00 ± 147.19) and the percentage reduction in mean abomasal worm count was 75.40%.

  11. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

    SciTech Connect

    Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

    2010-05-14

    To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

  12. Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein.

    PubMed

    Vijayanandraj, S; Yogita, M; Das, Amrita; Ghosh, Amalendu; Mandal, Bikash

    2013-09-01

    Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom.

  13. Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum.

    PubMed

    Deaglio, Silvia; Capobianco, Andrea; Calì, Angelita; Bellora, Francesca; Alberti, Federica; Righi, Luisella; Sapino, Anna; Camaschella, Clara; Malavasi, Fabio

    2002-11-15

    Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2 gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2(+) target cells and simultaneous negativity with TFR-1(+) cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern--it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2(+) cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

  14. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  15. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  16. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  17. A linker peptide with high affinity towards silica-containing materials.

    PubMed

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  18. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by...

  19. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by...

  20. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by...

  1. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by...

  2. Characterization of purified New Delhi metallo-β-lactamase-1.

    PubMed

    Thomas, Pei W; Zheng, Min; Wu, Shanshan; Guo, Hua; Liu, Dali; Xu, Dingguo; Fast, Walter

    2011-11-22

    New Delhi metallo-β-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used β-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-β-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of β-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 μM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 μM ZnSO(4), displaying k

  3. Apparatus and methods for purifying lead

    DOEpatents

    Tunison, Harmon M.

    2016-01-12

    Disclosed is an exemplary method of purifying lead which includes the steps of placing lead and a fluoride salt blend in a container; forming a first fluid of molten lead at a first temperature; forming a second fluid of the molten fluoride salt blend at a second temperature higher than the first temperature; mixing the first fluid and the second fluid together; separating the two fluids; solidifying the molten fluoride salt blend at a temperature above a melting point of the lead; and removing the molten lead from the container. In certain exemplary methods the molten lead is removed from the container by decanting. In still other exemplary methods the molten salt blend is a Lewis base fluoride eutectic salt blend, and in yet other exemplary methods the molten salt blend contains sodium fluoride, lithium fluoride, and potassium fluoride.

  4. Induction slag reduction process for purifying metals

    DOEpatents

    Traut, Davis E.; Fisher, II, George T.; Hansen, Dennis A.

    1991-01-01

    A continuous method is provided for purifying and recovering transition metals such as neodymium and zirconium that become reactive at temperatures above about 500.degree. C. that comprises the steps of contacting the metal ore with an appropriate fluorinating agent such as an alkaline earth metal fluosilicate to form a fluometallic compound, and reducing the fluometallic compound with a suitable alkaline earth or alkali metal compound under molten conditions, such as provided in an induction slag metal furnace. The method of the invention is advantageous in that it is simpler and less expensive than methods used previously to recover pure metals, and it may be employed with a wide range of transition metals that were reactive with enclosures used in the prior art methods and were hard to obtain in uncontaminated form.

  5. Method of separating and purifying gadolinium-153

    DOEpatents

    Bray, Lane A [Richland, WA; Corneillie, Todd M [Davis, CA

    2001-01-01

    The present invention is an improvement to the method of separating and purifying gadolinium from a mixture of gadolinium and europium having the steps of (a) dissolving the mixture in an acid; (b) reducing europium+3 to europium+2; and (c) precipitating the europium+2 with a sulfate ion in a superstoichiometric amount; wherein the improvement is achieved by using one or more of the following: (i) the acid is an anoic acid; (ii) the reducing is with zinc metal in the absence of a second metal or with an amount of the second metal that is ineffective in the reducing; (iii) adding a group IIA element after step (c) for precipitating the excess sulfate prior to repeating step (c); (iv) the sulfate is a sulfate salt with a monovalent cation; (v) adding cold europium+3 prior to repeating step (c).

  6. Some enzymatic activities associated with purified parapoxvirions.

    PubMed Central

    Caplen, H S; Holowczak, J A

    1983-01-01

    Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons. Images PMID:6188861

  7. Ozone emissions from a "personal air purifier".

    PubMed

    Phillips, T J; Bloudoff, D P; Jenkins, P L; Stroud, K R

    1999-01-01

    Ozone emissions were measured above a "personal air purifier" (PAP) designed to be worn on a lapel, shirt pocket, or neck strap. The device is being marketed as a negative ion generator that purifies the air. However, it also produces ozone within the person's immediate breathing zone. In order to assess worst-case potential human exposure to ozone at the mouth and nose, we measured ozone concentrations in separate tests at 1, 3, 5, and 6 in. above each of two PAPs in a closed office. One PAP was new, and one had been used slightly for 3 months. Temperature, relative humidity, atmospheric pressure, room ozone concentration, and outdoor ozone concentration also were measured concurrently during the tests. Average ozone levels measured directly above the individual PAPs ranged from 65-71 ppb at 6 in. above the device to 268-389 ppb at 1 in. above the device. Ozone emission rates from the PAPs were estimated to be 1.7-1.9 microg/minute. When house dust was sprinkled on the top grid of the PAPs, one showed an initial peak of 522 ppb ozone at 1 in., and then returned to the 200-400 ppb range. Room ozone levels increased by only 0-5 ppb during the tests. Even when two PAPs were left operating over a weekend, room ozone levels did not noticeably increase beyond background room ozone levels. These results indicate that this "PAP," even without significant background ozone, can potentially elevate the user's exposures to ozone levels greater than the health-based air quality standards for outdoor air in California (0.09 ppm, 1-hour average) and the United States (0.08 ppm, 8-hour average).

  8. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  9. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  10. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  11. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  12. Effect of polyclonal activators on cytokine production by blood cells and by malignant breast cancer cells.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  15. Histopathological and molecular features of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly.

    PubMed

    Del Giudice, Ilaria; Pileri, Stefano A; Rossi, Maura; Sabattini, Elena; Campidelli, Cristina; Starza, Irene Della; De Propris, Maria S; Mancini, Francesca; Perrone, Maria P; Gesuiti, Paola; Armiento, Daniele; Quattrocchi, Luisa; Tafuri, Agostino; Amendola, Angela; Mauro, Francesca R; Guarini, Anna; Foà, Robin

    2009-03-01

    Five cases of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly are reported; three were splenectomized. BCL2/IGH rearrangements were found in three cases; HLA-DRB1*07 in all. Bone marrow (BM) trephines showed a moderate lymphoid infiltrate with intrasinusoidal distribution resembling a splenic marginal-zone lymphoma. Splenic white pulp revealed an enlargement of the marginal-zone area; red pulp was infiltrated by the same lymphocytes engulfing the sinuses. Splenic and BM B-lymphocytes were CD79a(+)/CD20(+)/IgM(+)/IgD(+)/bcl-2(+)/CD27(+)/DBA.44(-)/CD31(-) and polyclonal by immunophenotype/polymerase chain reaction. PPBL features an expansion of splenic marginal-zone B-lymphocytes, which infiltrate BM sinusoids and circulate in the blood with no evidence of clonality, even in cases with progressive splenomegaly.

  16. Treatment of Uveitis by In Situ Administration of Ex Vivo-Activated Polyclonal Regulatory T Cells.

    PubMed

    Grégoire, Sylvie; Terrada, Céline; Martin, Gaelle H; Fourcade, Gwladys; Baeyens, Audrey; Marodon, Gilles; Fisson, Sylvain; Billiard, Fabienne; Lucas, Bruno; Tadayoni, Ramin; Béhar-Cohen, Francine; Levacher, Béatrice; Galy, Anne; LeHoang, Phuc; Klatzmann, David; Bodaghi, Bahram; Salomon, Benoît L

    2016-03-01

    CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell therapy is a promising approach for the treatment of autoimmune diseases. To be effective, Treg cells should be in an activated state in the target tissue. This can be achieved by systemic administration of Ag-specific Treg cells, which are difficult to produce in conditions that can be translated to the clinic. In this paper, we propose an alternative approach consisting of in situ injection of preactivated polyclonal Treg cells that would exert bystander suppression in the target tissue. We show that polyclonal Treg cells suppressed uveitis in mice as efficiently as Ag-specific Treg cells but only when preactivated and administered in the vitreous. Uveitis control was correlated with an increase of IL-10 and a decrease of reactive oxygen species produced by immune cell infiltrates in the eye. Thus, our results reveal a new mechanism of Treg cell-mediated suppression and a new Treg cell therapy approach.

  17. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    PubMed

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

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

    PubMed

    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

  19. Balancing speed and accuracy of polyclonal T cell activation: a role for extracellular feedback

    PubMed Central

    2012-01-01

    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. PMID:22925037

  20. Development of Polyclonal Antibodies for the Detection of Styrene Oxide Modified Proteins

    PubMed Central

    Yuan, Wei; Chung, Jouku; Gee, Shirley; Hammock, Bruce D.; Zheng, Jiang

    2008-01-01

    Styrene is widely used as one of the most important industrial materials for the production of synthetic rubbers, plastic, insulation, fiberglass, and automobile parts. Inhaled styrene has been reported to produce respiratory toxicity in humans and animals. Styrene oxide, a reactive metabolite of styrene formed via cytochrome P450 enzymes, has been reported to form covalent bonds with proteins, such as albumin and hemoglobin. Among all of the amino acids, cysteine is the most reactive amino acid to be modified by electrophilic species. The purpose of this study is to develop polyclonal antibodies for the detection of styrene oxide cysteinyl protein adducts. Two immunogens were designed, synthesized, and used to induce polyclonal antibodies in rabbits. Immune responses were observed from the raised antibodies by antiserum dilution tests. Competitive ELISA demonstrated that the resulting antibodies specifically recognized the styrene oxide-derived N-acetylcysteine adduct. Western blot results showed that the antibodies recognize styrene oxide-modified albumin. The binding was found to depend on the amount of protein adducts blotted and hapten loading in protein adducts. No cross reaction was observed from the native protein. Competitive Western blots further indicated that these antibodies specifically recognized styrene oxide cysteinyl–protein adducts. Immunoblots revealed the presence of several bands at a molecular weight ranging from 50 to 80 kDa in rat nasal mucosa treated with styrene. In conclusion, we successfully raised polyclonal antibodies to detect styrene oxide-derived protein/cysteine adducts. PMID:17266334

  1. Potent in vitro and in vivo effects of polyclonal anti-human-myeloma globulins

    PubMed Central

    Schieferdecker, Aneta; Shoshani, Ofer; Westner, Benedikt; Zipori, Dov; Fehse, Boris; Kröger, Nicolaus; Ayuk, Francis

    2016-01-01

    Introduction Multiple myeloma is still incurable in most cases. Polyclonal anti T lymphocyte globulins (ATG) have been reported to kill human myeloma cells in vitro and in mouse models. Methods Anti-human-myeloma globulins (AMG) were produced by immunizing rabbits with human myeloma cell lines RPMI-8226 (AMG-8226) or KMS-12-BM (AMG-12-BM). Cytotoxicity of the polyclonal antibodies was analyzed in vitro and in a xenograft NOD-SCID mouse model. Results Both AMG had stronger cytotoxicity against myeloma cells compared to ATG. In primary T cells, AMG-8226 showed greater complement-dependent cytotoxicity (CDC) than ATG, whereas complement-independent cytotoxicity did not differ. Effects on non-hematopoietic cell lines were also similar. Competitive blocking assays revealed fourfold more antibodies against CD38 in AMG-8226 compared to ATG. Low concentrations of AMG-8226 and ATG increased ADCC. At higher concentrations, ATG inhibited ADCC more potently than AMG-8226. Combinations of ATG and AMG-8226 with melphalan or bortezomib showed additive to synergistic cytotoxicity on myeloma cells. The cytotoxic effects of AMG and ATG were confirmed in the xenograft NOD-SCID mouse model. Conclusion Our data show more potent antimyeloma effects of AMG compared to ATG. These results lay the ground for the development of polyclonal antibodies for the treatment of multiple myeloma. PMID:27563813

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

    PubMed

    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.

  3. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  4. Contractions of affine spherical varieties

    SciTech Connect

    Arzhantsev, I V

    1999-08-31

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL{sub 2}-varieties are considered.

  5. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

    PubMed

    Thillaivinayagalingam, Pranavan; Newcombe, Anthony R; O'Donovan, Kieran; Francis, Richard; Keshavarz-Moore, Eli

    2007-12-01

    Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

  6. Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma.

    PubMed

    Nakano, Yasuko; Shoji, Ayako; Arakawa, Atsushi; Iizuka, Yumiko; Kikuchi, Yuriko; Kobayashi, Maya; Tobe, Takashi

    2010-01-01

    Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.

  7. Particle tracking microrheology of purified gastrointestinal mucins.

    PubMed

    Georgiades, Pantelis; Pudney, Paul D A; Thornton, David J; Waigh, Thomas A

    2014-04-01

    The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self-assembled semi-dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as η∼c(0.53±0.08) for MUC5AC and η∼c(0.53±0.06) for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (ce , the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, η∼c(3.92±0.38) for MUC5AC and η∼c(5.1±0.8) for MUC2. The dynamics of the self-assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred-fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi-dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol-gel transition in semi-dilute mucin solutions at neutral pH.

  8. Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

    PubMed

    Kosar-Hashemi, Behjat; Irwin, Jennifer A; Higgins, Jody; Rahman, Sadequr; Morell, Matthew K

    2006-05-01

    A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.

  9. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  10. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    SciTech Connect

    Baumgold, J.

    1986-05-01

    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  11. Hydrogen purifier module and method for forming the same

    SciTech Connect

    DeVries, Peter David

    2012-02-07

    A hydrogen purifier utilizing a hydrogen permeable membrane, and a gas-tight seal, where the seal is uses a low temperature melting point metal, which upon heating above the melting point subsequently forms a seal alloy with adjacent metals, where the alloy has a melting point above the operational temperature of the purifier. The purifier further is constructed such that a degree of isolation exists between the metal that melts to form the seal and the active area of the purifier membrane, so that the active area of the purifier membrane is not corrupted. A method of forming a hydrogen purifier utilizing a hydrogen permeable membrane with a seal of the same type is also disclosed.

  12. Isolation and characterization of an outer membrane protein of Salmonella paratyphi B: a mitogen and polyclonal activator of human B lymphocytes.

    PubMed Central

    Sager, S; Virella, G; Chen, W Y; Fudenberg, H H

    1984-01-01

    Salmonella paratyphi B (S. paratyphi B) has been previously characterized as a human T-independent polyclonal B cell activator. To define further the nature of the bacterial structure responsible for these properties, we studied the effects of autoclaving and enzyme treatment of S. paratyphi B on its stimulatory capacity. We found that both autoclaving and papain treatment decreased the ability of S. paratyphi B to induce B cell activation, while trypsin treatment did not affect this capacity. Neither type of treatment affected the binding of S. paratyphi B to lymphocytes, suggesting that binding and B cell stimulation are mediated by different structures. The observation that B cell stimulation was significantly reduced by papain treatment led us to attempt to purify membrane proteins so that we could investigate whether they shared the stimulating capacity of S. paratyphi B. A water-insoluble, 43-45,000 mol. wt. protein, rich in aspartic acid, glutamine, glycine, alanine and leucine, similar in mol. wt. and physicochemical chemical properties to the porins of other gram negative bacteria, was isolated and designated as outer membrane protein (OMP). This protein was equally efficient to S. paratyphi B in inducing T-independent B cell activation. By performing time-course studies of [3H]-thymidine incorporation we observed a burst of mitogenic activity after stimulation of PBL or purified B cells with both S. paratyphi B and OMP peaking at 48-96 hr of culture (compared to 96-120 hr for the PWM proliferation peak), and with a magnitude of roughly 10% of that observed after PWM stimulation. Given the fact that the proportion of B lymphocytes in PBL is 4-12%, it appears likely that the proliferation burst seen with S. paratyphi B and OMP corresponds to a mitogenic effect mainly restricted to the B cell population. Images Figure 1 PMID:6370841

  13. Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins.

