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Sample records for antibody variants produced

  1. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    PubMed

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  2. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    PubMed

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

  3. Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants.

    PubMed

    Lai, Huafang; He, Junyun; Hurtado, Jonathan; Stahnke, Jake; Fuchs, Anja; Mehlhop, Erin; Gorlatov, Sergey; Loos, Andreas; Diamond, Michael S; Chen, Qiang

    2014-10-01

    Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases. PMID:24975464

  4. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    PubMed

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. PMID:27214604

  5. Heterologous expressed toxic and non-toxic peptide variants of toxin CssII are capable to produce neutralizing antibodies against the venom of the scorpion Centruroides suffusus suffusus.

    PubMed

    Hernández-Salgado, Kenya; Estrada, Georgina; Olvera, Alejandro; Coronas, Fredy I; Possani, Lourival D; Corzo, Gerardo

    2009-08-15

    Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30microg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.

  6. Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast

    PubMed Central

    Xu, Ping; Robinson, Anne Skaja

    2009-01-01

    Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the unfolded protein response, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4−4−20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the unfolded protein response. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties. PMID:19415776

  7. Engineered antibody Fc variants with enhanced effector function

    NASA Astrophysics Data System (ADS)

    Lazar, Greg A.; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S.; Hyun, Linus; Chan, Cheryl; Chung, Helen S.; Eivazi, Araz; Yoder, Sean C.; Vielmetter, Jost; Carmichael, David F.; Hayes, Robert J.; Dahiyat, Bassil I.

    2006-03-01

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fc receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy. antibody-dependent cell-mediated cytotoxicity | FcR | protein engineering | cancer

  8. Exceptional Antibodies Produced by Successive Immunizations.

    PubMed

    Gearhart, Patricia J; Castiblanco, Diana P; Russell Knode, Lisa M

    2015-12-01

    Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations. PMID:26641938

  9. Exceptional Antibodies Produced by Successive Immunizations

    PubMed Central

    Gearhart, Patricia J.; Castiblanco, Diana P.; Russell Knode, Lisa M.

    2015-01-01

    Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations. PMID:26641938

  10. Engineered antibody Fc variants with enhanced effector function

    PubMed Central

    Lazar, Greg A.; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S.; Hyun, Linus; Chan, Cheryl; Chung, Helen S.; Eivazi, Araz; Yoder, Sean C.; Vielmetter, Jost; Carmichael, David F.; Hayes, Robert J.; Dahiyat, Bassil I.

    2006-01-01

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcγ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcγ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy. PMID:16537476

  11. Engineered antibody Fc variants with enhanced effector function.

    PubMed

    Lazar, Greg A; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S; Hyun, Linus; Chan, Cheryl; Chung, Helen S; Eivazi, Araz; Yoder, Sean C; Vielmetter, Jost; Carmichael, David F; Hayes, Robert J; Dahiyat, Bassil I

    2006-03-14

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

  12. Glycosylation of plant produced human antibodies.

    PubMed

    Kallolimath, Somanath; Steinkellner, Herta

    2015-12-23

    Human immunoglobulins circulate as highly heterogeneously glycosylated mixture of otherwise homogeneous protein backbones. A series of studies, mainly on IgG, have unequivocally proven that antibodies modulate their effector function through sugars present in the Fc domain. However, our limited technology in producing complex proteins such as antibodies, with defined glycan structures hamper in depths studies. This review introduces a plant based expression platform enabling engineering of antibody glycans. The procedure is based on the simultaneous delivery of appropriate constructs, carrying cDNAs of target proteins (e.g. heavy and light chain of antibodies) in combination with human glycosylation enzymes into plant leaves. Harvesting of recombinant proteins one week post construct delivery allows high speed and flexibility. Major achievements include the production of functional active slialylated pentameric IgMs in tobacco leaves. The system provides a viable approach to the generation of antibodies with defined glycoforms on demand, contributing to studies on antibody glycans and the development of novel antibody based drugs. PMID:27472861

  13. Prion-Specific Antibodies Produced in Wild-Type Mice.

    PubMed

    Heegaard, Peter M H; Bergström, Ann-Louise; Andersen, Heidi Gertz; Cordes, Henriette

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant.

  14. Prion-Specific Antibodies Produced in Wild-Type Mice.

    PubMed

    Heegaard, Peter M H; Bergström, Ann-Louise; Andersen, Heidi Gertz; Cordes, Henriette

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant. PMID:26424281

  15. Production and characterization of a peptide-based monoclonal antibody against CD44 variant 6.

    PubMed

    Zarei, Saeed; Bayat, Ali Ahmad; Hadavi, Reza; Mahmoudi, Ahmad R; Tavangar, Banafsheh; Vojgani, Yasaman; Jeddi-Tehrani, Mahmood; Amirghofran, Zahra

    2015-02-01

    The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 provides a powerful tool to monitor and trace CD44v6 function in different biological fluids. In this study, a synthetic peptide from CD44v6 was conjugated to keyhole limpet hemocyanin (KLH) and injected into BALB/c mice. Splenocytes from the immunized mice were fused with murine SP2/0 myeloma cells followed by selection of antibody producing hybridoma cells. After screening of hybridoma colonies by ELISA, high affinity antibodies were selected and purified by affinity chromatography. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibodies. Six stable hybridoma cell lines, designated as 1H1, 1H2, 2A12, 2G11, 3H3, and 3H7, were obtained. Flow cytometry and immunocytochemistry results showed that the new monoclonal antibodies recognized CD44v6 on the cell surface. This novel panel of anti-CD44v6 antibodies has the potential for investigating the role of CD44v6 in cancer pathogenesis. PMID:25723282

  16. Considerations in producing preferentially reduced half-antibody fragments.

    PubMed

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity. PMID:26779832

  17. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  18. A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

    PubMed Central

    Babcook, J S; Leslie, K B; Olsen, O A; Salmon, R A; Schrader, J W

    1996-01-01

    We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755564

  19. Functional properties and effect on growth suppression of human neuroblastoma tumors by isotype switch variants of monoclonal antiganglioside GD2 antibody 14.18.

    PubMed

    Mujoo, K; Kipps, T J; Yang, H M; Cheresh, D A; Wargalla, U; Sander, D J; Reisfeld, R A

    1989-06-01

    A complete family of IgG isotype switch variant hybridomas was generated from the anti-GD2 monoclonal IgG3-producing hybridoma, 14.18, with the aid of the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a monoclonal antibodies (Mabs) produced by respective isotype switch variant hybridomas 14G1, 14G2b, or 14G2a, have binding activities for the biochemically defined GD2 antigen and GD2-expressing neuroblastoma target cell lines identical to that of IgG3 Mabs produced by the 14.18 parent cell line. This permitted us to examine the relative in vitro and in vivo cytotoxic capacities of each of the anti-GD2 antibodies for GD2-expressing neuroblastoma cells independent of antibody binding affinity or specificity. Mabs produced by 14.18, 14G2a, or 14G2b, but not 14G1, can direct efficient complement-dependent cytotoxicity against neuroblastoma tumor cells in the presence of human complement. Mabs produced by the parent 14.18 or by 14G2a are more efficient in directing antibody-dependent cell-mediated cytotoxicity than Mabs produced by 14G2b, and Mabs of 14G1 are inactive. However, despite these noted in vitro differences, antibodies produced by each member of this switch variant family suppress the growth of human neuroblastoma tumor cells in BALB/c athymic nu/nu mice. These studies suggest that a mechanism(s) other than Fc-directed complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity may account for the in vivo antitumor effects of these particular antibodies. PMID:2720646

  20. Antibody

    MedlinePlus

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

  1. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.

    PubMed

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia; Murray, Christopher J

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

  2. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    PubMed

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

  3. Multivalent group A streptococcal vaccine elicits bactericidal antibodies against variant M subtypes.

    PubMed

    Dale, James B; Penfound, Thomas; Chiang, Edna Y; Long, Valerie; Shulman, Stanford T; Beall, Bernard

    2005-07-01

    Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.