    PubMed

    Sun, Yong; Feng, Xue; Qu, Jiao; Han, Wenqi; Liu, Zi; Li, Xu; Zou, Ming; Zhen, Yuhong; Zhu, Jie

    2015-06-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family. In this study, the whole extracellular domain gene of HER2 was amplified by RT-PCR from human breast cancer cell line SK-BR-3. The genes of membrane-distal region (A) and membrane proximal region (B) of HER2 extracellular domain were amplified from the cloned template, and then inserted into the expression vector pET-28a and pET-30a, respectively. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) cells and induced by isopropyl-b-D-thiogalactopyranoside (IPTG) for expression of proteins His-A and His-B. The expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The optimization of culture conditions led us to accomplish the recombinant protein induction with 1.0 mM IPTG at 37 °C for 8 h, and both proteins were expressed in the insoluble form. Both proteins were purified under the denaturing condition using Ni-NTA sepharose column. Balb/c mice were immunized with the purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by ELISA testing and had good specificity by western blot detection. The HER2 ECD proteins His-A and His-B could be expressed in E. coli and were suitable for production of high titer antibodies against HER2 ECD.

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

    PubMed

    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.

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

    SciTech Connect

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

    1982-05-01

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

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

    PubMed Central

    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.; McKee, Krisha; O’Dell, Sijy; Wang, Felicia; Binley, James M.; Connors, Mark; Haynes, Barton F.; 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. PMID:28052137

  17. Chemical binding affinity estimation using MSB

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Rauwerdink, Adam M.

    2011-03-01

    Binding affinity can be estimated in several ways in the laboratory but there is no viable way to estimate binding affinity in vivo without assumptions on the number of binding sites. Magnetic spectroscopy of nanoparticle Brownian motion, MSB, measures the rotational Brownian motion. The MSB signal is affected by nanoparticle binding affinity so it provides a mechanism to measure the chemical binding affinity. We present a possible mechanism to quantify the binding affinity and test that mechanism using viscous solutions.

  18. Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins.

    PubMed

    Fatima, Aiman; Husain, Qayyum

    2007-06-01

    Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

  19. Brucella fractions behave as nonspecific mitogens and polyclonal B-cell activators for human lymphocytes.

    PubMed Central

    Vendrell, J P; Rabesandratana, H; Huguet, M F; Cannat, A; Serre, A

    1985-01-01

    Two lipid-A-free fractions which were extracted from Brucella melitensis and were designated PI and SF stimulated human unsensitized mononuclear cells to proliferate and to secrete immunoglobulins. Both of these effects were observed in cultures of peripheral blood, tonsils, and cord blood lymphocytes. Neither B cells nor T cells alone proliferated in the presence of these fractions, whereas the proliferative response of T cells plus B cells was largely independent of accessory cells. Polyclonal activation was estimated by counting the cells which secreted immunoglobulins of different isotypes into culture supernatants. This phenomenon was strongly T dependent. PMID:3876286

  20. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  1. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  2. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  3. ELECTRON AFFINITIES OF INORGANIC RADICALS.

    DTIC Science & Technology

    energy in the latter compound is 110 kcals/mole, distinctly higher than in ammonia. Cyanogen (CN)2 and hydrocyanic acid (HCN) yield values for the...ions very readily, and the electron affinity is 49 kcals/mole. A comparison with the results from thiocyanic acid (HNCS) indicates that the H-N bond

  4. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  5. New method for purifying histidine-rich glycoprotein from human plasma redefines its functional properties.

    PubMed

    Patel, Kruti K; Poon, Ivan K H; Talbo, Gert H; Perugini, Matthew A; Taylor, Nicole L; Ralph, Troy J; Hoogenraad, Nicholas J; Hulett, Mark D

    2013-06-01

    Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ~1,000-fold purification with an overall yield of ~32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.

  6. Human Antibodies against a Purified Glucosylceramide from Cryptococcus neoformans Inhibit Cell Budding and Fungal Growth

    PubMed Central

    Rodrigues, Marcio L.; Travassos, Luiz R.; Miranda, Kildare R.; Franzen, Anderson J.; Rozental, Sonia; de Souza, Wanderley; Alviano, Celuta S.; Barreto-Bergter, Eliana

    2000-01-01

    A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a β-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the β-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal β-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro. PMID:11083830

  7. Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

    PubMed Central

    Callahan, L T; Martinez, D; Marburg, S; Tolman, R L; Galloway, D R

    1984-01-01

    For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C. Images PMID:6321348

  8. Enhanced Follicular Dendritic Cell-B Cell Interaction in HIV and SIV Infections and its Potential Role in Polyclonal B Cell Activation

    PubMed Central

    Lewis, Mark. G.; Kosco-Vilbois, Marie H.

    1998-01-01

    Human immunodeficiency virus (HIV) infections have been characterized by both polyclonal Bcell activation and enhanced responsiveness to B-cell growth factors on one hand and the loss of specific antibody (Ab) responses and refractoriness to the normal signals for B-cell activation on the other. Histopathological studies of lymph node from HIV- and simian immunodeficiency virus (SIV)-infected individuals have indicated initial follicular hyperplasia and the appearance of large irregular germinal centers that undergo progressive involution concomitant with follicular dendritic-cell (FDC) disruption. During this process, follicular dendritic-cell -enriched lymph-node-cell cultures exhibit increased ability to induce cluster formation (“in vitro germinal centers”), lymphocyte proliferation and antibody production compared to uninfected controls. This paper discusses how enhanced FDC-B-cell interaction within SIV-infected germinal centers may result in a reduced ability to select high-affinity B cells and alter the dynamics of antibodyproducing- cell and memory-cell generation resulting in the observed hyperactivity. PMID:9716906

  9. Enzyme-linked immunosorbent assay for human insulin-like growth factor-I using monoclonal and polyclonal antibodies with defined epitope recognition.

    PubMed

    Tamura, K; Kobayashi, M; Suzuki, S; Ishii, Y; Koyama, S; Yamada, H; Hashimoto, K; Niwa, M; Shibayama, F

    1990-05-01

    Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1-25 micrograms/l, compared with the range of 1-50 micrograms/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.

  10. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column.

  11. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet air purifier. 880.6500 Section... (CONTINUED) MEDICAL DEVICES GENERAL HOSPITAL AND PERSONAL USE DEVICES General Hospital and Personal Use Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet...

  12. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems.

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

    SciTech Connect

    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.

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

    USGS Publications Warehouse

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

    1989-01-01

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

  15. Polyclonal anti-Candida antibody improves phagocytosis and overall outcome in zebrafish model of disseminated candidiasis.

    PubMed

    Bergeron, Audrey C; Barker, Sarah E; Brothers, Kimberly M; Prasad, Brinda C; Wheeler, Robert T

    2017-03-01

    Fungal infections are a major cause of animal and plant morbidity and mortality worldwide. Effective biological therapeutics could complement current antifungal drugs, but understanding of their in vivo mechanisms has been hampered by technical barriers to intravital imaging of host-pathogen interactions. Here we characterize the fungal infection of zebrafish as a model to understand the mechanism-of-action for biological antifungal therapeutics through intravital imaging of these transparent animals. We find that non-specific human IgG enhances phagocytosis by zebrafish phagocytes in vivo. Polyclonal anti-Candida antibodies enhance containment of fungi in vivo and promote survival. Analysis suggests that early phagocytic containment is a strong prognostic indicator for overall survival. Although polyclonal anti-Candida antibodies protect against disease, this is not necessarily the case for individual monoclonal anti-Candida antibodies. Thus, the zebrafish appears to provide a useful model host for testing if a biological therapeutic promotes phagocytosis in vivo and enhances protection against candidemia.

  16. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human american visceral leishmaniasis.

    PubMed Central

    Galvão-Castro, B; Sá Ferreira, J A; Marzochi, K F; Marzochi, M C; Coutinho, S G; Lambert, P H

    1984-01-01

    In a prospective study, serum and plasma samples from 17 patients with kala-azar were collected in Rio de Janeiro. High levels of immune complexes (IC) were detected in serum by means of 125I-Clq and conglutinin binding assays. The Clq binding material had a sedimentation coefficient of 19-25S, as determined by ultracentrifugation on sucrose gradient. Plasma levels of C3 and C3 breakdown products were measured and the C3d levels were increased in six out of 11 patients. The occurrence of polyclonal B cell activation was suggested by (a) a marked increase of serum IgG and IgM levels and (b) of the presence of antibodies against various proteins and haptens (SRBC, DNP-BSA, FITC-BSA,KLH). There was a close association between the presence of IC and anti-immunoglobulin antibodies. Anti-smooth muscle antibodies were also observed. These data are consistent with a major role of polyclonal B cell activation in the induction of IC during visceral leishmaniasis. PMID:6424987

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

    PubMed

    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.

  18. Portable self-contained solar powered water purifier

    SciTech Connect

    Sherman, M.

    1991-10-22

    This patent describes a portable self-contained solar powered water purifier. It comprises housing means for buoyantly supporting the purifier; solar cell means supported by the housing means above water to be treated; purification means depending from the housing means so as to be positioned in water to be treated and including sacrificial anode means providing ionized metallic ions for purifying the water and cathode means providing abstraction of electrons to facilitate the release of oxygen into the water; means for electrically connecting the solar cell means to the electrolytic purification means to enable the electrolytic purification means to purify water when the purifier is placed therein; and diode means for preventing reverse current flow between the anode means and cathode means.

  19. Structure of Greyhound hemoglobin: origin of high oxygen affinity.

    PubMed

    Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

    2011-05-01

    This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

  20. Quantifying Bioactive P Pools in Fertilized and Manure-Amended Soils by Purified Phytic-Acid High Affinity Aspergillus phosphohydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In areas of intensive animal agriculture, repeated land application of manure resulted in elevated soil concentrations of inorganic and organic phosphorus (P) myo-Inositol hexaphosphate, lower phosphomonoesters are the most abundant organic P compounds. Such P-enriched soils are potential pollution...

  1. Dephosphorylation and Quantification of Organic Phosphorus in Poultry Litter by Purified Phytic-Acid High Affinity Aspergillus Phosphohydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extracellular phosphohydrolases have received increased attention as they are shown to influence the behavior and bioavailability of environmental phosphorus, which pose risks of impairment of surface waters and degradation of sensitive aquatic ecosystems. Induction and culture of five strains of A...

  2. Collagen-bound von Willebrand factor has reduced affinity for factor VIII.

    PubMed

    Bendetowicz, A V; Wise, R J; Gilbert, G E

    1999-04-30

    von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

  3. Polyclonal free light chain of Ig may interfere with interpretation of monoclonal free light chain κ/λ ratio.

    PubMed

    Levinson, Stanley S

    2010-01-01

    There is controversy about whether a sensitive assay for the serum Ig free light chain (FLC) κ/λ ratio can replace urine immunofixation electrophoresis (UIFE). This report describes two untreated patients in whom monoclonal FLCs were identified in urine despite normal serum FLC κ/λ ratios. Unlike the classical serum electrophoretic patterns in multiple myeloma, both serum samples showed adequate amounts of polyclonal Ig. The most likely explanation is a masking effect by polyclonal FLC on the serum κ/λ ratio when sufficient concentrations of polyclonal FLC exist. These cases illustrate this likely effect and attest to the continued importance of UIFE for initial screening of patients for Bence-Jones protein.

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

    PubMed Central

    Bounds, Callie E.; Kwilas, Steven A.; Kuehne, Ana I.; Brannan, Jennifer M.; Bakken, Russell R.; Dye, John M.; Hooper, Jay W.; Dupuy, Lesley C.; Ellefsen, Barry; Hannaman, Drew; Wu, Hua; Jiao, Jin-an; Sullivan, Eddie J.; Schmaljohn, Connie S.

    2015-01-01

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

    PubMed

    Bounds, Callie E; Kwilas, Steven A; Kuehne, Ana I; Brannan, Jennifer M; Bakken, Russell R; Dye, John M; Hooper, Jay W; Dupuy, Lesley C; Ellefsen, Barry; Hannaman, Drew; Wu, Hua; Jiao, Jin-an; Sullivan, Eddie J; Schmaljohn, Connie S

    2015-01-01

    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

  6. RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

    ERIC Educational Resources Information Center

    Bailey, Cheryl P.

    2009-01-01

    This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

  7. Highly efficient and low-cost purification of lysozyme: a novel tris(hydroxymethyl)aminomethane immobilized affinity column.

    PubMed

    Quan, Li; Cao, Qing; Li, Zhiyu; Li, Na; Li, Kean; Liu, Feng

    2009-03-01

    A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287 U/mg (activity of commercial LZM was 8171 U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white.

  8. Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-01-01

    Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

  9. Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

    PubMed Central

    Aubin, J E; Subrahmanyan, L; Kalnins, V I; Ling, V

    1976-01-01

    Several rabbit antisera have been prepared against reduced and alkylated, electrophoretically purified tubulin isolated from chick brain. These antisera give a single precipitin line in Ouchterlony double diffusion plates when tested against partially purified tubulin, and label specifically microtubule- and tubulin-containing structures, such as mitotic spindles, cilia, and vinblastine-induced crystals, in a variety of cells. The same antisera also display the unique ability to stimulate the colchicine-binding activity of tubulin preparations from chick brain and Chinese hamster ovary tissue culture cells. This specific stimulation of colchicine binding activity is also obtained with the gamma globulin fractions purified by ammonium sulfate precipitation of these antisera. Images PMID:57619

  10. Vacuolar H(+)-pyrophosphatase purified from pear fruit.

    PubMed

    Suzuki, Y; Kanayama, Y; Shiratake, K; Yamaki, S

    1999-02-01

    A vacuolar H(+)-translocating inorganic pyrophosphatase was purified from pear fruit through selective detergent treatments, Superose 6 and Mono Q column chromatography. The specific activity of the purified enzyme was 850 mumol h-1 mg protein-1. The Mr of V-PPase was 66 kDa by SDS-PAGE and the polypeptide cross-reacted with the antiserum against V-PPase of mung bean. The purified V-PPase was stimulated by potassium and inhibited by calcium and N, N'-dicyclohexylcarbodiimide.

  11. Study of gas purifiers for the CMS RPC detector

    NASA Astrophysics Data System (ADS)

    Benussi, L.; Bianco, S.; Colafranceschi, S.; Fabbri, F. L.; Felli, F.; Ferrini, M.; Giardoni, M.; Greci, T.; Paolozzi, A.; Passamonti, L.; Piccolo, D.; Pierluigi, D.; Russo, A.; Saviano, G.; Buontempo, S.; Cimmino, A.; de Gruttola, M.; Fabozzi, F.; Iorio, A. O. M.; Lista, L.; Paolucci, P.; Baesso, P.; Belli, G.; Pagano, D.; Ratti, S. P.; Vicini, A.; Vitulo, P.; Viviani, C.; Guida, R.; Sharma, A.

    2012-01-01

    The CMS RPC muon detector utilizes a gas recirculation system called closed loop (CL) to cope with large gas mixture volumes and costs. A systematic study of CL gas purifiers has been carried out over 400 days between July 2008 and August 2009 at CERN in a low-radiation test area, with the use of RPC chambers with currents monitoring, and gas analysis sampling points. The study aimed to fully clarify the presence of pollutants, the chemistry of purifiers used in the CL, and the regeneration procedure. Preliminary results on contaminants release and purifier characterization are reported.

  12. Polyclonal and monoclonal IgG binding on Protein A resins - Evidence of competitive binding effects.

    PubMed

    Weinberg, Justin; Zhang, Shaojie; Crews, Gillian; Healy, Edward; Carta, Giorgio; Przybycien, Todd

    2017-03-14

    Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, 'shrinking core' behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. This article is protected by copyright. All rights reserved.

  13. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    PubMed Central

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. Catalase<-->Abeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro. PMID:10567208

  14. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  15. Transient abnormal myelopoiesis in a phenotypically normal newborn with polyclonal trisomy 21.

    PubMed

    Corazza, Francesco; Astolfi, Annalisa; Libri, Virginia; Franzoni, Monica; Serravalle, Salvatore; Alessandroni, Rosina; Melchionda, Fraia; Pession, Andrea

    2014-06-01

    We report a rare case of transient abnormal myelopoiesis (TAM) in a phenotypically normal neonate. The presence of a palpable hepatomegaly prompted in-depth laboratory tests, which revealed the presence of severe hyperleukocytosis, with blast cells present in a peripheral blood smear. Although no signs of Down syndrome were present, we suspected TAM. Further analysis identified a mutation in GATA1 along with the unique finding of two different trisomic cell lines, detected upon karyotyping; one with trisomy 21 only, and one with trisomies 21 and 22, which was present in a subpopulation of peripheral blood cells. These genetic abnormalities disappeared by the age of 6 months. The presence of two different trisomic clones may be an evidence of the polyclonal nature of TAM in this patient.

  16. Polyclonal outbreak of bacteremia caused by Burkholderia cepacia complex and the presumptive role of ultrasound gel.

    PubMed

    Nannini, Esteban C; Ponessa, Adriana; Muratori, Rosa; Marchiaro, Patricia; Ballerini, Viviana; Flynn, Luis; Limansky, Adriana S

    2015-01-01

    A nosocomial polyclonal outbreak associated to bacteremia caused by different Burkholderia cepacia complex (BCC) species and clones is reported. Molecular characterization identified Burkholderia stabilis, Burkholderia contaminans, and Burkholderia ambifaria among BCC isolates obtained from patients in neonatal and adult intensive care units. BCC was also isolated from an intrinsically contaminated ultrasound gel, which constituted the presumptive BCC source. Prior BCC outbreak related to contaminated ultrasound gels have been described in the setting of transrectal prostate biopsy. Outbreak caused strains and/or clones of BCC have been reported, probably because BCC are commonly found in the natural environment; most BCC species are biofilm producers, and different species may contaminate an environmental source. The finding of multiple species or clones during the analysis of nosocomial BCC cases might not be enough to reject an outbreak from a common source.

  17. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

  18. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-04

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

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

    PubMed Central

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

    2010-01-01

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

  20. Detection of acute inflammation with /sup 111/In-labeled nonspecific polyclonal IgG

    SciTech Connect

    Fischman, A.J.; Rubin, R.H.; Khaw, B.A.; Callahan, R.J.; Wilkinson, R.; Keech, F.; Nedelman, M.; Dragotakes, S.; Kramer, P.B.; LaMuraglia, G.M.

    1988-10-01

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

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

    PubMed

    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.

  2. The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

    PubMed

    Leenaars, P P; Hendriksen, C F; de Leeuw, W A; Carat, F; Delahaut, P; Fischer, R; Halder, M; Hanly, W C; Hartinger, J; Hau, J; Lindblad, E B; Nicklas, W; Outschoorn, I M; Stewart-Tull, D E

    1999-01-01

    This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes

  3. Replication of adenovirus type 4 DNA by a purified fraction from infected cells.

    PubMed Central

    Temperley, S M; Hay, R T

    1991-01-01

    An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication. Images PMID:1829516

  4. STUDIES ON MEDIATOR PRODUCTION BY HIGHLY PURIFIED HUMAN T AND B LYMPHOCYTES

    PubMed Central

    Rocklin, Ross E.; MacDermott, Richard P.; Chess, Leonard; Schlossman, Stuart F.; David, John R.

    1974-01-01

    Highly purified populations of T and B lymphocytes obtained by affinity column separation were stimulated by antigen and their ability to produce two mediators, migration inhibitory factor (MIF) and lymphocyte mitogenic factor (LMF) was assessed. Both T- and B-cell populations made MIF; the production of MIF was antigen-specific using purified protein derivative of tuberculin, streptokinase-streptodornase, and Candida antigens. The MIF activity from both populations could not be attributed to antigen-antibody complexes as the inhibitory activity eluted from Sephadex G-100 columns in the same region corresponding to mol wt 23,000 daltons. Further studies indicate that the T cells producing MIF are proliferating cells whereas the B cells producing this mediator are not. In contrast, LMF was made only by T cells and not B cells when these populations were stimulated by antigen. The LMF induced the [3H]thymidine incorporation into both T and B cells obtained from donors lacking sensitivity to the antigens used to elicit the factor. Chromatographic studies indicate that LMF eluted from Sephadex G-100 in a fraction of mol wt 23,000 daltons where MIF is also found; however, since B cells produce MIF but not LMF, these two factors appear to be distinct from one another. Some of the implications of these findings are discussed. The explanation for the production or lack of production of MIF by lymphocytes obtained from patients with immunodeficiency disorders requires reinterpretation. PMID:4608321

  5. In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

    PubMed Central

    Arora, D J; Houde, M; Justewicz, D M; Mandeville, R

    1984-01-01

    The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins. PMID:6387178

  6. Identification of the Cardiac Beta-Adrenergic Receptor Protein: Solubilization and Purification by Affinity Chromatography

    PubMed Central

    Lefkowitz, Robert J.; Haber, Edgar; O'Hara, Donald

    1972-01-01

    A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified β-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 × 105 liters/mol (as compared to two association constants, 107 and 106 liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for β-adrenergic drugs and antagonists. Images PMID:4507606

  7. Polyclonal B-cell activation induced by extracts of Gram-negative bacteria isolated from periodontally diseased sites.

    PubMed Central

    Bick, P H; Carpenter, A B; Holdeman, L V; Miller, G A; Ranney, R R; Palcanis, K G; Tew, J G

    1981-01-01

    The objective of this research was to determine whether gram-negative bacteria frequently isolated from periodontally diseased sites contained polyclonal B-cell activators. Polyclonal B-cell activation, which results in nonspecific activation of multiple B-cell clones was analyzed by a hemolysis-in-gel assay designed to detect a broad range of antibody specificities. Extracts from numerous bacterial strains, including Bacteroides gingivalis, Bacteroides melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. intermedius, Fusobacterium nucleatum, Selenomonas sputigena, Capnocytophaga ochracea, and Actinobacillus actinomycetemcomitans, were tested. Extracts of the above organisms were found to stimulate polyclonal antibody responses in cultures of normal human peripheral blood lymphocytes, although the magnitude of stimulation varied among the extracts. Optimal antibody-forming cell responses were found at stimulator doses between 5 and 1,000 micrograms/ml. We conclude that the resident gram-negative subgingival flora associated with periodontal lesions possesses potent polyclonal B-cell activators. These activators may contribute to disease pathogenesis by inducing B lymphocytes to produce antibody, osteolytic factors, or both and possibly other mediators of inflammation. PMID:6975240

  8. The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme.

    PubMed

    Glass, G A; DeLisle, D M; DeTogni, P; Gabig, T G; Magee, B H; Markert, M; Babior, B M

    1986-10-05

    A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.

  9. Substrate Specificity of Purified Recombinant Chicken β-Carotene 9',10'-Oxygenase (BCO2).

    PubMed

    Dela Seña, Carlo; Sun, Jian; Narayanasamy, Sureshbabu; Riedl, Kenneth M; Yuan, Yan; Curley, Robert W; Schwartz, Steven J; Harrison, Earl H

    2016-07-08

    Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8'-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.

  10. Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus.

    PubMed

    Giraldo, Marielle Aleixo; Gonçalves, Heloísa Bressan; Furriel, Rosa Dos Prazeres Melo; Jorge, João Atílio; Guimarães, Luis Henrique Souza

    2014-05-01

    The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

  11. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-08

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  12. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

    PubMed

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-11-14

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

  13. Triazine dyes as inhibitors and affinity ligands of glycosyltransferases.

    PubMed

    Kamińska, J; Dziecioł, J; Kościelak, J

    1999-11-01

    The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activity of a purified preparation of alpha1,6fucosyltransferase (GDP-L-fucose: N-acetyl beta-glucosaminide 6-alpha-L-fucosyltransferase, EC 2.4.1.68) from human blood platelets. Cibacron Blue 3GA and Reactive Red 120 were examined for the nature of the inhibition and both were found to be competitive inhibitors of the enzyme, with Ki = 11 microM and 2 microM, respectively. The two dyes inhibited also serum glycosyltransferases: alpha1,2fucosyltransferase (GDP-L-fucose: beta-D-galactosyl-R2-alpha-L-fucosyltransferase, EC 2.4.1.69), beta1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase, EC 2.4.1.90) and beta1,3N-acetylglucosaminyltransferase (UDP-GlcNAc: 4-beta-D-galactosyl-D-glucose). Cibacron Blue 3GA was a more effective inhibitor of the glycosyltransferases that use UDP-linked sugar donors than Reactive Red 120 while the latter was a stronger inhibitor of the fucosyltransferases that use GDP-linked donor. All four glycosyltransferases could be affinity purified on Cibacron Blue 3GA-Agarose columns. The order of elution of glycosyltransferases from the columns with solutions of 0.25-1.0 M potassium iodide also depended upon the structure of nucleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransferases. The main advantages of the use of triazine dyes as affinity ligands for isolation of glycosyltransferases are their universal applicability regardless of enzyme specificity, low cost, and insensitivity to high concentration of other proteins present in the solution.

  14. Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria A. V.

    2016-12-01

    As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.

  15. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  16. Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

    PubMed

    Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

    2006-01-01

    Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

  17. Magnetic particles as affinity matrix for purification of antithrombin

    NASA Astrophysics Data System (ADS)

    Mercês, A. A. D.; Maciel, J. C.; Carvalho Júnior, L. B.

    2015-11-01

    Immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials. It provides advantages: enhanced stability and easy separation. In this work two different magnetic composites were synthesized (MAG-PANI-HS and mDAC-HS) to human antithrombin purification. The magnetic particles (MAG) were obtained by co-precipitation method of iron salts II and III and subsequently coated with polyaniline (MAG-PANI particles). Dacron (polyethylene terephthalate) suffered a hydrazinolysis reaction to obtain a powder (Dacron hydrazide) which was subsequently magnetized (mDAC particles) also by co-precipitation method. Heparan sulfate (HS) was immobilized to MAG-PANI and mDAC retained respectively 35μg and 38.6μg per of support. The magnetic composite containing HS immobilized (MAG-PANI-HS and mDAC-HS) was incubated with human blood plasma (1mL) and then washed with NaCl gradients. Electrophoresis of proteins present in eluates showed bands of antithrombin (58kDa). A reduction in the antithrombin activity was detected in plasma that were incubated in the composites magnetic with HS immobilized, suggesting that the antithrombin was removed of the human blood plasma and then purified. Therefore, the above results suggest that both preparations: MAG-PANI-HS and mDAC-HS are able to affinity purify antithrombin, an important component of blood coagulation.

  18. Affinity membrane introduction mass spectrometry

    SciTech Connect

    Xu, C.; Patrick, J.S.; Cooks, R.G. )

    1995-02-15

    A new technique, affinity membrane introduction mass spectrometry, is described. In this method, a chemically modified membrane is used to selectively adsorb analytes bearing a particular functional group and concentrate them from solution. Release of the bound analyte results in its transfer across the membrane and allows it to be monitored mass spectrometrically, using, in the present case, a benchtop ion trap instrument. Alkylamine-modified cellulose membranes are used to bind substituted benzaldehydes through imine formation at high pH. Release of the bound aldehyde is achieved by acid hydrolysis of the surface-bound imine. Benzaldehyde is detected with excellent specificity at 10 ppm in a complex mixture using this method. Using the enrichment capability of the membrane, a full mass spectrum of benzaldehyde can be measured at a concentration of 10 ppb. The behavior of a variety of other aldehydes is also discussed to illustrate the capabilities of the method. 21 refs., 5 figs., 2 tabs.

  19. Bioengineering of bacteria to assemble custom-made polyester affinity resins.

    PubMed

    Hay, Iain D; Du, Jinping; Burr, Natalie; Rehm, Bernd H A

    2015-01-01

    Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.

  20. Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

    PubMed Central

    Hay, Iain D.; Du, Jinping; Burr, Natalie

    2014-01-01

    Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

  1. Antisymmetric tensor generalizations of affine vector fields

    PubMed Central

    Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-01-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank-p antisymmetric affine tensor fields in n-dimensions is bounded by (n + 1)!/p!(n − p)!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes. PMID:26858463

  2. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, Kenneth Sherman

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  3. Strong purifying selection at genes escaping X chromosome inactivation.

    PubMed

    Park, Chungoo; Carrel, Laura; Makova, Kateryna D

    2010-11-01

    To achieve dosage balance of X-linked genes between mammalian males and females, one female X chromosome becomes inactivated. However, approximately 15% of genes on this inactivated chromosome escape X chromosome inactivation (XCI). Here, using a chromosome-wide analysis of primate X-linked orthologs, we test a hypothesis that such genes evolve under a unique selective pressure. We find that escape genes are subject to stronger purifying selection than inactivated genes and that positive selection does not significantly affect the evolution of these genes. The strength of selection does not differ between escape genes with similar versus different expression levels in males versus females. Intriguingly, escape genes possessing Y homologs evolve under the strongest purifying selection. We also found evidence of stronger conservation in gene expression levels in escape than inactivated genes. We hypothesize that divergence in function and expression between X and Y gametologs is driving such strong purifying selection for escape genes.

  4. Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model.

    PubMed

    Wising, Catharina; Svensson, Liselott A; Ahmed, Hinda J; Sundaeus, Vivianne; Ahlman, Karin; Jonsson, Ing-Marie; Mölne, Lena; Lagergård, Teresa

    2002-08-01

    The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10(6)CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100microg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed

  5. Functional analysis and regulation of purified connexin hemichannels

    PubMed Central

    Fiori, Mariana C.; Reuss, Luis; Cuello, Luis G.; Altenberg, Guillermo A.

    2014-01-01

    Gap-junction channels (GJCs) are aqueous channels that communicate adjacent cells. They are formed by head-to-head association of two hemichannels (HCs), one from each of the adjacent cells. Functional HCs are connexin hexamers composed of one or more connexin isoforms. Deafness is the most frequent sensineural disorder, and mutations of Cx26 are the most common cause of genetic deafness. Cx43 is the most ubiquitous connexin, expressed in many organs, tissues, and cell types, including heart, brain, and kidney. Alterations in its expression and function play important roles in the pathophysiology of very frequent medical problems such as those related to cardiac and brain ischemia. There is extensive information on the relationship between phosphorylation and Cx43 targeting, location, and function from experiments in cells and organs in normal and pathological conditions. However, the molecular mechanisms of Cx43 regulation by phosphorylation are hard to tackle in complex systems. Here, we present the use of purified HCs as a model for functional and structural studies. Cx26 and Cx43 are the only isoforms that have been purified, reconstituted, and subjected to functional and structural analysis. Purified Cx26 and Cx43 HCs have properties compatible with those demonstrated in cells, and present methodologies for the functional analysis of purified HCs reconstituted in liposomes. We show that phosphorylation of serine 368 by PKC produces a partial closure of the Cx43 HCs, changing solute selectivity. We also present evidence that the effect of phosphorylation is highly cooperative, requiring modification of several connexin subunits, and that phosphorylation of serine 368 elicits conformational changes in the purified HCs. The use of purified HCs is starting to provide critical data to understand the regulation of HCs at the molecular level. PMID:24611052

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

    PubMed Central

    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

  7. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  8. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  9. 76 FR 29194 - Purified Carboxymethylcellulose From Mexico and Sweden: Revocation of Antidumping Duty Orders

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-20

    ... International Trade Administration Purified Carboxymethylcellulose From Mexico and Sweden: Revocation of... duty orders on purified carboxymethylcellulose from Mexico and Sweden. Pursuant to section 751(c) of... of the existing antidumping duty orders on purified carboxymethylcellulose from Mexico and...

  10. 75 FR 57815 - Purified Carboxymethylcellulose From Finland, Mexico, Netherlands, and Sweden

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-22

    ... COMMISSION Purified Carboxymethylcellulose From Finland, Mexico, Netherlands, and Sweden AGENCY: United... antidumping duty orders on purified carboxymethylcellulose from Finland, Mexico, Netherlands, and Sweden... antidumping duty orders on purified carboxymethylcellulose from Finland, Mexico, Netherlands, and Sweden...

  11. Human secondary lymphoid organs typically contain polyclonally-activated proliferating regulatory T cells.

    PubMed

    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.

  12. Development and characterization of polyclonal peptide antibodies for the detection of Yellow fever virus proteins.

    PubMed

    Stock, N K; Escadafal, C; Achazi, K; Cissé, M; Niedrig, M

    2015-09-15

    There is still a considerable need for development of new tools and methods detecting specific viral proteins for the diagnosis and pathogenesis study of the Yellow fever virus (YFV). This study aimed to develop and characterize polyclonal peptide antisera for detection of YFV-C and -NS1 proteins. The antisera were used further to investigate NS1 protein expression during YFV infection in mammalian cells. YFV target proteins were detected by all antisera in western blot and immunofluorescence assays. No cross-reactivity was observed with Dengue virus, West Nile virus, Tick-borne encephalitis virus and Japanese encephalitis virus. Nuclear localization of the YFV-C protein was demonstrated for the first time. Experiments investigating NS1 expression suggested a potential use of the YFV-NS1 antisera for development of diagnostic approaches targeting the secreted form of the NS1 protein. The antisera described in this study offer new possibilities for use in YFV research and for the development of novel diagnostic tests.