  4. Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

    PubMed

    Edgeworth, Matthew J; Phillips, Jonathan J; Lowe, David C; Kippen, Alistair D; Higazi, Daniel R; Scrivens, James H

    2015-12-01

    Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

  5. Microchip zone electrophoresis for high-throughput analysis of monoclonal antibody charge variants.

    PubMed

    Wheeler, Tobias D; Sun, Jing Lucy; Pleiner, Sina; Geier, Holger; Dobberthien, Philine; Studts, Joey; Singh, Rajendra; Fathollahi, Bahram

    2014-06-01

    A high-throughput screening assay on a microfluidic chip was developed for the determination of charge variants of monocolonal antibodies (mAbs) in pI range of 7-10. This method utilizes microchip zone electrophoresis for rapid separation (<90 s) of mAb charge variants that are labeled fluorescently without altering the overall charge. The microfluidic assay achieves between 8- and 90-fold times faster separation time over conventional methods while maintaining comparable resolution and profiles of charge variant distributions. We further characterized the assay with respect to (i) the effect of pH on resolution, (ii) the effect of excipients and buffering agents, (iii) the performance of the assay compared to conventional methods, and (vi) the reproducibility of charge variant profiles. Finally, we explored the utility of the assay with four case studies: (i) monitoring C-terminal lysine modification of a mAb, (ii) quantifying the extent of deamidation of a mAb, (iii) providing charge variant information on which to base clone selection, and (iv) making process parameter-related decisions from a "design of experiment" (DoE) study. The results of these case studies demonstrate the applicability of the microfluidic assay for high-throughput monitoring of mAb quality in process development of biopharmaceuticals.

  6. Nucleotide changes in sequential variants of influenza virus hemagglutinin genes and molecular structures of corresponding monoclonal antibodies specific for each variant.

    PubMed Central

    Meek, K; Johansson, B; Schulman, J; Bona, C; Capra, J D

    1989-01-01

    We have generated four monoclonal antibodies specific for one or more members of a series of two sequentially derived PR8 influenza virus variants. Three of these antibodies share cross-reactive idiotypes. The amino acid sequences of these antibodies were determined, and it was found that two of these antibodies use genes from the VH7183 family, whereas the third uses a gene from the VHJ558 family. All four monoclonal antibodies derive from different families of genes encoding the variable region of the kappa chain. The RNA sequence of the parent PR8 virus as well as the RNA sequences of the sequential variants were also determined, and it was demonstrated that the variant hemagglutinin molecules differed from the parent molecule by only a single amino acid interchange. Despite these subtle differences in antigenic structures of hemagglutinin, and the cross-reactive idiotype of the antibodies, their primary structures were very different. These data reinforce the idea that a wide variety of antibody structures exist which are directed against subtly different structures in biologically important antigens. PMID:2471975

  7. Development of a stable phosphoarginine analog for producing phosphoarginine antibodies.

    PubMed

    Ouyang, Han; Fu, Chuan; Fu, Songsen; Ji, Zhe; Sun, Ying; Deng, Peiran; Zhao, Yufen

    2016-02-14

    Protein phosphorylation is one of the most common and extensively studied post-translational modifications (PTMs). Compared to the O-phosphorylation on Ser, Thr and Tyr residues, our understanding of arginine phosphorylation is relatively limited, both in prokaryotes and eukaryotes, due to the intrinsic instability of phosphoarginine (pArg) and the lack of a feasible method to produce anti-pArg antibodies. We report the design and synthesis of a stable pArg analog, in which the labile N-P bond is replaced with a non-hydrolyzable C-P bond. Significantly, this analog was successfully used as a hapten to raise an immune response and the first mouse polyclonal antibody that specifically recognizes pArg-containing peptides and proteins was produced using analog-KLH conjugated as the immunogen. The generated antibody shows excellent specificity towards pArg-containing peptides and proteins, and could be used for a variety of biological detection methods. This provides us an invaluable tool to unravel the mystery of the biological function of pArg.

  8. Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

    PubMed Central

    Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

    2012-01-01

    The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

  9. An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

    PubMed

    Rizk, Shahir S; Luchniak, Anna; Uysal, Serdar; Brawley, Crista M; Rock, Ronald S; Kossiakoff, Anthony A

    2009-07-01

    We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.

  10. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    SciTech Connect

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji )

    1991-07-02

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with {sup 13}C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with (1-{sup 13}C)Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how {sup 13}C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

  11. Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

    PubMed

    Huss, David J; Pellerin, Alex F; Collette, Brian P; Kannan, Arun K; Peng, Liaomin; Datta, Abhishek; Wipke, Brian T; Fontenot, Jason D

    2016-07-01

    Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

  12. Autoimmunity and antibody affinity maturation are modulated by genetic variants on mouse chromosome 12.

    PubMed

    Collin, Roxanne; Dugas, Véronique; Chabot-Roy, Geneviève; Salem, David; Zahn, Astrid; Di Noia, Javier M; Rauch, Joyce; Lesage, Sylvie

    2015-04-01

    Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

  13. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens.

    PubMed

    Tan, Joshua; Pieper, Kathrin; Piccoli, Luca; Abdi, Abdirahman; Foglierini, Mathilde; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Ndungu, Francis Maina; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro; Marsh, Kevin; Thathy, Vandana; Corti, Davide; Sallusto, Federica; Bull, Peter; Lanzavecchia, Antonio

    2016-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

  14. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens.

    PubMed

    Tan, Joshua; Pieper, Kathrin; Piccoli, Luca; Abdi, Abdirahman; Foglierini, Mathilde; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Ndungu, Francis Maina; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro; Marsh, Kevin; Thathy, Vandana; Corti, Davide; Sallusto, Federica; Bull, Peter; Lanzavecchia, Antonio

    2016-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. PMID:26700814

  15. A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens

    PubMed Central

    Abdi, Abdirahman; Perez, Mathilde Foglierini; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Maina Ndungu, Francis; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro; Marsh, Kevin; Thathy, Vandana; Corti, Davide; Sallusto, Federica

    2016-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies1–4. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here, we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 100 amino acid collagen-binding domain of LAIR-1, an Ig superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B cell clone and carry distinct somatic mutations in the LAIR-1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. PMID:26700814

  16. Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

    PubMed Central

    Mimoto, Futa; Igawa, Tomoyuki; Kuramochi, Taichi; Katada, Hitoshi; Kadono, Shojiro; Kamikawa, Takayuki; Shida-Kawazoe, Meiri; Hattori, Kunihiro

    2013-01-01

    Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (TM) of the CH2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the TM of the CH2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens. PMID:23406628

  17. An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

    PubMed

    Irving, James A; Miranda, Elena; Haq, Imran; Perez, Juan; Kotov, Vadim R; Faull, Sarah V; Motamedi-Shad, Neda; Lomas, David A

    2015-05-15

    A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.

  18. Mechanistic modeling of ion-exchange process chromatography of charge variants of monoclonal antibody products.

    PubMed

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

    2015-12-24

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

  19. Mechanistic modeling of ion-exchange process chromatography of charge variants of monoclonal antibody products.

    PubMed

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

    2015-12-24

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

  20. Proteomic differences in recombinant CHO cells producing two similar antibody fragments

    PubMed Central

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin

    2016-01-01

    ABSTRACT Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  1. Recognition of influenza H3N2 variant virus by human neutralizing antibodies

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.

    2016-01-01

    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  2. Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase.

    PubMed

    Zhang, Yamin; Wang, Wentao; Xiao, Xue; Jia, Li

    2016-09-30

    Monoclonal antibodies (mAbs) are highly heterogeneous and complex glycoproteins requiring powerful analytical tools for characterization and quality control. In this work, we utilize adsorbed bovine serum albumin (BSA) as a stationary phase in open tubular (OT) capillary electrochromatography for separation of charge state variants of mAbs. Poly(diallydimethylammonium chloride) (PDDA) was used to assist fabrication of BSA coated OT column by electrostatic self-assembly. Scanning electron microscopy and electroosmotic flow measurement were carried out to characterize the as-prepared BSA coated PDDA OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of basic proteins and different charge state variants of mAbs. The effects of background solution pH and concentration on separation were investigated. A rapid separation of charge state variants of mAbs was successfully achieved in the BSA coated PDDA OT column. Separation of seven variants of the mAb cetuximab was achieved using the prepared column. Two basic variants and one acidic variant of rituximab, and two basic variants and four acidic variants of trastuximab were successfully distinguished from the main forms. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-to-day and batch-to-batch relative standard deviations of migration times less than 3.7%.