  13. Detection, characterization and quantitation of coxsackievirus A16 using polyclonal antibodies against recombinant capsid subunit proteins.

    PubMed

    Liu, Qingwei; Ku, Zhiqiang; Cai, Yicun; Sun, Bing; Leng, Qibin; Huang, Zhong

    2011-04-01

    Coxsackievirus A16 (CVA16), together with enterovirus type 71 (EV71), is responsible for most cases of hand, foot and mouth disease (HFMD) worldwide. Recent findings suggest that the recombination between CVA16 and EV71, and co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years. Thus, for CVA16, further understanding of its virology, epidemiology and development of diagnostic tests and vaccines are of importance. The present study aimed to develop reagents and protocols for the detection, characterization and quantitation of CVA16. Recombinant CVA16 capsid subunit proteins VP0, VP3 and truncated VP1, were produced in Escherichia coli and used to immunize guinea pigs to generate polyclonal antibodies. The resultant three antisera detected specifically CVA16 propagated in Vero cells by immunostaining, ELISA and Western blotting. The antisera was used to show that CVA16 capsids were composed of correctly processed VP0, VP1 and VP3 subunits, and were present in the form of efficiently assembled particles. A method for the quantitation of the yield of CVA16 in Vero cells was established based on a Western blotting protocol using the recombinant VP0 as a reference standard and anti-VP0 as the detection antibody. This study shows the development and validation of reagents and methods, for qualitative and quantitative determination of CVA16, which are essential for the development of vaccines.

  14. Immunoperoxidase detection of ophidian paramyxovirus in snake lung using a polyclonal antibody.

    PubMed

    Homer, B L; Sundberg, J P; Gaskin, J M; Schumacher, J; Jacobson, E R

    1995-01-01

    In a retrospective study of proliferative interstitial pneumonia in viperid and nonviperid snakes, formalin-fixed, paraffin-embedded lungs from 52 snakes were screened for immunohistochemical reactivity to ophidian paramyxovirus. All snakes were from zoological collections that experienced mortalities attributed to paramyxovirus infection. Of the 52 snakes, 47 had pulmonary lesions compatible with ophidian paramyxovirus infection. Histologic changes in affected lungs included hyperplasia and hypertrophy of septal and faveolar epithelial cells, loss of ciliated cells, mixed leukocytic interstitial infiltrates, fibrinonecrotic exudate in the lumen of proximal and distal faveolar compartments, and occasional epithelial syncytial cell formation or intraepithelial eosinophilic intracytoplasmic inclusions. Lungs were immunohistochemically stained for paramyxovirus antigens by utilizing rabbit polyclonal antibodies against a paramyxovirus isolate from a black mamba (Dendroaspis polyepis polyepis). Virus infection in 6 snakes was confirmed by virus isolation from frozen lung tissue. Of the 6 lungs from which paramyxovirus was isolated, 5 lungs stained positively for viral antigens utilizing antisera to the black mamba isolate. Altogether, 36 lungs stained positively for paramyxovirus antigens. There was multifocal to diffuse linear staining of the lumenal surface of faveolar epithelium, and there were multiple foci of granular cytoplasmic staining. Immunohistochemical staining of formalin-fixed lungs from snakes with proliferative interstitial pneumonia was helpful as a routine diagnostic test for substantiating a diagnosis of ophidian paramyxovirus infection.

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

    PubMed Central

    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

  16. Production and application of polyclonal and monoclonal antibodies against Spiroplasma eriocheiris

    PubMed Central

    Zhang, Ying; Bao, Haixun; Miao, Fengqin; Peng, Yaqin; Shen, Yuqing; Gu, Wei; Meng, Qingguo; Wang, Wen; Zhang, Jianqiong

    2015-01-01

    A new species of spiroplasma, Spiroplasma eriocheiris (S. eriocheiris), was identified as a lethal pathogen of tremor disease (TD) in Chinese mitten crab recently. In order to acquire appropriate biological and diagnostic tools for characterizing this newly discovered pathogen, 5 monoclonal antibodies (mAbs) and a polyclonal antibody (pAb) against S. eriocheiris were produced. Among the mAbs, 6F5, 7C8 and 12H5 lead to the deformation of S. eriocheiris. A peptide sequence, YMRDMQSGLPRY was identified as a mimic motif of MreB that is the cell shape determining protein of S. eriocheiris interacting with 3 mAbs. Furthermore, a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of S. eriocheiris was established using the mAb and pAb we prepared. It detected as low as 0.1 μg/mL of S. eriocheiris. No cross-reaction was observed with three other common bacteria (Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis) and the hemolymph samples of healthy Eriocheir sinensis. Collectively, our results indicated that the mAbs and pAb we prepared could be used in the analysis of S. eriocheiris membrane proteins mimotope and development of a diagnostic kit for S. eriocheiris infections. PMID:26639364

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

    PubMed

    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.

  18. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    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 (Flu) 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. PMID:23049532

  19. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  20. Selection and characterization of human respiratory syncytial virus escape mutants resistant to a polyclonal antiserum raised against the F protein.

    PubMed

    Tomé, Lorena; Frabasile, Sandra; Candia, Claudia; Pittini, Alvaro; Farina, Natalia; Melero, José Antonio; Arbiza, Juan

    2012-06-01

    A human respiratory syncytial virus (HRSV) neutralization escape mutant was obtained after 56 serial passages in the presence of a polyclonal antiserum raised against the F protein. Nucleotide sequence analysis of this escape mutant virus revealed two amino acid substitutions: Asn268Ile and Val533Met. When this virus was allowed to grow in the absence of the anti-F polyclonal serum, only the mutation Asn268Ile was stably maintained. Both the double and single escape mutant viruses lost reactivity with mAbs belonging to antigenic site II of the fusion protein of RSV. Mutation Asn268Ile has already been reported in RS viruses that are resistant to mAbs 47F and 11 and palivizumab (PZ). We have thus identified a novel mutation (Val533Met) in the transmembrane domain of the F protein that was selected under immune pressure.

  1. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Medical ultraviolet water purifier. 880.6710 Section 880.6710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL HOSPITAL AND PERSONAL USE DEVICES General Hospital and Personal Use Miscellaneous Devices § 880.6710...

  2. Obtaining partial purified xylose reductase from Candida guilliermondii

    PubMed Central

    Tomotani, Ester Junko; de Arruda, Priscila Vaz; Vitolo, Michele; de Almeida Felipe, Maria das Graças

    2009-01-01

    The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40%, 7% and 67%, respectively. PMID:24031408

  3. Small-Scale Evaluation of the Expeditionary Unit Water Purifier

    EPA Science Inventory

    The U.S. EPA’s Technology Testing and Evaluation Program has been charged by EPA to evaluate the performance of commercially available water security-related technologies. Throughout 2007, an evaluation the Expeditionary Unit Water Purifier (EUWP), a mobile water treatment techno...

  4. Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA

    PubMed Central

    Stewart, James Bruce; Freyer, Christoph; Elson, Joanna L; Wredenberg, Anna; Cansu, Zekiye; Trifunovic, Aleksandra; Larsson, Nils-Göran

    2008-01-01

    There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity. PMID:18232733

  5. Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes.

    PubMed

    Pimplikar, S W; Reithmeier, R A

    1986-07-25

    Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

  6. Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

    PubMed Central

    Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

    2002-01-01

    Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

  7. Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue.

    PubMed

    Wilson, J E

    1989-01-01

    The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.

  8. New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes.

    PubMed

    Melissis, S C; Rigden, D J; Clonis, Y D

    2001-05-11

    Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg

  9. Analysis of in vitro polyclonal B cell differentiation responses to bacterial peptidoglycan and pokeweed mitogen in rheumatoid arthritis.

    PubMed Central

    Pardo, I; Carafa, C; Dziarski, R; Levinson, A I

    1984-01-01

    To gain insight into possible determinants of in vivo polyclonal B cell activation seen in rheumatoid arthritis (RA), we enumerated immunoglobulin secreting cells appearing in cultures of peripheral blood mononuclear cells that were stimulated with pokeweed mitogen (PWM) or a newly described polyclonal B cell activator, bacterial peptidoglycan. Peptidoglycan, the major constituent of the cell wall of gram positive bacteria, has properties which warrant its consideration in the pathogenesis of RA; including the ability to induce rheumatoid factor production as well as a RA like syndrome in experimental animals. RA patients as a group had similar immunoglobulin secreting cell responses in PWM stimulated cultures compared to arthritis controls and showed moderately depressed responses compared to healthy volunteers. However, their in vitro responses to peptidoglycan were markedly depressed when compared to those of both control groups. Of note, severely reduced peptidoglycan-induced responses were seen in 26 of 55 rheumatoid patients who demonstrated intact PWM-induced responses. These impaired responses to peptidoglycan were not due to (1) aberrant kinetic response; (2) shift in the dose-response pattern; (3) decreased cell survival in culture or (4) the inability of peptidoglycan to activate RA cells. Cell fractionation studies indicated that peptidoglycan reactive B cells were present in the blood of some patients but their reactivity was abrogated by suppressor T cells. These studies provide evidence of aberrant in vitro polyclonal B cell activation in patients with RA and provide a basis for further investigation of peptidoglycan as an immunopathogenetic agent in this disease. PMID:6610510

  10. Molecular epidemiology of HIV-associated tuberculosis in Dar es Salaam, Tanzania: strain predominance, clustering, and polyclonal disease.

    PubMed

    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.

  11. Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts.

    PubMed

    Ahmad, Niaz; Michoux, Franck; McCarthy, James; Nixon, Peter J

    2012-04-01

    Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.

  12. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  13. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  14. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  15. Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes

    SciTech Connect

    Kanngiesser, U.; Nalik, P.; Pongs, O.

    1988-05-01

    Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca/sup 2 +/ channel) has been purified as a 340-kDa protein complex to approx.80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two step-chromatographic purification procedure. The equilibrium dissociation constant of (/sup 3/H) (+) -PN200-110 binding (K/sub d/; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approx.300 kDa. The 195-kDa subunit was affinity-labeled with (/sup 3/H)azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

  16. A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

    PubMed

    Honey, S; Schneider, B L; Schieltz, D M; Yates, J R; Futcher, B

    2001-02-15

    A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

  17. Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins.

    PubMed

    Qi, Y; Yan, Z; Huang, J

    2001-10-30

    A high-performance liquid-chromatographic method based on coupled DEAE anion-exchange and Protein G affinity columns has been developed for the simultaneous separation and purification of immunoglobulin G and albumin from mouse serum. The diluted mouse serum was injected directly into this system, and the proteins were eluted separately from the DEAE and Protein G columns, coupled in series, by the column-switching technique. The advantages of this method are that IgG and albumin can be separated and purified simultaneously, the expensive affinity column is protected from contamination by the impurities in the mouse serum, and it is fast, selective, robust, and reproducible.

  18. HLA class I alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity.

    PubMed

    Paul, Sinu; Weiskopf, Daniela; Angelo, Michael A; Sidney, John; Peters, Bjoern; Sette, Alessandro

    2013-12-15

    Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ~30,000 dengue virus-derived peptides only 0.3% were predicted to bind HLA A*0101, whereas nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles.

  19. Properties of binding of partially purified glucocorticoid receptor from rat liver with glucocorticoids of different biopotencies.

    PubMed

    Izawa, M; Satoh, Y; Yoshida, A; Ichii, S

    1985-06-01

    To elucidate the relationship between binding parameters and biopotencies of glucocorticoids, we partially purified the receptor from the liver cytosol of rats in a dexamethasone-bound and unactivated form by precipitation with protamine sulfate, gel filtration and DEAE-cellulose chromatography (approximately 100-fold) and examined the interaction of the preparation with 3 glucocorticoids of different biopotencies (dexamethasone; Dex, corticosterone; Cort and prednisolone; Pred). The partially purified receptor (PPR) was stable at -20 degrees C for at least 2 months in the presence of bovine serum albumin, glycerol, molybdate and dithiothreitol. Treatment of the PPR with p-hydroxymercuribenzoate liberated the ligands and the treated PPR reassociated 3H-glucocorticoids efficiently following the addition of dithiothreitol. The reassociated PPR was bound to the DNA-cellulose after a brief heating. Metabolic activity on ligands and inactivation of the binding sites in the PPR were insignificant under the conditions used. Kd's were approximately 0.9, approximately 3 and approximately 6 nM for Dex, Cort and Pred, respectively (at 0 degree C). Relative binding affinity of ligands to the PPR which was estimated by competitions was higher in the order of triamcinolone acetonide greater than Dex greater than Cort greater than Pred greater than progesterone greater than cortexolone. Association of Dex and Cort was relatively rapid and significantly accelerated by raising the incubation temperature, while the association of Pred was slower and effects of the temperature was moderate. The rate of dissociations was also varied with ligands. The rate of dissociation of Dex was the lowest among the 3 ligands and was elevated by raising the temperature. Because the effect of temperature was more pronounced in the dissociation than in the association, apparent Ka's decreased at higher temperature. Thermodynamic examinations of glucocorticoid binding in the PPR revealed that the

  20. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  1. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  2. A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

    PubMed Central

    Moses, A C; Rosen, H N; Moller, D E; Tsuzaki, S; Haddow, J E; Lawlor, J; Liepnieks, J J; Nichols, W C; Benson, M D

    1990-01-01

    In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4. Images PMID:1979335

  3. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  4. Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

    2014-09-07

    In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

  5. B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens.

    PubMed

    Akerlund, Janie; Getahun, Andrew; Cambier, John C

    2015-08-01

    Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsiveness referred to as anergy. Reversibility of anergy indicates that chronically occupied BCR must transduce non-durable regulatory signals that maintain unresponsiveness. Consistent with such a mechanism, studies of immunoglobulin transgenic, as well as naturally occurring polyclonal autoreactive B cells demonstrate activation of the inositol 5-phosphatase SHIP-1 in anergic cells, and low affinity chromatin autoantigen-reactive B cells have been shown to require expression of this phosphatase to maintain anergy. However, it has been reported that anergy of B cells recognizing high affinity soluble antigen may not require SHIP-1, and is instead mediated by upregulation of the inositol 3-phosphatase PTEN. To further explore this apparent difference in mechanism we analyzed the effect of B cell-targeted SHIP-1 deletion on immune tolerance of high affinity anti-HEL B cells in mice expressing soluble HEL (MD4.ML-5). We report that SHIP-1 functions to dampen responses of naïve and low-dose antigen-primed B cells in vitro, and is required for induction of B cell tolerance. Thus, while anergy of B cells reactive with low affinity and likely polyvalent chromatin antigens is maintained by activation of inhibitory signaling circuitry involving SHIP-1, anergy of B cells recognizing soluble self antigen with high affinity also requires increased activity of SHIP-1.

  6. Comparative evaluation of purified Taenia solium glycoproteins and crude metacestode extracts by immunoblotting for the serodiagnosis of human T. solium cysticercosis.

    PubMed Central

    Rodriguez-Canul, R; Allan, J C; Fletes, C; Sutisna, I P; Kapti, I N; Craig, P S

    1997-01-01

    A lentil-lectin purified glycoprotein (LL-Gp) and a crude saline extract of Taenia solium metacestodes were compared for the immunodiagnosis of human cysticercosis by immunoblotting. The LL-Gp preparation was 95% sensitive for antibodies against a range of seven antigens with molecular masses of 50 to 13 kDa, whereas the sensitivity of the crude saline extract for the detection of antibodies against two major polypeptide molecules (26 and 8 kDa) was 91%. Specificity was 100% with both sets of diagnostic antigens. Affinity-purified antibodies against the 26-kDa molecule from the crude saline extract recognized the 24-kDa diagnostic region in the LL-Gp-purified extract and vice versa, suggesting that the antigens had common epitopes recognized by cysticercotic sera. In addition, in a preliminary community study of 115 randomly selected people from Bali (Indonesia), seroprevalence by immunoblot assay varied from 7.8% (with the crude saline antigen extract) to 9.6% (with the LL-Gp-purified extract). The results of this study demonstrate that both antigenic preparations are applicable for the immunodiagnosis of T. solium cysticercosis. The crude T. solium metacestode antigen extract was as specific as the purified LL-Gp T. solium metacestode extract and simpler to produce but slightly less sensitive. PMID:9302208

  7. Use of anionic denaturing detergents to purify insoluble proteins after overexpression

    PubMed Central

    2012-01-01

    Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology. PMID:23231964

  8. Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

    PubMed

    Mittermayr, Stefan; Lê, Giao N; Clarke, Colin; Millán Martín, Silvia; Larkin, Anne-Marie; O'Gorman, Peter; Bones, Jonathan

    2017-02-03

    The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.

  9. Mechanisms of polyclonal B-cell activation in autoimmune B6-lpr/lpr mice.

    PubMed

    Warren, R W; Roths, J B; Murphy, E D; Pisetsky, D S

    1984-03-01

    The influence of the lpr gene on spontaneous and lipopolysaccharide (LPS)-induced immunoglobulin production was studied in B6 mice homozygous for the mutant lpr gene (B6-lpr/lpr). Male and female mice of this congenic strain were followed for 1 year and sera serially tested by the enzyme-linked immunosorbent assay (ELISA) for the production of antibodies to single-stranded DNA (anti-sDNA), immunoglobulin (anti-IgG), and keyhole limpet hemocyanin (anti-KLH), models of autoantibody and non-autoantibody responses, respectively. Female B6-lpr/lpr mice demonstrated marked spontaneous responses to all three antigens; the responses of male B6-lpr/lpr mice were significantly lower but still exceeded those of the congenic B6-+/+ controls. These results demonstrate a generalized influence of sex on lpr associated responses. To determine whether this sex difference could be demonstrated with other forms of B-cell activation, young B6-+/+ and B6-lpr/lpr male and female mice were immunized with lipopolysaccharide and the induced responses determined. This immunization caused significant increases in the IgM response only. The levels of the induced responses produced after LPS treatment were comparable for +/+ and lpr/lpr mice. These results indicate that the enhanced responsiveness of female mice to lpr action is not reflected in the polyclonal response to LPS, which, furthermore, was unaffected by the presence of lpr. The differential influence of sex on lpr and LPS-induced responses and their apparent independence suggests that lpr and LPS promote B-cell activation by dissimilar mechanisms.

  10. Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G.

    PubMed

    Prasanna, Rajasekar R; Vijayalakshmi, Mookambeswaran A

    2010-06-04

    Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu(2+), Ni(2+) and Zn(2+) were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu(2+) was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14-16 mg/ml with Cu(2+) and 7-9 mg/ml with Ni(2+) for human IgG with MMA buffer. In the case of CIM-IDA-Zn(2+) column, the binding capacity was only about 0.5mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.

  11. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  12. Reconstitution and Characterization of a Calmodulin-Stimulated Ca2+-Pumping ATPase Purified from Brassica oleracea L. 1

    PubMed Central

    Askerlund, Per; Evans, David E.

    1992-01-01

    Purification and functional reconstitution of a calmodulin-stimulated Ca2+-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca2+, together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca2+ uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 μmol min−1 (mg protein)−1, and the Ca2+/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca2+-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca2+) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca2+-pumping ATPase in Brassica oleracea inflorescences. Images Figure 2 Figure 3 PMID:16653183

  13. Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

    PubMed

    Wang, Yanlin; Murray-Stewart, Tracy; Devereux, Wendy; Hacker, Amy; Frydman, Benjamin; Woster, Patrick M; Casero, Robert A

    2003-05-16

    The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

  14. Hydrolysis of cellulose by purified cellulase components: Synergistic effects

    SciTech Connect

    Woodward, J.; Lee, N.E.

    1987-01-01

    The hydrolysis of cellulose by purified cellulase components is reported. The adsorption of purified cellobiohydrolases (CBH I and II) and endoglucanases (EG I and II) from Trichoderma reesei strain L27 to microcrystalline cellulose (Avicel) has been studied. Scatchard analysis of the adsorption data gave the maximum amount of each component that bound to Avicel at saturation. Hydrolysis of Avicel was thus carried out by saturating and non-saturating (50% saturation) concentrations of cellulase components alone and in combination with each other, and it was found that the greatest amount of synergism between them was observed when Avicel was incubated with non-saturating concentrations of enzyme. Synergism was observed between CBH I and CBH II, as well as between EG I and CBH I; however, inhibition of hydrolysis occurred using a combination of EG I and EG II. Synergism between cellulase components may be significant during cellulose hydrolysis only when non-saturating enzyme concentrations are used. 4 refs., 3 figs.

  15. Purifying food-grade, naturally occurring CO/sub 2/

    SciTech Connect

    Nobles, J.E.; Stancik, J.W.

    1983-12-26

    Technology to purify naturally occurring CO/sub 2/ into products suitable for enhanced oil recovery (EOR) and food grade CO/sub 2/ markets has been commercially demonstrated in a 20 MMscfd (1,100 ton/day) CO/sub 2/ processing facility owned and operated by Columbia Hydrocarbon Corp. The unit demonstrates that it is technically and economically feasible to process low grade natural gas to produce pipeline specification natural gas, raw CO/sub 2/, and food grade CO/sub 2/. The Selexol treating facility is located on a reclaimed coal strip mine with very restricted plot area. Only compression and dehydration equipment for the CO/sub 2/ is located there. The CO/sub 2/ is transported by pipeline to the Marmet site, approx. 7 miles away. This site contains the facilities for purifying the CO/sub 2/ and for storage.

  16. Application of paramagnetic beads for purifying Bacillus anthracis protective antigen.

    PubMed

    Zarzecka, A; Bartoszcze, M

    2006-10-01

    Paramagnetic beads coated with Protein G and Tosylactivated-280 dynabeads have been used to purify Bacillus anthracis protective antigen from a liquid culture. The obtained protein was used in the enzyme-linked immunosorbent assay test to detect B. anthracis protective antigen antibodies in human sera collected from immunized individuals. The purification method using paramagnetic beads is very effective. It is fast, easy and may be carried out practically in any laboratory.

  17. Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

    DOEpatents

    Tucker, Melvin P.; Grohmann, Karel; Himmel, Michael E.; Mohagheghi, Ali

    1992-01-01

    A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

  18. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, William S.; Thomas, Steven R.; Nieves, Rafael A.; Himmel, Michael E.

    1994-01-01

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C.sub.1, and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81.degree. C. at pH's from about 2 to about 9, and at a inactivation temperature of about 100.degree. C. at pH's from about 2 to about 9.

  19. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, W.S.; Thomas, S.R.; Nieves, R.A.; Himmel, M.E.

    1994-11-22

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C[sub 1], and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81 C at pH's from about 2 to about 9, and at a inactivation temperature of about 100 C at pH's from about 2 to about 9. 9 figs.

  20. [Proteomic Analyses of Purified Particles of the Rabies Virus].

    PubMed

    Tu, Zhongzhong; Gong, Wenjie; Zhang, Yan; Feng, Ye; Li, Nan; Tu, Changchun

    2015-05-01

    The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.

  1. THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

    PubMed Central

    Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

    1944-01-01

    1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

  2. Emergent properties of nanosensor arrays: applications for monitoring IgG affinity distributions, weakly affined hypermannosylation, and colony selection for biomanufacturing.

    PubMed

    Reuel, Nigel F; Grassbaugh, Brittany; Kruss, Sebastian; Mundy, J Zachary; Opel, Cary; Ogunniyi, Adebola O; Egodage, Kamal; Wahl, Ramon; Helk, Bernhard; Zhang, Jingqing; Kalcioglu, Z Ilke; Tvrdy, Kevin; Bellisario, Darin O; Mu, Bin; Blake, Steven S; Van Vliet, Krystyn J; Love, J Christopher; Wittrup, Karl Dane; Strano, Michael S

    2013-09-24

    It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG (KD μ = 19 μM, σ(2) = 1000 mM(2)), murine IgG (KD μ = 4.3 nM, σ(2) = 3 μM(2)), and human IgG from CHO cells (KD μ = 2.5 nM, σ(2) = 0.01 μM(2)). Second, we show that an array of nanosensors can uniquely monitor weakly affined analyte interactions via the increased number of observed interactions. One application involves monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example, we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself.

  3. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  4. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  5. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  6. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  7. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    PubMed Central

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  8. Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis.

    PubMed Central

    Young, D; Kent, L; Rees, A; Lamb, J; Ivanyi, J

    1986-01-01

    A 38-kilodalton (kDa) protein antigen from Mycobacterium tuberculosis was purified by monoclonal antibody TB71-based affinity chromatography. This molecule carries two nonoverlapping epitopes recognized by monoclonal antibodies TB71 and TB72, which are expressed substantially more strongly by M. tuberculosis than by Mycobacterium bovis. However, cross-reactive determinants between these two species were revealed on the 38-kDa protein by a rabbit anti-BCG serum. An immunoradiometric assay based on the TB71 and TB72 antibody pair specifically determined 38-kDa-antigen concentrations in mycobacterial extracts. Antibodies in sera from tuberculosis patients estimated by binding to 38-kDa-antigen-coated microtiter plates were positively correlated with TB72 competing titers. Unlike antibodies, T-cell proliferative responses to the 38-kDa protein were expressed equally by 60% of tuberculosis patients and healthy BCG-vaccinated subjects. Similarly, delayed-type hypersensitivity skin reactions were elicited in both M. tuberculosis- and M. bovis-sensitized guinea pigs. The results suggest the immunodominance of the species-specific B-cell and cross-reactive T-cell stimulatory epitopes. Images PMID:2428751

  9. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  10. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

    PubMed Central

    Mehta, R J; Diefenbach, B; Brown, A; Cullen, E; Jonczyk, A; Güssow, D; Luckenbach, G A; Goodman, S L

    1998-01-01

    The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity. PMID:9480902

  11. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    PubMed

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  12. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  13. Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

    PubMed Central

    Berman, Jonathan Dembitz; Young, Michael

    1971-01-01

    Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more. Images PMID:5277092

  14. Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

    PubMed Central

    Lingwood, C A; Wasfy, G; Han, H; Huesca, M

    1993-01-01

    Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism. Images PMID:8500882

  15. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  16. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

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

    PubMed

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

    2015-01-01

    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.

  18. Evaluation of the polyclonal ELISA HPV serology assay as a biomarker for HPV exposure

    PubMed Central

    Coseo, Sarah E.; Porras, Carolina; Dodd, Lori E.; Hildesheim, Allan; Rodriguez, Ana Cecilia; Schiffman, Mark; Herrero, Rolando; Wacholder, Sholom; Gonzalez, Paula; Sherman, Mark E.; Jimenez, Silvia; Solomon, Diane; Bougelet, Catherine; van Doorn, Leen-Jan; Quint, Wim; Safaeian, Mahboobeh

    2011-01-01

    Background Seropositivity to HPV16 and 18 antibodies is used as a measure of cumulative HPV exposure and as a stratifier of HPV exposure for vaccine efficacy analyses. Overall performance of these assays, as a measure of HPV exposure, has not been evaluated. Methods Using data from the enrollment phase of the HPV16/18 vaccine trial in Costa Rica, we evaluated the performance of the polyclonal ELISA HPV16 and 18 serological assays as a measure of HPV exposure. Biological (for eg. HPV infection at the cervix) and behavioral characteristics (for eg. lifetime number of sexual partners) with known associations with current and past HPV infection were used to define cases and controls (HPV exposed vs. not exposed). Pre-vaccination serum was measured for antibodies against HPV16 and HPV18 by ELISA; cervical samples were tested for HPV DNA using PCR SPF10/LiPA25. ELISA results were analyzed using receiver-operator-characteristic curves (ROC); performance was evaluated at the manufacturer set cutpoint (HPV16 =8, HPV18 =7) and at cutpoints chosen to optimize sensitivity and specificity (HPV16 =34, HPV18 =60). Results Defining cases as type-specific HPV DNA positive with high-grade abnormal cytolzogy (i.e. combined molecular and microscopic markers of infection), HPV16-ELISA gave sensitivity that was lower at the optimal cutpoint than the manufacturer cutpoint (62.2 compared with 75.7, respectively; p=0.44). However, specificity was higher (85.3 compared with 70.4, respectively; p<0.0001). Similarly, HPV18-ELISA gave sensitivity that was lower at the optimal cutpoint than the manufacturer cutpoint (34.5 compared with 51.7, respectively; p=0.40), with higher specificities (94.9 compared with 72.6, respectively; p<0.0001). Conclusions Modifying cutpoints did not improve the low sensitivity. The low sensitivity of this assay does not support its use for risk stratification or clinical settings. PMID:21934576

  19. Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.

    PubMed Central

    Poruchynsky, M S; Atkinson, P H

    1991-01-01

    Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process. Images PMID:1651404

  20. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    PubMed

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.

  1. Penem derivatives: beta-lactamase stability and affinity for penicillin-binding proteins in Escherichia coli.

    PubMed

    Ohya, S; Utsui, Y; Sugawara, S; Yamazaki, M

    1982-03-01

    Penem derivatives, a new group of beta-lactam antibiotics with potent activities against a wide range of bacteria, including Pseudomonas aeruginosa, were tested for their stability against hydrolysis by beta-lactamases purified from clinical isolates of Morganella morganii. Proteus vulgaris, and Escherichia coli and by a penicillinase from Bacillus cereus. Penems having 6 alpha substituents, such as hydroxyethyl, hydroxymethyl, and ethyl groups, were very stable against hydrolysis by each of the enzymes. Penems having no 6 alpha substituents were easily hydrolyzed by P. vulgaris and E. coli enzymes, whereas they were rather stable against hydrolysis by M. morganii and B. cereus enzymes, a typical cephalosporinase and penicillinase, respectively. Affinity of the penems for E. coli penicillin-binding proteins (PBPs) was also tested. beta-Lactamase-stable penems having a 6 alpha-hydroxyethyl group showed high affinity for PBP-4, -5, and -6 as well as for PBP-1A, -1Bs, and -2. However, the penems having no 6 alpha substituents showed a far lower affinity for PBP-4, -5, and -6 than that shown by the corresponding 6 alpha-hydroxyethyl penems. Among the penems tested, affinity for PBP-4, -5, and -6 was closely related to their beta-lactamase stability, as was the case among cephamycins and cephalosporins. Effects of the penems on the morphology of a strain of E. coli are also described.

  2. Generation of antisera to purified prions in lipid rafts.

    PubMed

    Hnasko, Robert; Serban, Ana V; Carlson, George; Prusiner, Stanley B; Stanker, Larry H

    2010-01-01

    Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP(C) into alternately folded PrP(Sc). Immunoassays toward pre-clinical detection of infectious PrP(Sc) have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP(Sc) selective antibodies. We report a method to purify infectious PrP(Sc) from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP(Sc) was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM <20-fold and purified DRM immunogen <40-fold) relative to 1% crude brain homogenate. Purification of PrP(Sc) from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP(Sc) antigen was performed using wild-type (wt) and Prnp(0/0) mice, both on Balb/cJ background. A robust immune response against PrP(Sc) was observed in all inoculated Prnp(0/0) mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP(C) and PrP(Sc), while binding to other brain-derived protein was not observed. In contrast, the PrP(Sc) inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.