  3. Adherence of Staphylococcus aureus slime-producing strain variants to biomaterials used in orthopaedic surgery.

    PubMed

    Gracia, E; Fernández, A; Conchello, P; Laclériga, A; Paniagua, L; Seral, F; Amorena, B

    1997-01-01

    The adherence of Staphylococcus aureus to biomaterials used in orthopaedic surgery (polymethylmethacrylate, fresh bone, steel and titanium alloys) and to glass was studied in vitro at 1, 2, 6, 24 and 48 h of incubation. Nonslime-producing strains (72, 80 and 510) and slime-producing variants of these strains were used. An automated and fast method of ATP-bioluminiscence was applied to determine bacterial viability. The lowest adherence corresponded to polymethylmethacrylate and bone, and the highest to metals. Significant adherence was detected in all cases after 6 h and was strain dependent, being lowest for strain 72. In most cases, adherence of nonslime-producing variants was not significant compared with controls, and slime-producing were more adherent than nonslime-producing variants. These differences were maximal at 6 h or 48 h, depending on the strain and the material. The findings suggest that the appearance of slime-producing cells within a given nonslime-producing bacterial population may jeopardise postoperative immune systems and antibiotic efficacy as a consequence of biofilm formation on implants and prostheses.

  4. Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant.

    PubMed

    Mimoto, Futa; Igawa, Tomoyuki; Kuramochi, Taichi; Katada, Hitoshi; Kadono, Shojiro; Kamikawa, Takayuki; Shida-Kawazoe, Meiri; Hattori, Kunihiro

    2013-01-01

    Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T(M)) of the C(H)2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T(M) of the C(H)2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.

  5. Genetic variants are major determinants of CSF antibody levels in multiple sclerosis

    PubMed Central

    Pauwels, Ine; Gustavsen, Marte W.; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D.; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D’Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A.; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H.; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G.; Gourraud, Pierre-Antoine; Sawcer, Stephen J.; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F.

    2015-01-01

    Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index—the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10−16). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10−7). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10−37). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10−22), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10−6). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

  6. Genetic variants are major determinants of CSF antibody levels in multiple sclerosis.

    PubMed

    Goris, An; Pauwels, Ine; Gustavsen, Marte W; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D'Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G; Gourraud, Pierre-Antoine; Sawcer, Stephen J; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F

    2015-03-01

    Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

  7. Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies

    PubMed Central

    Bhiman, Jinal N.; Anthony, Colin; Doria-Rose, Nicole A.; Karimanzira, Owen; Schramm, Chaim A.; Khoza, Thandeka; Kitchin, Dale; Botha, Gordon; Gorman, Jason; Garrett, Nigel J.; Abdool Karim, Salim S.; Shapiro, Lawrence; Williamson, Carolyn; Kwong, Peter D.; Mascola, John R.; Morris, Lynn; Moore, Penny L.

    2015-01-01

    The elicitation of broadly neutralizing antibodies (bNAbs) is likely to be essential for a preventative HIV-1 vaccine, but this has not yet been achieved by immunization. In contrast some HIV-1-infected individuals naturally mount bNAb responses during chronic infection, suggesting that years of maturation are required for breadth1-6. Recent studies have shown that viral diversification precedes the emergence of bNAbs but the significance of this observation is unknown7,8. Here, we delineate the key viral events that drove neutralization breadth within the CAP256-VRC26 family of 33 monoclonal antibodies (mAbs) isolated from a superinfected individual. First, we identified minority viral variants that were distinct from both transmitted/founder (T/F) viruses and efficiently engaged the bNAb precursor, termed bNAb-initiating envelopes. Second, deep sequencing revealed a pool of diverse epitope variants (immunotypes) that were preferentially neutralized by broader members of the antibody lineage. In contrast, a “dead-end” antibody sublineage unable to neutralize these immunotypes showed limited evolution and failed to develop breadth. Thus, early viral escape at key antibody-virus contact sites selects for sublineages that can tolerate these changes, providing a new mechanism for the generation of neutralization breadth within a developing antibody lineage. PMID:26457756

  8. Is there a difference among human populations in the rate with which mutation produces electrophoretic variants?

    PubMed Central

    Neel, J V; Rothman, E

    1981-01-01

    Data are summarized that suggest that tropical-zone/tribal/nonindustrialized populations have higher frequencies of certain types of protein variants than temperate-zone/civilized/industrial populations, and it is demonstrated that these differences are not an artifact produced by the contagious type of sampling used with respect to tribal populations. Evidence is reviewed that suggests that a possible explanation of this difference is higher mutation rates in the tribal populations studied. PMID:6942419

  9. Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Funk, J.; Biber, N.; Schneider, M.; Hauser, E.; Enzenmüller, S.; Förtsch, C.

    2015-01-01

    In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death. PMID:25824835

  10. Evidence for trisulfide bonds in a recombinant variant of a human IgG2 monoclonal antibody.

    PubMed

    Pristatsky, Pavlo; Cohen, Steven L; Krantz, Debra; Acevedo, Jillian; Ionescu, Roxana; Vlasak, Josef

    2009-08-01

    The hinge region of human IgG2 contains four cysteine residues involved in disulfide linkages between the heavy chains, as well as the heavy and light chains. These linkages provide the fundamental framework of three distinct IgG2 disulfide isoforms recently described. Here, we detail another, disulfide-related post-translational modification in a recombinant variant of human IgG2. Heterogeneity associated with this antibody was separated into several fractions by anion-exchange chromatography (AEX), which is an important initial step that highlights the resolving power of surface charge-based HPLC techniques. Mass spectrometry of the intact antibody revealed weakly resolved discrete covalent additions of 25-35 Da in one of the two main AEX fractions. Digestion by endoproteinase Lys-C performed under nonreducing conditions, as well as tandem MS experiments, narrowed the modification to the peptide-containing disulfide-bridged hinge structure. High mass resolution and accuracy measurements of the peptide strongly suggested an addition of one or two S atoms. The modification could be eliminated by a mild reducing treatment of the intact antibody. Overall, these findings are consistent with the replacement of up to two disulfide bridges (S-S) with a like number of trisulfides (S-S-S) in the antibody hinge. The trisulfide modification is rather uncommon for proteins and its possible origins in the IgG2 variant are discussed.

  11. Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

    PubMed Central

    Nara, P L; Robey, W G; Pyle, S W; Hatch, W C; Dunlop, N M; Bess, J W; Kelliher, J C; Arthur, L O; Fischinger, P J

    1988-01-01

    Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV. PMID:3392769

  12. Should charge variants of monoclonal antibody therapeutics be considered critical quality attributes?

    PubMed

    Singh, Sumit Kumar; Narula, Gunjan; Rathore, Anurag S

    2016-09-01

    Charge variants, namely acidic and basic variants, are typically found in mAb therapeutics. Charge heterogeneity is typically not regarded to affect safety and efficacy of the product. As a result, the commonly followed approach involves assignment of a specification for the variants based on statistical analysis of variability in levels that is seen during commercial manufacturing. This is followed by monitoring of product quality to demonstrate consistency. This paper aims to demonstrate that this perception of charge variants warrants a more in-depth investigation to evaluate the role charge variants play in safety and efficacy of a mAb therapeutic. In addition, a novel procedure has been suggested for making this assessment and alleviate the problems that are traditionally faced when isolating these variants for characterization. The suggested procedure utilizes the principles of bioseparations, cell biology, and statistics and it is demonstrated that this is significantly more efficient than the approach practiced today. PMID:27387433

  13. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  14. Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency

    PubMed Central

    Chen, Long; Cohen, Justin; Song, Xiaoda; Zhao, Aishan; Ye, Zi; Feulner, Christine J.; Doonan, Patrick; Somers, Will; Lin, Laura; Chen, Peng R.

    2016-01-01

    Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme’s poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation. PMID:27534437

  15. Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency.