  3. Generation of antisera to purified prions in lipid rafts

    PubMed Central

    Hnasko, Robert; Serban, Ana V; Carlson, George; Prusiner, Stanley B

    2010-01-01

    Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrPC into alternately folded PrPSc. Immunoassays toward pre-clinical detection of infectious PrPSc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrPSc selective antibodies. We report a method to purify infectious PrPSc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrPSc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrPSc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrPSc antigen was performed using wild-type (wt) and Prnp0/0 mice, both on Balb/cJ background. A robust immune response against PrPSc was observed in all inoculated Prnp0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrPC and PrPSc, while binding to other brain-derived protein was not observed. In contrast, the PrPSc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein. PMID:20647769

  4. Comparative study of crude and purified cellulose from wheat straw.

    PubMed

    Sun, Xiao-Feng; Sun, Run-Cang; Su, Yinquan; Sun, Jing-Xia

    2004-02-25

    A sequential totally chlorine-free procedure for isolation of cellulose from wheat straw was proposed in this study. The dewaxed straw was pretreated with 0.5 M NaOH in 60% methanol at 60 degrees C for 2.5 h under ultrasonic irradiation for 0-35 min and sequentially posttreated with 2% H(2)O(2)-0.2% TAED at pH 11.8 for 12 h at 48 degrees C, which together solubilized 85.3-86.1% of the original hemicelluloses and 91.7-93.2% of the original lignin, respectively. The yield of crude cellulose ranged between 46.2 and 49.2% on a dry weight basis related to wheat straw, which contained 11.2-12.2% residual hemicelluloses and 2.5-2.9% remaining lignin. Further treatment of the corresponding crude cellulosic preparations with 80% acetic acid-70% nitric acid under the condition given yielded 36.8-37.7% of the purified cellulose, which contained minor amounts of bound hemicelluloses (2.5-2.8%) and was relatively free of associated lignin (0.1-0.2%). The isolated crude and purified cellulose samples were comparatively studied by FT-IR and CP/MAS (13)C NMR spectroscopy, and the relative crystallinity was also estimated. The final stage treatment with 80% acetic acid-70% nitric acid decreased the hemicelluloses and lignin associated in the crude cellulose but led to 3.1-5.4% degradation of the original cellulose; in addition, the purity of the obtained cellulose was high. However, it was found that the final stage treatment is not severe enough to cause decrystallization of cellulose. The thermal stability of the purified cellulose is higher than that of the corresponding crude cellulose.

  5. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  6. [Importance of air purifiers in the prevention of respiratory allergy].

    PubMed

    Sabbah, A

    2000-06-01

    Prevention of respiratory allergy and thus diminution of its prevalence consists of the application not only of preventative measures on the allergens themselves, but also on atmospheric pollutants such as NO, CO and diesel particles. These modify the allergens and increase synthesis of allergen-specific IgE. Use of air purifiers, on condition that they have HEPA or ULPA filters may contribute to reduction of allergenicity linked to pollutants and are one of the not-negligible means of protection from respiratory allergy and asthma in particular.

  7. Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, Michael E.; Adney, William S.; Tucker, Melvin P.; Grohmann, Karel

    1994-01-01

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.

  8. Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, M.E.; Adney, W.S.; Tucker, M.P.; Grohmann, K.

    1994-01-04

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068) is presented. The cellulase is water soluble, possesses both C[sub 1] and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83 C at pH's from about 2 to about 9, and in inactivation temperature of about 110 C at pH's from about 2 to about 9. 7 figures.

  9. Growth of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; De Vellis, J.

    1981-11-01

    Astrocytes purified from primary cultures of neonatal rat cerebrum can not be grown in a synthetic medium supplemented with putrescine, prostaglandin F/sub 2//sub ..cap alpha../, insulin, fibroblast growth factor, and hydrocortisone. These five supplements have a marked synergistic effect on growth when used in combination but have little effect when used individually. Astrocytes grown in the defined medium exhibit dramatic changes in morphological characteristics in comparison to cells grown in serum-free or serum-supplemented medium. In addition, these cells express the astrocyte-specific marker glial fibrillary acidic protein and are estimated by several criteria to be greater than 95% astrocytes.

  10. Spectrophotometric assay for amikacin using purified kanamycin acetyltransferase.

    PubMed

    Scarbrough, E; Williams, J W; Northrop, D B

    1979-08-01

    A rapid spectrophotometric assay has been developed for measuring the concentrations of amikacin and related antibiotics in serum. The assay uses a purified enzyme from R-factor E. coli which acetylates amikacin with the production of coenzyme A, the latter in turn being reacted with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, that is measured in the visible spectrum. The system complements an earlier assay for gentamicin-related antibiotics thereby facilitating the rapid measurement of the concentrations of all clinically important aminoglycosides in serum.

  11. Substrate specificity of fucosyltransferase purified from human parotid saliva.

    PubMed

    Tamagawa, H; Iwakura, K; Amano, A; Shizukuishi, S; Tsunemitsu, A

    1987-03-01

    The purified fucosyltransferase from human parotid saliva was shown to transfer fucose from GDP-fucose onto the oligosaccharide chains containing the Gal beta 1----3GlcNAc or Gal beta 1----4GlcNAc/Glc sequences. Competition studies between asialotransferrin and either lacto-N-fucopentaose 1 or 2'-fucosyllactose provided evidence that both the substrates competed for a common enzyme active site. These results suggest that the fucosyltransferase activities for the three acceptors may be catalyzed by the same enzyme.

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

    PubMed

    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

  13. New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

    PubMed

    Watanabe, Eiki; Hoshino, Ryoko; Kanzaki, Yukiko; Tokumoto, Hiroshi; Kubo, Hiroaki; Nakazawa, Hiroyuki

    2002-06-19

    The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed

  14. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  15. Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.

    PubMed

    Cleveland, Thomas E; McCabe, Jacqueline M; Leahy, Daniel J

    2014-12-01

    Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.

  16. Gas-phase nitronium ion affinities.

    PubMed Central

    Cacace, F; de Petris, G; Pepi, F; Angelelli, F

    1995-01-01

    Evaluation of nitronium ion-transfer equilibria, L1NO2+ + L2 = L2NO2+ + L1 (where L1 and L2 are ligands 1 and 2, respectively) by Fourier-transform ion cyclotron resonance mass spectrometry and application of the kinetic method, based on the metastable fragmentation of L1(NO2+)L2 nitronium ion-bound dimers led to a scale of relative gas-phase nitronium ion affinities. This scale, calibrated to a recent literature value for the NO2+ affinity of water, led for 18 ligands, including methanol, ammonia, representative ketones, nitriles, and nitroalkanes, to absolute NO2+ affinities, that fit a reasonably linear general correlation when plotted vs. the corresponding proton affinities (PAs). The slope of the plot depends to a certain extent on the specific nature of the ligands and, hence, the correlations between the NO2+ affinities, and the PAs of a given class of compounds display a better linearity than the general correlation and may afford a useful tool for predicting the NO2+ affinity of a molecule based on its PA. The NO2+ binding energies are considerably lower than the corresponding PAs and well below the binding energies of related polyatomic cations, such as NO+, a trend consistent with the available theoretical results on the structure and the stability of simple NO2+ complexes. The present study reports an example of extension of the kinetic method to dimers, such as L1(NO2+)L2, bound by polyatomic ions, which may considerably widen its scope. Finally, measurement of the NO2+ affinity of ammonia allowed evaluation of the otherwise inaccessible PA of the amino group of nitramide and, hence, direct experimental verification of previous theoretical estimates. PMID:11607578

  17. Magnetism for understanding catalyst analysis of purified carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Bellouard, Christine; Mercier, Guillaume; Cahen, Sébastien; Ghanbaja, Jaafar; Medjahdi, Ghouti; Gleize, Jérôme; Lamura, Gianrico; Hérold, Claire; Vigolo, Brigitte

    2016-08-01

    The precise quantification of catalyst residues in purified carbon nanotubes is often a major issue in view of any fundamental and/or applicative studies. More importantly, since the best CNTs are successfully grown with magnetic catalysts, their quantification becomes strictly necessary to better understand intrinsic properties of CNT. For these reasons, we have deeply analyzed the catalyst content remained in nickel-yttrium arc-discharge single walled carbon nanotubes purified by both a chlorine-gas phase and a standard acid-based treatment. The study focuses on Ni analysis which has been investigated by transmission electron microscopy, X-ray diffraction, thermogravimetry analysis, and magnetic measurements. In the case of the acid-based treatment, all quantifications result in a decrease of the nanocrystallized Ni by a factor of two. In the case of the halogen gas treatment, analysis and quantification of Ni content is less straightforward: a huge difference appears between X-ray diffraction and thermogravimetry results. Thanks to magnetic measurements, this disagreement is explained by the presence of Ni2+ ions, belonging to NiCl2 formed during the Cl-based purification process. In particular, NiCl2 compound appears under different magnetic/crystalline phases: paramagnetic or diamagnetic, or well intercalated in between carbon sheets with an ordered magnetic phase at low temperature.

  18. PURIFY: a new approach to radio-interferometric imaging

    NASA Astrophysics Data System (ADS)

    Carrillo, R. E.; McEwen, J. D.; Wiaux, Y.

    2014-04-01

    In a recent paper series, the authors have promoted convex optimization algorithms for radio-interferometric imaging in the framework of compressed sensing, which leverages sparsity regularization priors for the associated inverse problem and defines a minimization problem for image reconstruction. This approach was shown, in theory and through simulations in a simple discrete visibility setting, to have the potential to outperform significantly CLEAN and its evolutions. In this work, we leverage the versatility of convex optimization in solving minimization problems to both handle realistic continuous visibilities and offer a highly parallelizable structure paving the way to significant acceleration of the reconstruction and high-dimensional data scalability. The new algorithmic structure promoted relies on the simultaneous-direction method of multipliers (SDMM) and contrasts with the current major-minor cycle structure of CLEAN and its evolutions, which in particular cannot handle the state-of-the-art minimization problems under consideration where neither the regularization term nor the data term are differentiable functions. We release a beta version of an SDMM-based imaging software written in C and dubbed PURIFY (http://basp-group.github.io/purify/) that handles various sparsity priors, including our recent average sparsity approach sparsity averaging reweighted analysis (SARA). We evaluate the performance of different priors through simulations in the continuous visibility setting, confirming the superiority of SARA.

  19. Synthesis of gold and silver nanoparticles using purified URAK.

    PubMed

    Deepak, Venkataraman; Umamaheshwaran, Paneer Selvam; Guhan, Kandasamy; Nanthini, Raja Amrisa; Krithiga, Bhaskar; Jaithoon, Nagoor Meeran Hasika; Gurunathan, Sangiliyandi

    2011-09-01

    This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV-visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 μg/ml.

  20. Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen.

    PubMed

    Hellmich, R L; Siegfried, B D; Sears, M K; Stanley-Horn, D E; Daniels, M J; Mattila, H R; Spencer, T; Bidne, K G; Lewis, L C

    2001-10-09

    Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

  1. Tea Derived Galloylated Polyphenols Cross-Link Purified Gastrointestinal Mucins

    PubMed Central

    Georgiades, Pantelis; Pudney, Paul D. A.; Rogers, Sarah; Thornton, David J.; Waigh, Thomas A.

    2014-01-01

    Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria. PMID:25162539

  2. Activation of purified calcium channels by stoichiometric protein phosphorylation

    SciTech Connect

    Nunoki, K.; Florio, V.; Catterall, W.A. )

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

  3. Life cycle assessment comparison of photocatalytic coating and air purifier.

    PubMed

    Tichá, Marie; Žilka, Miroslav; Stieberová, Barbora; Freiberg, František

    2016-07-01

    This article presents a comparison of 2 very different options for removal of undesirable microorganisms and airborne pollutants from the indoor environment of hospitals, schools, homes, and other enclosed spaces using air purifiers and photocatalytic coatings based on nano titanium dioxide (TiO2 ). Both products were assessed by life cycle assessment (LCA) methodology from cradle-to-grave. The assessment also includes comparison of 2 different nano TiO2 production technologies, one by continuous hydrothermal synthesis and the other by a sulfate process. Results of the study showed a relatively large contribution of photocatalytic coatings to reducing the effects of selected indices in comparison with an air purifier, regardless of which nano TiO2 production method is used. Although the impacts of the sulfate process are significantly lower compared to those of hydrothermal synthesis when viewed in terms of production alone, taken in the context of the entire product life cycle, the net difference becomes less significant. The study has been elaborated within the Sustainable Hydrothermal Manufacturing of Nanomaterials (SHYMAN) project, which aims to develop competitive and sustainable continuous nanoparticle (NP) production technology based on supercritical hydrothermal synthesis. Integr Environ Assess Manag 2016;12:478-485. © 2016 SETAC.

  4. Ethanol increases affinity of protein kinase C for phosphatidylserine

    SciTech Connect

    Chin, J.H.

    1986-03-01

    Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition of calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.

  5. Purification of baculovirus vectors using heparin affinity chromatography

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; van der Loo, Johannes CM; Malik, Punam

    2016-01-01

    Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications. PMID:27933303

  6. [Procedure for purifying RNA polymerase II from human placenta].

    PubMed

    Kandyba, L V; Matsanova, V R; Shamovskiĭ, I V; Raĭt, V K

    1994-12-01

    DNA-dependent RNA polymerase IIB having a specific activity of 320 u./mg has been isolated from the term placenta homogenate using extraction performed at 4-6 degrees C in the presence of 75 mM ammonium sulfate and 1.5% nonidet P40, fractionation on DEAE-cellulose DE 23, desalting and heparin-agarose chromatography, resulting in 330-fold purification and a 18% yield. Technical details have been determined which are of crucial importance for reproducibility of affinity chromatography. The possibility of proteolysis of the IIc subunit during enzyme purification has been demonstrated.

  7. Stability of flavin semiquinones in the gas phase: the electron affinity, proton affinity, and hydrogen atom affinity of lumiflavin.

    PubMed

    Zhang, Tianlan; Papson, Kaitlin; Ochran, Richard; Ridge, Douglas P

    2013-11-07

    Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

  8. Proton Affinity Calculations with High Level Methods.

    PubMed

    Kolboe, Stein

    2014-08-12

    Proton affinities, stretching from small reference compounds, up to the methylbenzenes and naphthalene and anthracene, have been calculated with high accuracy computational methods, viz. W1BD, G4, G3B3, CBS-QB3, and M06-2X. Computed and the currently accepted reference proton affinities are generally in excellent accord, but there are deviations. The literature value for propene appears to be 6-7 kJ/mol too high. Reported proton affinities for the methylbenzenes seem 4-5 kJ/mol too high. G4 and G3 computations generally give results in good accord with the high level W1BD. Proton affinity values computed with the CBS-QB3 scheme are too low, and the error increases with increasing molecule size, reaching nearly 10 kJ/mol for the xylenes. The functional M06-2X fails markedly for some of the small reference compounds, in particular, for CO and ketene, but calculates methylbenzene proton affinities with high accuracy.

  9. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  10. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

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

    PubMed

    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.

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

    PubMed

    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.

  13. Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5.

    PubMed

    Yang, Jie; Guo, Shi-Ying; Pan, Fei-Yan; Geng, Hui-Xia; Gong, Yi; Lou, Dan; Shu, Yong-Qian; Li, Chao-Jun

    2007-05-01

    Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Defective disposal of immune complexes and polyclonal B cell activation persist long after exposure to bacterial lipopolysaccharide in mice

    SciTech Connect

    Granholm, N.A.; Cavallo, T. )

    1989-11-01

    Patients with systemic lupus erythematosus experience clinical exacerbation during superimposed bacterial infection. Previous studies in mice indicated that heightened immune phenomena during exposure to bacterial lipopolysaccharide (LPS) appear to be related, in part, to polyclonal B cell activation, to abnormal disposal of immune complexes (IC), and to increased localization of IC in tissues. To investigate whether such effects were reversible, we administered bacterial LPS to C57BL/6 mice for 5 weeks. Control mice received vehicle alone. We then discontinued LPS, and 6 weeks later LPS and control mice were challenged with a subsaturating dose of radiolabeled IC; the removal of IC from the circulation, their localization in the liver, spleen, and kidney were determined. In comparison to values in control mice, in mice previously exposed to LPS, serologic features of polyclonal B cell activation persisted; liver uptake of pathogenic IC (greater than Ag2Ab2) was normal, but removal of small size IC (less than or equal to Ag2Ab2) from the circulation was delayed; localization of IC in the kidneys was enhanced, and pathologic proteinuria developed. The effects of repeated exposure to bacterial LPS are partially reversible, but they last long after LPS is discontinued and may contribute to altered disposal of IC, enhanced organ localization of IC, and organ dysfunction.

  16. Redesigning Protein Cavities as a Strategy for Increasing Affinity in Protein-Protein Interaction: Interferon-γ Receptor 1 as a Model

    PubMed Central

    Biedermannová, Lada; Mikulecký, Pavel; Zahradník, Jiří; Charnavets, Tatsiana; Šebo, Peter

    2015-01-01

    Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity. PMID:26060819

  17. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

  18. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  19. Physicochemical properties and amino acid composition of highly purified preparation of distinctive estrogen-binding protein from rat liver

    SciTech Connect

    Shchelkunova, T.A.; Smirnov, A.N.; Rozen, V.B.

    1986-12-10

    The structure and properties of a distinctive estrogen-binding protein (DEBP) from the livers of male rats, purified with the aid of an affinity sorbent, was investigated. A high degree of purification of the DEBP obtained (> 99%), associated with the pronounced microheterogeneity, was found. Apparently, this microheterogeneity is the result of partial proteolysis of the protein from the N-end during isolation. Purified DEBP molecules have the following parameters: molecular weight 31,000 (according to the data of electrophoresis in polyacrylamide gel in the presence of SDS), sedimentation coefficient 3.765, Stokes' radius 25.6 A, frictional ratio 1.11. Absorption maximum of the protein in the ultraviolet region 276 nm; extinction coefficient 26; content of ..cap alpha..-helical segments 25-30%. The equilibrium constant of the association with estradiol is 5 x 10/sup 7/ M/sup -1/. Estriol (> 100%) and, to a lesser degree, estrone and testosterone (approx. 10%) compete for the protein-binding sites on (/sup 3/H) estradiol, whereas androsterone has practically no competitive effect. A study of the amino acid composition of the DEBP showed that the protein contains a large number of residues with hydrophobic side groups (34.4%), it has more acidic than basic amino acids, and possesses a low content of cysteine, threonine, and histidine.

  20. Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.

    PubMed

    Geada, Déborah; Valdés, Rodolfo; Escobar, Arturo; Ares, Dulce M; Torres, Edel; Blanco, Reinaldo; Ferro, Williams; Dorta, Dayamí; González, Marcos; Alemán, María R; Padilla, Sigifredo; Gómez, Leonardo; Del Castillo, Norma; Mendoza, Otto; Urquiza, Dioslaida; Soria, Yordanka; Brito, José; Leyva, Alberto; Borroto, Carlos; Gavilondo, Jorge V

    2007-10-01

    Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.

  1. Affine coherent states and Toeplitz operators

    NASA Astrophysics Data System (ADS)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  2. Non-affine elasticity in jammed systems

    NASA Astrophysics Data System (ADS)

    Maloney, Craig

    2006-03-01

    Symmetry dictates that perfect crystals should deform homogeneously, or affinely, under external load, and computing the elastic moduli from the underlying interaction potential is then straightforward. For disordered materials no such simple procedure exists, and recent numerical works have demonstrated that non-affine corrections can dramatically reduce the naive expectation for the shear modulus in a broad class of disordered systems and may control rigidity loss in the zero pressure limit in purely repulsive systems, i.e. the unjamming transition (c.f. [O'Hern et. al. PRE 68, 011306 (2003)]). We present numerical results and an analytical framework for the study of these non-affine corrections to the elastic response of disordered packings.

  3. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  4. Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

    PubMed

    Pearce, Lesley A; Yu, Meng; Waddington, Lynne J; Barr, Jennifer A; Scoble, Judith A; Crameri, Gary S; McKinstry, William J

    2015-12-01

    Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.

  5. Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.

    PubMed

    Longstaff, Colin; Hogwood, John; Gray, Elaine; Komorowicz, Erzsebet; Varjú, Imre; Varga, Zoltán; Kolev, Krasimir

    2016-03-01

    Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.

  6. CHARACTERIZATION OF THE PURIFIED HYALURONAN SYNTHASE FROM STREPTOCOCCUS EQUISIMILIS*

    PubMed Central

    Tlapak-Simmons, Valarie L.; Baron, Christina A.; Weigel, Paul H.

    2006-01-01

    Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg2+ to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP and UTP. Purified seHAS was ~40% more active in 25 mM, compared to 50 mM, PO4 in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5–11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pKa values at pH 6.6 and 11.8. As the pH was increased from ~6.5, both Km and Vmax increased until pH ~10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/sec) was essentially unchanged from pH 6.5 to pH 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Δ9) fatty acids. The apparent Ea for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 Kg/mol caused 50% inhibition of Vmax at 21, 6.5, and 3.5 mM, respectively. The apparent Ki values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3 and 1.2 mM. PMID:15248781

  7. Use of Affinity Diagrams as Instructional Tools in Inclusive Classrooms.

    ERIC Educational Resources Information Center

    Haselden, Polly G.

    2003-01-01

    This article describes how the affinity diagram, a tool for gathering information and organizing it into natural groupings, can be used in inclusive classrooms. It discusses how students can be taught to use an affinity diagram, how affinity diagrams can be used to reflect many voices, and how affinity diagrams can be used to plan class projects.…

  8. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

    PubMed

    Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

    2013-07-01

    Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

  9. 77 FR 14733 - Purified Carboxymethylcellulose From Finland and the Netherlands: Extension of Time Limit for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE International Trade Administration Purified Carboxymethylcellulose From Finland and the Netherlands: Extension..., inter alia, purified carboxymethylcellulose from Finland and the Netherlands covering the period July...

  10. 42 CFR 84.170 - Non-powered air-purifying particulate respirators; description.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... that contain adequate oxygen to support life. (b) Non-powered air-purifying particulate respirators are... includes oil-based liquid particulates. (c) Non-powered air-purifying particulate respirators...

  11. Cation affinity numbers of Lewis bases.

    PubMed

    Lindner, Christoph; Tandon, Raman; Maryasin, Boris; Larionov, Evgeny; Zipse, Hendrik

    2012-01-01

    Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  12. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  13. On the electron affinity of B2

    SciTech Connect

    Glezakou, Vanda A.; Taylor, Peter

    2009-02-02

    We present the results of high-level ab initio calculations on the electron affinity of B2. Our new best estimate of 1.93±0.03 eV is in agreement with previous calculations as well as the sole existing experimental estimate of 1.8 eV, as derived from quantities with an uncertainty of 0.4 eV. The electron affinity of atomic boron, which is much smaller, is also calculated for comparison, and again found to be in good agreement with experiment. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  14. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  15. Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol, phospholipids, and synthetic detergents.

    PubMed

    Holm, Jan; Hansen, Steen Ingemann

    2002-01-01

    Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.

  16. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    PubMed

    Mol, J A; van den Berg, T P; Visser, T J

    1988-02-01

    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  17. Regulator of insulin receptor affinity in rat skeletal muscle sarcolemmal vesicles

    SciTech Connect

    Whitson, R.H.; Barnard, K.J.; Kaplan, S.A.; Itakura, K.

    1986-05-01

    Wheat germ agglutinin (WGA) affinity purification of detergent solubilized insulin receptors (IR) from rat skeletal muscle sarcolemmal vesicles resulted in an apparent increase in total insulin binding activity of greater than 5-fold, suggesting that an inhibitory component had been removed. This was verified when the flow-through fraction from the WGA column was dialyzed and added back to the partially purified receptor. The addition of a 100-fold dilution of the inhibitor preparation caused a 50% reduction in binding to trace amounts of /sup 125/I-insulin. Scatchard analysis revealed that the effect of the inhibitor was to decrease the affinity of the muscle IR. The inhibitor appeared to be tissue specific, inasmuch as the I/sub 50/'s for WGA-purified IR from rat fat and liver were 10 times the I/sub 50/ for muscle IR. The I/sub 50/ for insulin binding to intact IM-9 cells was 30 times the value for muscle IR. The inhibitor eluted in the void volume of Sephadex G-50 columns. Its activity was not destroyed by heating at 90/sup 0/C for 10 minutes, or by prolonged incubation with trypsin or dithiothreitol. The inhibitor described here may have a role in modulating insulin sensitivity in skeletal muscle.

  18. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-08-08

    Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

  19. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  20. Evidence of multi-affinity in the Japanese stock market

    NASA Astrophysics Data System (ADS)

    Katsuragi, Hiroaki

    2000-04-01

    Fluctuations of the Japanese stock market (Tokyo Stock Price Index: TOPIX) are analyzed using a multi-affine analysis method. In the research to date, only some simulated self-affine models have shown multi-affinity. In most experiments using observations of self-affine fractal profiles, multi-affinity has not been found. However, we find evidence of multi-affinity in fluctuations of the Japanese stock market (TOPIX). The qth-order Hurst exponent Hq varies with changes in q. This multi-affinity indicates that there are plural mechanisms that affect the same time scale as stock market price fluctuation dynamics.

  1. Method and apparatus for removing residual hydrogen from a purified gas

    SciTech Connect

    Briesacher, J.L.; Applegarth, C.H.; Lorimer, D.H.

    1993-08-24

    A method is described for removing residual hydrogen from a purified gas comprising the steps of: (a) heating an impure gas; (b) contacting the heated impure gas with an impurity sorbing material to produce a purified gas having trace amounts of residual hydrogen; (c) cooling the purified gas to a temperature less than about 100 C; and (d) contacting the cooled purified gas with a hydrogen sorbing material to at least partially remove said residual hydrogen.

  2. X-ray diffraction study of highly purified human ceruloplasmin

    SciTech Connect

    Samygina, V. R.; Sokolov, A. V.; Pulina, M. O.; Bartunik, H. D.; Vasil'ev, V. B.

    2008-07-15

    The three-dimensional structure of ceruloplasmin (CP) with unoccupied labile metal-binding sites and the structure of CP containing Ni{sup 2+} in the labile sites were solved for the first time at 2.6 and 2.95 A resolution, respectively. Crystallization was performed with the use of storage-stable CP, which was prepared in the presence of proteinase inhibitors and purified from (pre)proteinases. Ceruloplasmin with Ni{sup 2+} crystallized in the orthorhombic space group, which had been earlier unknown for CP. Ceruloplasmin with the unoccupied labile sites crystallized in the trigonal crystal form. The differences in intermolecular contacts observed in the trigonal and orthorhombic crystal structures of CP are considered. The conformational changes attendant upon Ni{sup 2+} binding are described. It was suggested that the labile sites are multifunctional and can both bind metal ions potentially toxic to organisms and be involved in electron transfer from substrates to the active site.

  3. The Use of Detergents to Purify Membrane Proteins.

    PubMed

    Orwick-Rydmark, Marcella; Arnold, Thomas; Linke, Dirk

    2016-04-01

    Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included.

  4. X-ray diffraction study of highly purified human ceruloplasmin

    NASA Astrophysics Data System (ADS)

    Samygina, V. R.; Sokolov, A. V.; Pulina, M. O.; Bartunik, H. D.; Vasil'Ev, V. B.

    2008-07-01

    The three-dimensional structure of ceruloplasmin (CP) with unoccupied labile metal-binding sites and the structure of CP containing Ni2+ in the labile sites were solved for the first time at 2.6 and 2.95 Å resolution, respectively. Crystallization was performed with the use of storage-stable CP, which was prepared in the presence of proteinase inhibitors and purified from (pre)proteinases. Ceruloplasmin with Ni2+ crystallized in the orthorhombic space group, which had been earlier unknown for CP. Ceruloplasmin with the unoccupied labile sites crystallized in the trigonal crystal form. The differences in intermolecular contacts observed in the trigonal and orthorhombic crystal structures of CP are considered. The conformational changes attendant upon Ni2+ binding are described. It was suggested that the labile sites are multifunctional and can both bind metal ions potentially toxic to organisms and be involved in electron transfer from substrates to the active site.

  5. Purifying Greenberger-Horne-Zeilinger states using degenerate quantum codes

    NASA Astrophysics Data System (ADS)

    Ho, K. H.; Chau, H. F.

    2008-10-01

    Degenerate quantum codes are codes that do not reveal the complete error syndrome. Their ability to conceal the complete error syndrome makes them powerful resources in certain quantum-information processing tasks. In particular, the most error-tolerant way to purify depolarized Bell states using one-way communication known to date involves degenerate quantum codes. Here we study three closely related purification schemes for depolarized Greenberger-Horne-Zeilinger states shared among m⩾3 players by means of degenerate quantum codes and one-way classical communications. We find that our schemes tolerate more noise than all other one-way schemes known to date, further demonstrating the effectiveness of degenerate quantum codes in quantum-information processing.

  6. DNA helicase activity in purified human RECQL4 protein.

    PubMed

    Suzuki, Takahiro; Kohno, Toshiyuki; Ishimi, Yukio

    2009-09-01

    Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

  7. Drosophilia spectrin. I. Characterization of the purified protein.

    PubMed

    Dubreuil, R; Byers, T J; Branton, D; Goldstein, L S; Kiehart, D P

    1987-11-01

    We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.

  8. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

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

    PubMed Central

    Smedley, Rebecca C; Patterson, Jon S; Miller, RoseAnn; Massey, Jeffrey P; Wise, Annabel G; Maes, Roger K; Wu, Ping; Kaneene, John B; Kiupel, Matti

    2007-01-01

    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%, compared to 100% when

  10. Measurement of Ozone Emission and Particle Removal Rates from Portable Air Purifiers

    ERIC Educational Resources Information Center

    Mang, Stephen A.; Walser, Maggie L.; Nizkorodov, Sergey A.; Laux, John M.

    2009-01-01

    Portable air purifiers are popular consumer items, especially in areas with poor air quality. Unfortunately, most users of these air purifiers have minimal understanding of the factors affecting their efficiency in typical indoor settings. Emission of the air pollutant ozone (O[subscript 3]) by certain air purifiers is of particular concern. In an…

  11. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  12. On modality and complexity of affine embeddings

    SciTech Connect

    Arzhantsev, I V

    2001-08-31

    Let G be a reductive algebraic group and let H be a reductive subgroup of G. The modality of a G-variety X is the largest number of the parameters in a continuous family of G-orbits in X. A precise formula for the maximum value of the modality over all affine embeddings of the homogeneous space G/H is obtained.

  13. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  14. Stabilization of the Motion of Affine Systems

    NASA Astrophysics Data System (ADS)

    Babenko, E. A.; Martynyuk, A. A.

    2016-07-01

    Sufficient conditions for the stability of a nonlinear affine system subject to interval initial conditions are established. These conditions are based on new estimates of the norms of the solutions of the systems of perturbed equations of motion. This stabilization method is used to analyze an electromechanical system with permanent magnet

  15. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  16. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  17. Activity of purified hepatitis C virus protease NS3 on peptide substrates.

    PubMed Central

    Steinkühler, C; Urbani, A; Tomei, L; Biasiol, G; Sardana, M; Bianchi, E; Pessi, A; De Francesco, R

    1996-01-01

    The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation. PMID:8794305

  18. Purification and characterization of a Cytisus-type Ulex europeus hemagglutinin II by affinity chromatography.

    PubMed

    Konami, Y; Tsuji, T; Matsumoto, I; Osawa, T

    1981-07-01

    Ulex europeus hemagglutinin II [Cytisus-type anti-H(O) hemagglutinin] inhibited most by di-N-acetylchitobiose has been purified by affinity chromatography on a column of chitobiose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. The purified hemagglutinin was homogeneous by ultracentrifugal analysis and gave a single band by electrophoresis on polyacrylamide gel, and had a molecular weight of 105 000 by sedimentation equilibrium and an isoelectric point of pH 6.66. This hemagglutinin was found to be composed of four, apparently identical, subunits of a molecular weight of 25 000 +/- 2 000 by dodecyl sulphate-polyacrylamide gel electrophoresis, and to contain 10.3% carbohydrate in which mannose (3.7%) was the predominant sugar, with smaller amounts of glucose, glucosamine, xylose, fucose and galactose. Amino acid analysis of the purified hemagglutinin II showed a large amount of aspartic acid and serine, but as little as 0.1 mol/100 mol of cystine or methionine could be detected.

  19. A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

    PubMed Central

    Wu, Sau-Ching; Wang, Chris; Hansen, Dave; Wong, Sui-Lam

    2017-01-01

    SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged β-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months. PMID:28220817

  20. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  1. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  2. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

    PubMed

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C; Van Bergen En Henegouwen, Paul M P; Fernández, Luis Ángel

    2016-10-01

    Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

  3. Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

    PubMed Central

    Wu, Sau-Ching; Wong, Sui-Lam

    2013-01-01

    Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for

  4. Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

    PubMed

    Wu, Sau-Ching; Wong, Sui-Lam

    2013-01-01

    Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for

  5. Matrix effects in applying mono- and polyclonal ELISA systems to the analysis of weathered oils in contaminated soil.

    PubMed

    Pollard, S J T; Farmer, J G; Knight, D M; Young, P J

    2002-01-01

    Commercial mono- and polyclonal enzyme-linked immunosorbent assay (ELISA) systems were applied to the on-site analysis of weathered hydrocarbon-contaminated soils at a former integrated steelworks. Comparisons were made between concentrations of solvent extractable matter (SEM) determined gravimetrically by Soxhlet (dichloromethane) extraction and those estimated immunologically by ELISA determination over a concentration range of 2000-330,000 mg SEM/kg soil dry weight. Both ELISA systems tinder-reported for the more weathered soil samples. Results suggest this is due to matrix effects in the sample rather than any inherent bias in the ELISA systems and it is concluded that, for weathered hydrocarbons typical of steelworks and coke production sites, the use of ELISA requires careful consideration as a field technique. Consideration of the target analyte relative to the composition of the hydrocarbon waste encountered appears critical.