    PubMed

    Chen, Long; Cohen, Justin; Song, Xiaoda; Zhao, Aishan; Ye, Zi; Feulner, Christine J; Doonan, Patrick; Somers, Will; Lin, Laura; Chen, Peng R

    2016-01-01

    Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme's poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation. PMID:27534437

  16. Emergence of immunoglobulin variants following treatment of a B cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin

    PubMed Central

    1987-01-01

    The potency and specificity of immunotoxins consisting of monoclonal antiidiotype conjugated to the ribosome-inactivating protein, saporin, have been evaluated in the treatment of guinea pig L2C B lymphocytic leukemia. The immunotoxins were therapeutically much more effective than their parent antibodies. Their specificity reflected that of their antiidiotype component. Although the leukemia emerged eventually in most animals treated with these conjugates, most of the cells showed altered Ig expression, which rendered them resistant to the therapy. Commonly, the emerging cells had lost mu heavy chain production, leaving them negative for intracellular, surface, and secreted IgM, but still positive for lambda light chain production. In addition, a minor group of L2C variants was identified in a protocol designed to detect mutants at very low frequency: here the cells were exposed in vitro to immunotoxin and, while still viable as judged by dye-exclusion, inoculated in large numbers into animals. In tumor that emerged under these circumstances, the majority of cells were again immunoglobulin- negative; however a minority exhibited IgM with an altered idiotype (Idiotope-loss variants), rendering them unreactive with immunotoxin. Immunotherapy with unmodified anti-Id antibody alone does not reveal these variants, and we suggest it is the increased selective force exerted by the highly potent immunotoxins that allow these minor nonreactive populations to emerge. PMID:3110351

  17. Protein adsorption on ion exchange resins and monoclonal antibody charge variant modulation.

    PubMed

    Guélat, Bertrand; Khalaf, Rushd; Lattuada, Marco; Costioli, Matteo; Morbidelli, Massimo

    2016-05-20

    A novel multicomponent adsorption equilibrium model for proteins on ion-exchange resins is developed on a statistical thermodynamic basis including surface coverage effects and protein-resin and protein-protein interactions. The resulting model exhibits a general competitive Langmuirian behavior and was applied to the study and optimization of the separation of monoclonal antibody charge variants on two strong cation exchangers. The model accounts explicitly for the effect of both pH and salt concentration, and its parameters can be determined in diluted conditions, that is, through physically sound assumptions, all model parameters can be obtained using solely experiments in diluted conditions, and be used to make predictions in overloaded conditions. The parameterization of the model and optimization of the separation is based on a two-step approach. First, gradient experiments in diluted conditions are undertaken in order to determine the model parameters. Based on these experiments and on information about the proteins of interest and the stationary phase used, all the model parameters can be estimated. Second, using the parameterized model, an initial Pareto optimization is undertaken where overloaded operating conditions are investigated. Experiments from this Pareto set are then used to refine the estimation of the model parameters. A second Pareto optimization can then be undertaken, this time with the refined parameters. This can be repeated until a satisfactory set of model parameters is found. This iterative approach is shown to be extremely efficient and to provide large amounts of knowledge based on only a few experiments. It is shown that due to the strong physical foundation of the model and the very low number of adjustable parameters, the number of iterations is expected to be at most two or three. Furthermore, the model based tool is improved as more experimental knowledge is provided, allowing for better estimations of the chromatographic

  18. Protein adsorption on ion exchange resins and monoclonal antibody charge variant modulation.

    PubMed

    Guélat, Bertrand; Khalaf, Rushd; Lattuada, Marco; Costioli, Matteo; Morbidelli, Massimo

    2016-05-20

    A novel multicomponent adsorption equilibrium model for proteins on ion-exchange resins is developed on a statistical thermodynamic basis including surface coverage effects and protein-resin and protein-protein interactions. The resulting model exhibits a general competitive Langmuirian behavior and was applied to the study and optimization of the separation of monoclonal antibody charge variants on two strong cation exchangers. The model accounts explicitly for the effect of both pH and salt concentration, and its parameters can be determined in diluted conditions, that is, through physically sound assumptions, all model parameters can be obtained using solely experiments in diluted conditions, and be used to make predictions in overloaded conditions. The parameterization of the model and optimization of the separation is based on a two-step approach. First, gradient experiments in diluted conditions are undertaken in order to determine the model parameters. Based on these experiments and on information about the proteins of interest and the stationary phase used, all the model parameters can be estimated. Second, using the parameterized model, an initial Pareto optimization is undertaken where overloaded operating conditions are investigated. Experiments from this Pareto set are then used to refine the estimation of the model parameters. A second Pareto optimization can then be undertaken, this time with the refined parameters. This can be repeated until a satisfactory set of model parameters is found. This iterative approach is shown to be extremely efficient and to provide large amounts of knowledge based on only a few experiments. It is shown that due to the strong physical foundation of the model and the very low number of adjustable parameters, the number of iterations is expected to be at most two or three. Furthermore, the model based tool is improved as more experimental knowledge is provided, allowing for better estimations of the chromatographic

  19. Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody

    PubMed Central

    Shymanets, Aliaksei; Prajwal; Vadas, Oscar; Czupalla, Cornelia; LoPiccolo, Jaclyn; Brenowitz, Michael; Ghigo, Alessandra; Hirsch, Emilio; Krause, Eberhard; Wetzker, Reinhard; Williams, Roger L.; Harteneck, Christian; Nürnberg, Bernd

    2015-01-01

    Class IB phosphoinositide 3-kinases (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled-receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody (mAb(A)p110γ) to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ-interface, hydrogen-deuterium exchange coupled to mass spectrometry measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30 times more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs. PMID:26173259

  20. Surface plasmon resonance-based competition assay to assess the sera reactivity of variants of humanized antibodies.

    PubMed

    Gonzales, Noreen R; Schuck, Peter; Schlom, Jeffrey; Kashmiri, Syed V S

    2002-10-15

    While clinical trials are the only way to evaluate the immunogenicity, in patients, of murine or genetically engineered humanized variants of a potentially therapeutic or diagnostic monoclonal antibody (MAb), ethical and logistical considerations of clinical trials do not permit the evaluation of variants of a given MAb that are generated to minimize its immunogenicity. The most promising variant could be identified by comparing the reactivities of the parental antibody (Ab) and its variants to the sera of patients containing anti-variable region (anti-VR) Abs to the administered parental Ab. We have developed a surface plasmon resonance (SPR) biosensor-based assay to monitor the binding of the sera anti-VR Abs to the parental Ab and the inhibition of this binding by the variants. SPR biosensors allow the real-time detection and monitoring of the binding between an immobilized protein and its soluble ligand without the need for prior purification and labeling of the mobile analyte. This new assay requires no radiolabeling, is relatively less time-consuming, and uses only small amounts of serum (5-20 microl of diluted serum) through a new microfluidic sample handling technique. To validate the assay, we have tested the relative reactivities of the CDR-grafted anti-carcinoma Ab, HuCC49, and its two variants, designated V5 and V10, to the sera of patients who were earlier administered radiolabeled murine CC49 in a clinical trial. A comparison of IC(50)s (the concentrations of the competitor Abs required for 50% inhibition of the binding of sera to immobilized HuCC49) showed that V5 and V10 were less reactive than HuCC49 to the three patients' sera tested. We have also demonstrated, for the first time, the specific detection and comparison of relative amounts of anti-VR Abs present in the sera of different patients without prior removal of anti-murine Fc Abs and/or circulating antigen. This may facilitate the rapid screening, for the presence of anti-VR Abs, of the

  1. Antibody producing capacity to the bacteriophage phi X174 in rheumatoid arthritis.

    PubMed Central

    Bucknall, R; Bacon, P; Elson, C; Jones, J V

    1987-01-01

    A study of antibody production in response to a primary immunogen, the bacteriophage phi X174, was performed in 27 patients with rheumatoid arthritis and 15 controls. All patients produced a primary (IgM) response to initial immunisation. The frequency distribution of peak antibody titres after secondary immunisation showed a marked difference between the patients and controls, with 10 patients having peak titres below 5000. The IgG component of the antibody response expressed as a percentage of total phage antibody on the 10th day after secondary immunisation was less in the patients than in the control group. There was no correlation between antibody titres and indices of disease activity, rheumatoid factor titres, or the presence of DRw4, DRw3, and DRw2. After secondary immunisation the patients with rheumatoid arthritis were treated with D-penicillamine, azathioprine, levamisole, or maintained on a non-steroidal anti-inflammatory drug. Assessment of response to tertiary immunisation again showed an impairment of antibody production in the rheumatoid group receiving non-steroidal anti-inflammatory drugs compared with the controls. None of the drugs, D-penicillamine, azathioprine, or levamisole, produced further suppression or augmentation of antibody production in response to the immunogen. Images PMID:2962541

  2. Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

    PubMed Central

    Cook, R F; Berger, S L; Rushlow, K E; McManus, J M; Cook, S J; Harrold, S; Raabe, M L; Montelaro, R C; Issel, C J

    1995-01-01

    Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were

  3. Cells that produce deleterious autoreactive antibodies are vulnerable to suicide.

    PubMed

    Niu, Haitao; Leung, Danny T M; Ma, Chun Hung; Law, Eric C Y; Tam, Frankie C H; Lim, Pak-Leong

    2008-08-01

    It is puzzling how autoreactive B cells that escape self-tolerance mechanisms manage to produce Abs that target vital cellular processes without succumbing themselves to the potentially deleterious effects of these proteins. We report that censorship indeed exists at this level: when the Ab synthesis in the cell is up-regulated in IL-6-enriched environments (e.g., adjuvant-primed mouse peritoneum), the cell dies of the increased intracellular binding between the Ab and the cellular autoantigen. In the case in which telomerase is the autoantigen, mouse hybridoma cells synthesizing such an autoantibody, which appeared to grow well in culture, could not grow in syngeneic BALB/c mice to form ascites, but grew nevertheless in athymic siblings. Culture experiments demonstrated that peritoneal cell-derived IL-6 (and accessory factors) affected the growth and functions of the hybridoma cells, including the induction of mitochondria-based apoptosis. Electron microscopy revealed an abundance of Abs in the nuclear chromatin of IL-6-stimulated cells, presumably piggy-backed there by telomerase from the cytosol. This nuclear presence was confirmed by light microscopy analysis of isolated nuclei. In two other cases, hybridoma cells synthesizing an autoantibody to GTP or osteopontin also showed similar growth inhibition in vivo. In all cases, Ab function was crucial to the demise of the cells. Thus, autoreactive cells, which synthesize autoantibodies to certain intracellular Ags, live delicately between life and death depending on the cytokine microenvironment. Paradoxically, IL-6, which is normally growth-potentiating for B cells, is proapoptotic for these cells. The findings reveal potential strategies and targets for immunotherapy. PMID:18641365

  4. Viral hemorrhagic septicemia of rainbow trout: relation between the G polypeptide and antibody production in protection of the fish after infection with the F25 attenuated variant.

    PubMed

    Bernard, J; de Kinkelin, P; Bearzotti-Le Berre, M

    1983-01-01

    A nonpathogenic variant of viral hemorrhagic septicemia virus has been selected which immunizes fish against a subsequent challenge with the wild-type virus strain. In this paper, we demonstrate that both the variant and the wild-type virus strains multiplied in spleen and kidney of infected fish, but the virus yield was lower for the variant and soon dropped below the sensitivity of our titration technique, which indicates that an early mechanism prevents the establishment of septicemia. This early mechanism could also be responsible for early protection since fish were already immunized 48 h postinfection with the variant. In a second step antibodies relayed that first defence mechanism to ensure long-lasting immunity. Antisera collected after immunization by the variant or the wild-type virus strain cross-reacted poorly in neutralization tests, which is in agreement with results obtained with plant lectins and proves that the variant is modified in its antigenic properties.

  5. Complete assignment of the methionyl carbonyl carbon resonance in switch variant anti-dansyl antibodies labeled with (1- sup 13 C)methionine

    SciTech Connect

    Kato, Koichi; Matsunaga, C.; Igarashi, Takako; Kim, Hahyung; Odaka, Asano; Shimada, Ichio; Arata, Yoji )

    1991-01-01

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire C{sub H}1 domain is deleted. The switch variant antibodies were specifically labeled with (1-{sup 13}C)methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating {sup 15}N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of ({alpha}-{sup 15}N)lysine and ({sup 15}N)threonine, both of which cannot become the substrate of transaminases. It was found that {beta}-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with {sup 15}N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val. On the basis of the results of the present {sup 13}C study, possible use of the assigned carbonyl carbon resonances for the elucidation of the structure-function relationship in the antibody system has been briefly discussed.

  6. IgG subclass distributions of anti-horse serum antibodies and natural venom-antibodies produced in response to antivenom injection or snake bite in humans.

    PubMed

    Ameno, S; Ameno, K; Fuke, C; Kiryu, T; Ijiri, I

    1990-01-01

    The Japanese Mamushi (Agkistrodon halys blomhoffi, BOIE) is the most common snake in Japan. Bite victims treated with antivenom (horse serum) can produce antibodies against the horse serum and the snake venom. We studied distributions of the IgG subclasses of both these antibodies produced in response to antivenom injection and snake bite. We found that IgG1 and IgG4 of each antibody in the victims' serum were present for a long period of time. PMID:2343468

  7. A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds.

    PubMed

    Sawayama, Andrew M; Chen, Michael M Y; Kulanthaivel, Palaniappan; Kuo, Ming-Shang; Hemmerle, Horst; Arnold, Frances H

    2009-11-01

    Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant. PMID:19774562

  8. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

    PubMed Central

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

  9. Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

    PubMed

    Ishii, Yoichi; Imamoto, Yasufumi; Yamamoto, Rie; Tsukahara, Masayoshi; Wakamatsu, Kaori

    2015-01-01

    Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation. PMID:25501618

  10. Immunological features of idiopathic Addison's disease: an antibody to cells producing steroid hormones

    PubMed Central

    Anderson, J. R.; Goudie, R. B.; Gray, Kathleen; Stuart-Smith, D. A.

    1968-01-01

    Antibodies to adrenocortical cells, occurring in the serum of patients with idiopathic Addison's disease, were investigated by the indirect immunofluorescence technique. With selected human adrenal tissue obtained post mortem, staining was brightest in the innermost cells of the adrenal cortex. Strongly positive sera were observed to react with all thirty specimens of adrenal tissue examined, but lipid-depleted adrenocortical tissue provided the most suitable reagent for detecting weak antibody. Immunofluorescence tests upon twenty positive sera, using a wide range of human tissues, revealed in one serum an antibody, or antibodies, which reacted with all types of cells producing steroid hormones, namely testicular Leydig cells, hilus cells of the ovary and testis, theca interna and corpus luteal cells of the ovary, and placental trophoblast. A second, weaker example of `steroid-cell antibody' was detected among the twenty sera. The significance of steroid-cell antibody is discussed briefly in relation to steroid hormone biosynthesis and gonadal failure in idiopathic Addison's disease. PMID:4868174

  11. A monoclonal antibody to VIII:C produced by a mouse hybridoma.

    PubMed

    Muller, H P; van Tilburg, N H; Derks, J; Klein-Breteler, E; Bertina, R M

    1981-11-01

    Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000-10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.

  12. Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission

    PubMed Central

    Gregory, James A.; Li, Fengwu; Tomosada, Lauren M.; Cox, Chesa J.; Topol, Aaron B.; Vinetz, Joseph M.; Mayfield, Stephen

    2012-01-01

    Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus

  13. Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

    PubMed Central

    Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

    2016-01-01

    anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines. PMID:26819303

  14. A neutralization-resistant Theiler's virus variant produces an altered disease pattern in the mouse central nervous system.