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

    PubMed Central

    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

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

    PubMed

    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

    2016-04-01

    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.

  8. Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

    PubMed

    França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

    2016-10-15

    A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.

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

    PubMed

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

    2003-09-01

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

  10. Ice-active characteristics of soil bacteria selected by ice-affinity.

    PubMed

    Wilson, Sandra L; Kelley, Deborah L; Walker, Virginia K

    2006-10-01

    As an initial screen for microorganisms that produce ice-active macromolecules, ice-affinity was used to select microorganisms from soil consortia originating from three temperate regions. Once selected and subsequently purified to single colonies, these microbes were putatively identified by 16S ribosomal RNA sequencing and assayed for various ice-active properties. Ice-affinity selection appeared to select for bacteria with ice-associating activities: inhibition of ice recrystallization; ice nucleation; ice shaping. Although none of these activities were observed in Paenibacillus amyloliticus C8, others such as Chryseobacterium sp. GL8, demonstrated both ice recrystallization inhibition and ice-shaping activities. Pseudomonas borealis DL7 was classified as a type I ice nucleator, Flavobacterium sp. GL7, was identified as a type III ice nucleator and Acinetobacter radioresistens DL5 demonstrated ice recrystallization inhibition. In all, 19 different culturable bacteria were selected from the thousands of microbes in late-summer collected soil samples. Many of the selected microbes have been previously reported in glacial ice cores or polar sea ice, and of five isolates that were further characterized, four showed ice-associating activities. These results indicate the significant potential of ice-affinity selection even with temperate climate soils, suggesting that sampling in more extreme and remote areas is not required for the isolation of ice-active bacteria.

  11. Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein.

    PubMed

    Mali, Sujina; Moree, Wilna J; Mitchell, Morgan; Widger, William; Bark, Steven J

    2016-12-15

    Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.

  12. Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

    PubMed

    Shinya, Tomonori; Osada, Tomohiko; Desaki, Yoshitake; Hatamoto, Masahiro; Yamanaka, Yuko; Hirano, Hisashi; Takai, Ryota; Che, Fang-Sik; Kaku, Hanae; Shibuya, Naoto

    2010-02-01

    The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

  13. A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.

    PubMed

    Chen, Chen; El Khoury, Graziella; Zhang, Peiqing; Rudd, Pauline M; Lowe, Christopher R

    2016-04-29

    One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.

  14. Widespread purifying selection on RNA structure in mammals.

    PubMed

    Smith, Martin A; Gesell, Tanja; Stadler, Peter F; Mattick, John S

    2013-09-01

    Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies >4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5-22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1. This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes.

  15. Widespread purifying selection on RNA structure in mammals

    PubMed Central

    Smith, Martin A.; Gesell, Tanja; Stadler, Peter F.; Mattick, John S.

    2013-01-01

    Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies >4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5–22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1. This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes. PMID:23847102

  16. Differentiation of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; de Vellis, J.

    1981-01-01

    Homogeneous cultures of astrocytes and oligodendrocytes provide an excellent model system for studying the regulation of glial structure and function. Recently, a chemically defined (CD) medium was developed for purified cultures of astrocytes, thus eliminating the requirement for serum and providing a controlled system for the study of astroglial properties. Due to the widespread use of astrocyte cultures and the potential benefits to be gained from using a defined medium, astrocyte cultures raised in CD medium were analyzed for purity as well as morphological and biochemical properties. Purity was assessed using immunocytochemical staining for glial fibrillary acidic protein (GFAP) and fibronectin. Astrocytes raised in CD medium are 95% pure using the expression of GFAP as a criterion. Fewer than 1% of the cells in CD medium stained positive for fibronectin eliminating the possibility that CD medium is selective for meningeal or endothelial cells. Astrocytes raised in CD medium exhibit a striking degree of morphological differentiation as seen in scanning electron micrographs. They also exhibit a high degree of biochemical differentiation illustrated by increases in the specific activity of S-100 protein and the induction of glutamine synthetase by glucocorticoids. A defined medium that supports the proliferation of rat astrocytes and enhances numerous morphological and biochemical properties should greatly facilitate the study of factors controlling glial proliferation and differentiation.

  17. Simple, fast, and efficient process for producing and purifying trehalulose.

    PubMed

    Wei, Yutuo; Liang, Jiayuan; Huang, Ying; Lei, Panxian; Du, Liqin; Huang, Ribo

    2013-06-01

    A new property of recombinant trehalose synthase (GTase) from Thermus thermophilus HB-8 (ATCC 27634) was found and described in this study. GTase can act on sucrose and catalyze trehalulose formation without isomaltose, isomaltulose, or isomelezitose, releasing small amounts of glucose and fructose as byproducts. Maximum trehalulose yield (approximately 81%) was obtained at an optimum temperature of 65°C and was independent of substrate concentration. A simple, fast, and efficient method of producing and purifying trehalulose is then described. In the first step, GTase catalyzed trehalulose formation using a 20% sucrose substrate. Miscellaneous sugars were then rapidly removed, while trehalulose was completely preserved by Saccharomyces cerevisiae cells. Finally, the cells were separated by centrifugation, and salt ions were removed by an ion-exchange resin, subsequently obtaining a high-purity trehalulose solution. A trehalulose recovery rate of over 95% was achieved using this process. This method has a simple process, fast separation efficiency, and low investment in production equipment, so greatly to improve production efficiency and reduce production costs.

  18. Aflatoxin detoxification by manganese peroxidase purified from Pleurotus ostreatus

    PubMed Central

    Yehia, Ramy Sayed

    2014-01-01

    Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81 U mL−1, specific activity 78 U mg−1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4–5 and the optimum temperature was 25 °C. The pure MnP activity was enhanced by Mn2+, Cu2+, Ca2+ and K+ and inhibited by Hg+2 and Cd+2. H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL−1 enzyme activities. PMID:24948923

  19. Bioavailability of purified subcellular metals to a marine fish.

    PubMed

    Guo, Feng; Yao, Jie; Wang, Wen-Xiong

    2013-09-01

    In the present study, the authors used a supply of naturally contaminated oysters to investigate how the subcellular metal distribution and the metal burden in prey affected the transfer of metals to a marine fish, the grunt Terapon jarbua. The oysters, Crassostrea hongkongensis, each with different contamination histories, were collected and separated into 3 subcellular fractions: 1) metal-rich granules, 2) cellular debris, and 3) a combined fraction of organelles, heat-denatured proteins, and metallothionein-like proteins, defined as the trophically available metal (TAM). These purified fractions showed a wide range of metal concentrations and were fed to the fish for a period of 7 d at a daily comparable feeding rate of 3% of fish body weight. After 7 d exposure, the newly absorbed metals were mainly distributed in the intestine and liver, indicating a significant tissue-specific trophic transfer, especially for Cd and Cu. The trophic transfer factors (TTFs) showed a sequence of cellular debris >TAM > metal-rich granules, suggesting the impact of subcellular distribution in prey on metal bioavailability. However, significant inverse relationships between the TTFs and the metal concentrations in diets were also found in the present study, especially for Cd and Zn. The subcellular metal compartmentalization might be less important than the metal concentration in prey influencing the trophic transfer. The authors' results have important implications for bioavailability and environmental assessment of dietary metals.

  20. [Micronized purified flavonoid fraction in treatment of pelvic varicose veins].

    PubMed

    Gavrilov, S G; Karalkin, A V; Moskalenko, E P; Beliaeva, E S; Ianina, A M; Kirienko, A I

    2012-01-01

    Presented herein are the results of studying efficacy of micronized purified flavonoid fraction (MPFF) in treatment of pelvic varicose veins (PVV) using reference ray-tracing methods of study. We examined a total of 85 patients with PVV. Of these, 65 subjects were found to have isolated dilatation of pelvic venous plexuses (study group), and 20 were diagnosed as having combined dilation of gonadal veins and venous plexuses of the pelvis (control group). Besides clinical examination, the patients were subjected to ultrasonographic angioscanning (USAS) and emission computed tomography (ECT) of pelvic veins before treatment and 2, 6, 12, 24, 36 and 60 months after the beginning of phlebotrophic therapy. Based on the findings of the clinical and instrumental studies, it was determined that MPFF was most efficient in patients with isolated dilatation of uterine and parametrial veins. In this group of patients, pelvic pain and other symptoms of the disease disappeared completely and the clinical effect persisted for a long time (up to 6-9 months). In the control group, venotonic therapy had a positive effect which was less pronounced as compared to the control group, and pelvic pain reappeared in the nearest time (up to 3 weeks) after withdrawal of MPFF.

  1. Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphoro thioyloxy) benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-l...

  2. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

    PubMed

    Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

    2017-01-01

    Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

  3. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Phillips, Tsaiyun I.; Shapiro, Linda G.

    1989-03-01

    The affine-transformation matching scheme proposed by Hummel and Wolfson (1988) is very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. This paper addresses the implementation of the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. It points out errors that can occur with this method due to quantization, stability, symmetry, and noise problems. By beginning with an explicit noise model which the Hummel and Wolfson technique lacks, we can derive an optimal approach which overcomes these problems. We show that results obtained with the new algorithm are clearly better than the results from the original method.

  4. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  5. Negative affinity X-ray photocathodes

    NASA Technical Reports Server (NTRS)

    Vanspeybroeck, L.; Kellogg, E.; Murray, S.; Duckett, S.

    1974-01-01

    A new X-ray image intensifier is described. The device should eventually have a quantum efficiency which is an order of magnitude greater than that of presently available high spatial resolution X-ray detectors, such as microchannel plates. The new intesifier is based upon a GaAs crystal photocathode which is activated to achieve negative electron affinity. Details concerning the detector concept are discussed together with the theoretical relations involved, X-ray data, and optical data.

  6. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency

    PubMed Central

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  7. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  8. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

  9. Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

    PubMed

    Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

    2000-12-15

    The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

  10. Purified Shigella enterotoxin does not alter intestinal motility.

    PubMed Central

    Fernandez, A; Sninsky, C A; O'Brien, A D; Clench, M H; Mathias, J R

    1984-01-01

    A purified Shigella enterotoxin (pST) and a cell-free lysate with pST removed (CFL-pST) from the whole-cell lysate of Shigella dysenteriae 60 R were used to study their effect on the myoelectric activity and mucosal integrity of rabbit ileal segments. We have previously defined two myoelectric patterns: the migrating action potential complex and repetitive bursts of action potentials that occur in response to certain bacteria and their enterotoxins. The in vivo model consisted of isolated ileal segments in male New Zealand White rabbits. The segments were infused with sterile saline (1 ml/h), pST (2.4-micrograms injection), or CFL-pST (1 ml/h). Myoelectric activity in the segments exposed to pST was similar to that with the saline infusion, but CFL-pST induced significant alterations in myoelectric activity in the form of repetitive bursts of action potentials. The mucosa of the segments exposed to pST showed only mild inflammatory changes. In contrast, CFL-pST caused moderate to severe inflammatory changes with enterocyte necrosis. These studies show that pST, a known enterotoxin, did not alter myoelectric activity and had no significant effect on the integrity of ileal mucosa, as determined by light microscopy. CFL-pST caused both inflammation and tissue necrosis with significant alterations in motor activity. These studies suggest that S. dysenteriae 60 R produces a substance or substances other than pST that cause florid in vivo cytotoxicity and alter myoelectric activity. Images PMID:6363286

  11. Development of a Microwave Regenerative Sorbent-Based Hydrogen Purifier

    NASA Technical Reports Server (NTRS)

    Wheeler, Richard R., Jr.; Dewberry, Ross H.; McCurry, Bryan D.; Abney, Morgan B.; Greenwood, Zachary W.

    2016-01-01

    This paper describes the design and fabrication of a Microwave Regenerative Sorbent-based Hydrogen Purifier (MRSHP). This unique microwave powered technology was developed for the purification of a hydrogen stream produced by the Plasma Pyrolysis Assembly (PPA). The PPA is a hydrogen recovery (from methane) post processor for NASA's Sabatier-based carbon dioxide reduction process. Embodied in the Carbon dioxide Reduction Assembly (CRA), currently aboard the International Space Station (ISS), the Sabatier reaction employs hydrogen to catalytically recover oxygen, in the form of water, from respiratory carbon dioxide produced by the crew. This same approach is base-lined for future service in the Air Revitalization system on extended missions into deep space where resupply is not practical. Accordingly, manned exploration to Mars may only become feasible with further closure of the air loop as afforded by the greater hydrogen recovery permitted by the PPA with subsequent hydrogen purification. By utilizing the well-known high sorbate loading capacity of molecular sieve 13x, coupled with microwave dielectric heating phenomenon, MRSHP technology is employed as a regenerative filter for a contaminated hydrogen gas stream. By design, freshly regenerated molecular sieve 13x contained in the MRSHP will remove contaminants from the effluent of a 1-CM scale PPA for several hours prior to breakthrough. By reversing flow and pulling a relative vacuum the MRSHP prototype then uses 2.45 GHz microwave power, applied through a novel coaxial antenna array, to rapidly heat the sorbent bed and drive off the contaminants in a short duration vacuum/thermal contaminant desorption step. Finally, following rapid cooling via room temperature cold plates, the MRSHP is again ready to serve as a hydrogen filter.

  12. Correcting for Purifying Selection: An Improved Human Mitochondrial Molecular Clock

    PubMed Central

    Soares, Pedro; Ermini, Luca; Thomson, Noel; Mormina, Maru; Rito, Teresa; Röhl, Arne; Salas, Antonio; Oppenheimer, Stephen; Macaulay, Vincent; Richards, Martin B.

    2009-01-01

    There is currently no calibration available for the whole human mtDNA genome, incorporating both coding and control regions. Furthermore, as several authors have pointed out recently, linear molecular clocks that incorporate selectable characters are in any case problematic. We here confirm a modest effect of purifying selection on the mtDNA coding region and propose an improved molecular clock for dating human mtDNA, based on a worldwide phylogeny of > 2000 complete mtDNA genomes and calibrating against recent evidence for the divergence time of humans and chimpanzees. We focus on a time-dependent mutation rate based on the entire mtDNA genome and supported by a neutral clock based on synonymous mutations alone. We show that the corrected rate is further corroborated by archaeological dating for the settlement of the Canary Islands and Remote Oceania and also, given certain phylogeographic assumptions, by the timing of the first modern human settlement of Europe and resettlement after the Last Glacial Maximum. The corrected rate yields an age of modern human expansion in the Americas at ∼15 kya that—unlike the uncorrected clock—matches the archaeological evidence, but continues to indicate an out-of-Africa dispersal at around 55–70 kya, 5–20 ky before any clear archaeological record, suggesting the need for archaeological research efforts focusing on this time window. We also present improved rates for the mtDNA control region, and the first comprehensive estimates of positional mutation rates for human mtDNA, which are essential for defining mutation models in phylogenetic analyses. PMID:19500773

  13. Affinity labelling and identification of the high-affinity choline carrier from synaptic membranes of Torpedo electromotor nerve terminals with [3H]choline mustard.

    PubMed

    Rylett, R J

    1988-12-01

    The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.

  14. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    PubMed

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  15. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  16. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, C.R.; Ito, Takashi; Smith, C.L.

    1996-01-09

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

  17. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

    1996-01-01

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

  18. A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

    PubMed Central

    Man-Kupisinska, Aleksandra; Michalski, Mateusz; Maciejewska, Anna; Swierzko, Anna S.; Cedzynski, Maciej; Lugowski, Czeslaw; Lukasiewicz, Jolanta

    2016-01-01

    Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3. PMID:27232184

  19. Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

    PubMed

    Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

    2016-11-01

    We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.

  20. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    SciTech Connect

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.