    PubMed Central

    Zurbriggen, A; Fujinami, R S

    1989-01-01

    Theiler's murine encephalomyelitis virus infection of mice is an animal model for human demyelinating diseases. To further define the role of this virus in the disease process, we selected a virus variant resistant to neutralization by a monoclonal antibody to VP-1. This virus variant was then injected into SJL/J mice. Central nervous system tissue was compared between variant virus- and wild-type virus-infected mice. Within the brain, no large differences were observed between the two groups as to the distribution of inflammatory infiltrates around the injection site and the number of viral antigen-positive cells during the first weeks of the observation period. In contrast, in the spinal cord major differences were found between variant virus- and wild-type virus-infected mice regarding the number of inflammatory lesions, infected cells, and the size of the areas involved with time. By immunohistochemistry, equivalent numbers of infected cells could be found in the spinal cord 1 week postinfection (p.i.): however, after that time, the number of infected cells in the wild-type virus-infected mice continued to increase, whereas the virus-positive cells from the variant virus-infected mice gradually decreased. Thus, the number of viral antigen-containing cells peaked by 1 week p.i. in the variant virus-infected animals. Conversely, the number of infected cells in the spinal cords from mice inoculated with wild-type virus steadily increased until 8 weeks p.i. At this time (8 weeks p.i.), no more variant virus antigen-positive cells could be observed within the spinal cord. Plaque assay of central nervous system tissue confirmed these differences between the two groups observed by immunohistochemistry. No infectious variant virus could be isolated after 2 weeks p.i. from the brain and 4 weeks p.i. from the spinal cord, whereas infectious wild-type virus could be detected up to the end of the observation period (12 weeks p.i.). Virus which was isolated from variant

  15. A neutralization-resistant Theiler's virus variant produces an altered disease pattern in the mouse central nervous system.

    PubMed

    Zurbriggen, A; Fujinami, R S

    1989-04-01

    Theiler's murine encephalomyelitis virus infection of mice is an animal model for human demyelinating diseases. To further define the role of this virus in the disease process, we selected a virus variant resistant to neutralization by a monoclonal antibody to VP-1. This virus variant was then injected into SJL/J mice. Central nervous system tissue was compared between variant virus- and wild-type virus-infected mice. Within the brain, no large differences were observed between the two groups as to the distribution of inflammatory infiltrates around the injection site and the number of viral antigen-positive cells during the first weeks of the observation period. In contrast, in the spinal cord major differences were found between variant virus- and wild-type virus-infected mice regarding the number of inflammatory lesions, infected cells, and the size of the areas involved with time. By immunohistochemistry, equivalent numbers of infected cells could be found in the spinal cord 1 week postinfection (p.i.): however, after that time, the number of infected cells in the wild-type virus-infected mice continued to increase, whereas the virus-positive cells from the variant virus-infected mice gradually decreased. Thus, the number of viral antigen-containing cells peaked by 1 week p.i. in the variant virus-infected animals. Conversely, the number of infected cells in the spinal cords from mice inoculated with wild-type virus steadily increased until 8 weeks p.i. At this time (8 weeks p.i.), no more variant virus antigen-positive cells could be observed within the spinal cord. Plaque assay of central nervous system tissue confirmed these differences between the two groups observed by immunohistochemistry. No infectious variant virus could be isolated after 2 weeks p.i. from the brain and 4 weeks p.i. from the spinal cord, whereas infectious wild-type virus could be detected up to the end of the observation period (12 weeks p.i.). Virus which was isolated from variant

  16. Crystal structure of a 3B3 variant - A broadly neutralizing HIV-1 scFv antibody

    SciTech Connect

    Clark, K. Reed; Walsh, Scott T.R.

    2009-12-10

    We present the crystal structure determination of an anti-HIV-1 gp120 single-chain variable fragment antibody variant, 3B3, at 2.5 {angstrom} resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site-directed mutagenesis of the variable heavy chain (V{sub H}) complementary-determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross-clade primary isolates of HIV-1 by interaction with the recessed CD4-binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR-H3 of the V{sub H} domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR-H3 of 3B3, in light of the b12-gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR-L3 of the variable light (VL) domain triggered by a point mutation in CDR-H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3-gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water.

  17. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    PubMed Central

    Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

  18. Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans

    PubMed Central

    Gersbach, Philipp; Hug, Melanie N.; Bieri, Raphael; Bomio, Claudio; Li, Jun; Huber, Sylwia; Altmann, Karl-Heinz; Pluschke, Gerd

    2016-01-01

    Background Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. Methodology/Principal Findings Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. Conclusions/Significance The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target. PMID:27351976

  19. Characterization of C-strain “Riems” TAV-epitope escape variants obtained through selective antibody pressure in cell culture

    PubMed Central

    2012-01-01

    Classical swine fever virus (CSFV) C-strain “Riems” escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo. It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining. PMID:22515281

  20. Immunoglobulin classes of antibodies produced in the primary and secondary responses in man*

    PubMed Central

    Bandilla, K. K.; McDuffie, F. C.; Gleich, G. J.

    1969-01-01

    A method, radio-immunoprecipitation, is described which measures the antibody in the different classes in whole sera by its antigen binding capacity. In nineteen of twenty-four individuals immunized with Limulus polyphemus haemocyanin, haemagglutinating antibodies were produced in the primary response, but in only five were amounts significant enough to measure by radio-immunoprecipitation. In these five individuals, the primary response was characterized by an early appearance of IgM and IgA antibodies; IgG antibody was detectable but only in small amounts and not before the 18th day after immunization. All twenty-four individuals responded to a secondary injection. A sharp rise in IgG antibody after the secondary injection is typical of the secondary response. A definite but much lower anamnestic response for IgM and IgA is observed. The difference is so uniform and striking that it may well be characteristic for some primary responses in man. PMID:4189127

  1. DIFFERENTIATION AND FUNCTIONAL EXPRESSION OF POTENTIAL ANTIBODY-PRODUCING CELLS IN THE PRESENCE OF CHLORAMPHENICOL

    PubMed Central

    Schoenberg, Melvin D.; Moore, Richard D.; Weisberger, Austin S.

    1967-01-01

    Rabbits were immunized with diphtheria toxoid combined with complete Freund's adjuvant. Half of the animals were started on intramuscular injections of chloramphenicol 24 hr before the injection of the antigens. There was a general depression of protein synthesis in the immune system in the presence of chloramphenicol, but a greater effect on the synthesis of antibody than on the synthesis of proteins necessary for reproduction and maturation. In contrast to the finding of antibody in cells of the spleen and in the circulation of the control animals, those animals receiving chloramphenicol did not have measurable circulating antibody, and their spleens contained only a few cells with intracytoplasmic antibody late in the course of the experiment. Cytologically there was maturation of potential antibody-producing cells in the red pulp and nonfollicular white pulp of the spleen while the animals were receiving chloramphenicol. These cells developed more slowly, and were fewer and smaller than those of the control animals. They had numerous small, electron-opaque particles in their cytoplasm early in development. Ribosomes were synthesized, though fewer in number. The endoplasmic reticulum formed more slowly. PMID:10976231

  2. An animal model of pain produced by systemic administration of an immunotherapeutic anti-ganglioside antibody.

    PubMed

    Slart, R; Yu, A L; Yaksh, T L; Sorkin, L S

    1997-01-01

    For the management of pediatric neuroblastoma, a promising experimental treatment includes slow systemic infusion of a human/ mouse chimeric monoclonal antibody against the GD2 ganglioside. Beneficial actions are however, accompanied by severe pain and altered cardiovascular tone. The pain is conventionally controllable with moderate to relatively high doses of intravenous morphine. An animal model was established to examine the change in nociceptive threshold produced by anti-GD2-antibody. Rats given bolus injections of antibody through an in-dwelling jugular catheter developed a quantifiable mechanical allodynia. At higher doses, allodynia and touch evoked agitation began within the first 15-min test interval, was maximal within the first hour, and for some doses was still present, although greatly reduced at 24 and 48 h. Rapid administration of antibody led to an increase in mean resting blood pressure of 12 mmHg +/- 1.8 (P < or = 0.02) and the development of a prolonged cardiovascular response to an otherwise innocuous stimulus. These observations demonstrate that the pain associated with monoclonal antibody treatment can be modeled in animals. This approach has potential for defining the pharmacology of the allodynia and ways in which the pain state may be ameliorated.

  3. Pituitary prolactinoma, pancreatic glucagonomas, and aldosterone-producing adrenal cortical adenoma: a suggested variant of multiple endocrine neoplasia type I.

    PubMed

    Gould, E; Albores-Saavedra, J; Shuman, J

    1987-12-01

    A case of a pituitary prolactinoma, pancreatic glucagonoma, and an aldosterone-producing adrenal cortical adenoma coexisting in a 65-year-old man is reported. This case may represent a sporadic variant of the multiple endocrine neoplasia syndrome type I first manifested by hyperaldosteronism.

  4. Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131).

    PubMed

    Mimoto, F; Katada, H; Kadono, S; Igawa, T; Kuramochi, T; Muraoka, M; Wada, Y; Haraya, K; Miyazaki, T; Hattori, K

    2013-10-01

    Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the C(H)2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.

  5. Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIaR131 and FcγRIIaH131

    PubMed Central

    Mimoto, F.; Katada, H.; Kadono, S.; Igawa, T.; Kuramochi, T.; Muraoka, M.; Wada, Y.; Haraya, K.; Miyazaki, T.; Hattori, K.

    2013-01-01

    Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIaR131 allotype to a similar degree because FcγRIIb and FcγRIIaR131 are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIaR131 and FcγRIIaH131. This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIaR131 and FcγRIIaH131 is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the CH2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics. PMID:23744091

  6. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    PubMed

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  7. Semiautomated pH gradient ion-exchange chromatography of monoclonal antibody charge variants.

    PubMed

    Talebi, Mohammad; Shellie, Robert A; Hilder, Emily F; Lacher, Nathan A; Haddad, Paul R

    2014-10-01

    A new approach using a chromatography system equipped with isocratic pumps and an electrolytic eluent generator (EG) is introduced, replacing external pH gradient delivery using conventional gradient systems, in which bottled buffers with preadjusted pH are mixed using a gradient pump. The EG is capable of generating high purity base or acid required for online preparation of the buffer at the point of use, utilizing deionized water as the only carrier stream. Typically, the buffer was generated from online titration of a reagent composed of low molecular weight amines. The reagent was delivered isocratically into a static mixing tee, where it was titrated to the required pH with electrolytically generated base or acid. The required pH gradient was thus conveniently generated by electrically controlling the concentration of titrant. Also, since the pH was adjusted at the point of use, this approach offered enhanced throughput in terms of eluent preparation time and labor, and with a more reproducible pH profile. The performance of the system was demonstrated by running pH gradients ranging from pH 8.2 to 10.9 on a polymer monolith cation-exchange column for high throughput profiling of charge heterogeneity of intact, basic therapeutic monoclonal antibodies. A high degree of flexibility in modulating the key parameters of the pH gradient, including the buffer concentration, the pH gradient slope and the operating pH range was demonstrated. This enabled fine-tuning of the separation conditions for each individual antibody in order to enhance the chromatographic resolution. PMID:25199803

  8. Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing.

    PubMed

    Grosse-Hovest, Ludger; Müller, Sigrid; Minoia, Rosa; Wolf, Eckhard; Zakhartchenko, Valeri; Wenigerkind, Hendrik; Lassnig, Caroline; Besenfelder, Urban; Müller, Mathias; Lytton, Simon D; Jung, Gundram; Brem, Gottfried

    2004-05-01

    Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing.

  9. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    SciTech Connect

    Iwasaki, Ryohei; Kiuchi, Hiroki; Ihara, Masaki; Mori, Toshihiro; Kawakami, Masayuki; Ueda, Hiroshi

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  10. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach.

    PubMed

    Fekete, Szabolcs; Beck, Alain; Fekete, Jenő; Guillarme, Davy

    2015-01-01

    Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach. PMID:25240157

  11. Anti-human AFP variant monoclonal antibody in radioimmunodetection of primary hepatocellular carcinoma

    PubMed Central

    Liu, Yang; Wu, Meng-Chao; Chen, Han; Zhang, Bai-He; Qian, Guang-Xiang; Pan, Wen-Zhou; Qiang, Mei-Yu

    1997-01-01

    AIM: To investigate the affinity of AFP-R-LCA monoclonal antibody (AFP-R-LCA McAb) for AFP-positive primary hepatocellular carcinoma (HCC) cells. METHODS: AFP-R-LCA McAb was labeled by 131I. Eleven cases of HCC with AFP positivity, 6 with AFP negativity, and 4 with hepatitis B-related cirrhosis were investigated by radioimmunodetection. RESULTS: The 131I-AFP-R-LCA McAb immunoreacted with 9 of the HCC AFP-positive cases (9/11), but with none of the 6 AFP negative HCC cases or of the 4 cirrhosis patients. 131I-AFP-R-LCA McAb at a small dose (7.4 × 107 Bq/300 μg) was associated with no side effects as determined by the liver function test, prothrombin time (Pt) test and thyroid gland function test (P > 0.05). Two cases of AFP-positive HCC were not imaged because of large tumor size (diameter > 10 cm) and higher AFP concentration in serum (20000 μg/L). CONCLUSION: AFP-R-LCA McAb has a strong and special affinity to AFP-positive HCC cells and may be useful as a carrier for radioimmunodetection and radioimmunotherapy. PMID:27053873

  12. Parkinson-associated risk variant in enhancer element produces subtle effect on target gene expression

    PubMed Central

    Soldner, Frank; Stelzer, Yonatan; Shivalila, Chikdu S.; Abraham, Brian J.; Latourelle, Jeanne C.; Barrasa, M. Inmaculada; Goldmann, Johanna; Myers, Richard H.; Young, Richard A.; Jaenisch, Rudolf

    2016-01-01

    Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases but mechanistic insights are impeded by the lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis1. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale chromatin mapping studies have highlighted the enrichment of GWAS variants in regulatory DNA elements of disease-relevant cell types2–6. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor (TF) binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression7–10. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells (hPSCs). By generating a genetically precisely controlled experimental system we identify a common Parkinson’s disease (PD)-associated risk variant in a non-coding distal enhancer element that regulates the expression of alpha-synuclein (SNCA), a key gene implicated in the pathogenesis of PD. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific TFs EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease relevant phenotypes. PMID:27096366

  13. Anthracycline metabolites from Streptomyces violaceus A262. III. New anthracycline obelmycins produced by a variant strain SE2-2385.

    PubMed

    Johdo, O; Watanabe, Y; Ishikura, T; Yoshimoto, A; Naganawa, H; Sawa, T; Takeuchi, T

    1991-10-01

    New anthracycline antibiotics, designated as obelmycins A, D, E, F and G, were isolated from the culture broth of a variant strain of beta-rhodomycin-producing Streptomyces violaceus A262, identified as beta-isorhodomycinone glycosides and gamma-isorhodomycinone glycosides and assayed for their in vitro cytotoxicities against murine leukemic L1210 cell culture and the antimicrobial activities in comparison with some known anthracyclines. PMID:1955396

  14. Hepatitis C Virus Hypervariable Region 1 Variants Presented on Hepatitis B Virus Capsid-Like Particles Induce Cross-Neutralizing Antibodies

    PubMed Central

    Bankwitz, Dorothea; Osburn, William; Viazov, Sergei; Brovko, Olena; Zekri, Abdel-Rahman; Khudyakov, Yury; Nassal, Michael; Pumpens, Paul; Pietschmann, Thomas; Timm, Jörg; Roggendorf, Michael; Walker, Andreas

    2014-01-01

    Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses. PMID:25014219

  15. IMMUNODOMINANT EPITOPE AND PROPERTIES OF PYROGLUTAMATE-MODIFIED Aβ-SPECIFIC ANTIBODIES PRODUCED IN RABBITS

    PubMed Central

    Acero, G.; Manoutcharian, K.; Vasilevko, V.; Munguia, M.E.; Govezensky, T.; Coronas, G.; Luz-Madrigal, A.; Cribbs, DH.; Gevorkian, G.

    2009-01-01

    N-truncated and N-modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer’s disease (AD) and Down’s syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Aβ is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full-length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 3 (AβN3(pE)). We demonstrated that AβN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AβN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AβN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AβN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:19545911

  16. Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits.

    PubMed

    Acero, G; Manoutcharian, K; Vasilevko, V; Munguia, M E; Govezensky, T; Coronas, G; Luz-Madrigal, A; Cribbs, D H; Gevorkian, G

    2009-08-18

    N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.

  17. Feasibility studies of using the Catfish Immune System to produce monoclonal antibodies

    SciTech Connect

    Poston, T.M.

    1987-03-01

    The objective of these studies was to determine the feasibility of using a teleost cell line to produce monoclonal antibodies. Studies were undertaken to demonstrate the production of a polyclonal response of channel catfish (Icatalurus punctatus) challenged with mycotoxins coupled to a protein carrier. Companion studies were also performed to induce a permanent cell line with catfish lymphocytes. Attempts to demonstrate a polyclonal response to haptenized mycotoxins were inconclusive. Tests to induce an immortal, permanent cell line with benzene and x-ray irradiated cells were also inconclusive. 3 refs., 13 tabs.

  18. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing

    PubMed Central

    Zhang, Siyan; Hughes, Jason D.; Murgolo, Nicholas; Levitan, Diane; Chen, Janice; Liu, Zhong

    2016-01-01

    Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. PMID:27088091

  19. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  20. Risk of squamous cell skin cancer after organ transplant associated with antibodies to cutaneous papillomaviruses, polyomaviruses, and TMC6/8 (EVER1/2) variants.

    PubMed

    Madeleine, Margaret M; Carter, Joseph J; Johnson, Lisa G; Wipf, Gregory C; Davis, Connie; Berg, Daniel; Nelson, Karen; Daling, Janet R; Schwartz, Stephen M; Galloway, Denise A

    2014-10-01

    Squamous cell skin cancer (SCSC) disproportionately affects organ transplant recipients, and may be related to increased viral replication in the setting of immune suppression. We conducted a nested case-control study among transplant recipients to determine whether SCSC is associated with antibodies to cutaneous human papillomaviruses (HPV), to genes associated with a rare genetic susceptibility to HPV (TMC6/TMC8), or to human polyomaviruses (HPyV). Cases (n = 149) had histologically confirmed SCSC, and controls (n = 290) were individually matched to cases on time since transplant, type of transplant, gender, and race. All subjects had serum drawn immediately prior to transplant surgery. Antibodies to 25 cutaneous HPVs and six HPyVs were assayed by detection of binding to virus-like particles, and 11 TMC6/8 variants were genotyped. After correction for multiple comparisons, only antibodies to HPV37 were associated with SCSC (OR 2.0, 95% CI 1.2-3.4). Common genetic variants of TMC6/8 were not associated with SCSC, but three variants in TMC8 (rs12452890, rs412611, and rs7208422) were associated with greater seropositivity for species 2 betapapillomaviruses among controls. This study suggests that some betaHPVs, but not polyomaviruses, may play a role in the excess risk of SCSC among transplant recipients.

  1. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    PubMed Central

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  2. Antibodies to Capsular Polysaccharide and Clumping Factor A Prevent Mastitis and the Emergence of Unencapsulated and Small-Colony Variants of Staphylococcus aureus in Mice▿

    PubMed Central

    Tuchscherr, Lorena P. N.; Buzzola, Fernanda R.; Alvarez, Lucía P.; Lee, Jean C.; Sordelli, Daniel O.

    2008-01-01

    The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated “escape mutants” and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections. PMID:18809660

  3. Verotoxins in Bovine and Meat Verotoxin-Producing Escherichia coli Isolates: Type, Number of Variants, and Relationship to Cytotoxicity▿

    PubMed Central

    Krüger, Alejandra; Lucchesi, Paula M. A.; Parma, Alberto E.

    2011-01-01

    In this study, we determined vt subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing Escherichia coli (VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a vt2 gene. Moreover, the vt2 subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other vt subtypes detected were vt1, vt1d, vt2vha, vt2vhb, vt2O118, vt2d (mucus activatable), and vt2g. A total of 41 (22%) of the isolates possessed more than one vt subtype in its genome, and among them the most frequent combination was vt1/vt2, but we also observed multiple combinations among vt2 subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single vt2 variant, those carrying the vt2 subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying vt2vha or vt2hb variants. Notably, the isolates carrying the vt1 subtype showed a lesser increase than that of most of the vt2-positive VTEC strains. Furthermore, the presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of vt variants more than with the serotype or origin of the isolate. PMID:21037301

  4. Bioengineering of a Nisin A-producing Lactococcus lactis to create isogenic strains producing the natural variants Nisin F, Q and Z.

    PubMed

    Piper, Clare; Hill, Colin; Cotter, Paul D; Ross, R Paul

    2011-05-01

    Nisin is the prototypical example of the lantibiotic family of antimicrobial peptides and has been employed as a food preservative for over half a century. It has also attracted attention due to its potency against a number of multidrug-resistant clinical pathogens. Nisin A is the originally isolated form of Nisin and a further five natural variants have been described which differ by up to 10 amino acids (of 34 in total in Nisin A). Nisins A, Z, F and Q are produced by Lactococcus lactis, while Nisins U and U2 are produced by Streptococcus sp. In this study we bioengineered the nisA gene of a Nisin A producer to generate genes encoding Nisins Z, F, Q, U and U2. We determined that while active Nisin Z, F and Q can be produced against this genetic background, active forms of Nisin U and U2 are not generated. Minimum inhibitory concentration studies with Nisin A, Z, F and Q variants against a series of different clinically significant pathogens establish differences in specific activities against selected targets. Nisin F was most impressive, being the most active, or one of the most active, against the MRSA strain ST 525, EC 676, EC 725, VISA 22900, VISA 22781, hVISA 35197, Staphylococcus aureus 8325-4 and L. lactis HP. Nisin Z was most active against ST 299, hVISA 32683 and, together with Nisin F, HP but had contrastingly poor activity against ST 525, EC 676 and 8325-4. Nisin F, Q and A exhibited similar potency against VISA 22900. This was the only target against which Nisin Q and Nisin A were among the most active variants. PMID:21375711

  5. Association of Human Immunoglobulin G1 Heavy Chain Variants With Neutralization Capacity and Antibody-Dependent Cellular Cytotoxicity Against Human Cytomegalovirus.

    PubMed

    Vietzen, Hannes; Görzer, Irene; Puchhammer-Stöckl, Elisabeth

    2016-10-15

    Human cytomegalovirus (HCMV) infection is limited by HCMV-specific antibody functions. Here the association between the genetic marker (GM) 3/17 variants in the immunoglobulin G1 (IgG1) heavy chain constant region, virus neutralization, and natural killer (NK)-cell activation was investigated. In 100 HCMV-seropositive individuals, the GM3/17 polymorphism, serum 50% HCMV antibody neutralization titer (NT50), and in vitro HCMV-specific antibody NK-cell activation were assessed. The HCMV NT50 was higher in heterozygous GM3/17 persons than in GM3/3 persons (P = .0276). Furthermore, individuals expressing GM3/17 exhibited significantly higher NK-cell activation than persons carrying GM3/3 (P < .0001) or GM17/17 (P = .0095). Thus, persons expressing GM3/17 have potentially a selective advantage in HCMV defense.

  6. Antigen Identification and Characterization of Lung Cancer Specific Monoclonal Antibodies Produced by mAb Proteomics

    PubMed Central

    Wang, Dongdong; Hincapie, Marina; Guergova-Kuras, Mariana; Kadas, Janos; Takacs, Laszlo; Karger, Barry L.

    2010-01-01

    A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with non-small cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment and polyclonal antibody affinity chromatography normalization. In this paper, in order to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail in order to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a KD of roughly 10−9 M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the β-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state. PMID:20146545

  7. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

    PubMed Central

    Tada, Minoru; Tatematsu, Ken-Ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. PMID:26261057

  8. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis. PMID:26803706

  9. Immunoblot for densitometric estimation of antibodies (IDEA): a useful method for quantification of intrathecally produced antibodies against individual antigens in infectious diseases of the central nervous system.

    PubMed

    Neumann, B; Ritter, K; Felgenhauer, K

    1991-11-01

    Intrathecally produced antibodies specific for the infectious agent can be shown by immunoblot. A quantification is practicable by titration. Densitometric evaluation of the immunoblot by a new technique, IDEA (immunoblot for densitometric estimation of antibodies), does not only render titration unnecessary, but also has the advantage of presenting the differentiated local immune response against individual antigens of the infectious agent. Preliminary studies for the densitometric evaluation were performed with a measles virus immunoblot which had been developed with a cerebrospinal fluid (CSF) sample of a patient suffering from multiple sclerosis containing high antibody titers. A peroxidase-labeled anti-immunoglobulin antibody had been added. The intensity of the color reaction on the membrane dependent on the antibody concentration follows a saturation kinetics comparable to the Michaelis-Menten kinetics in enzymology. Thus, indices for the intrathecal antibody synthesis of each individual viral antigen can be calculated by taking the permeability of the blood-CSF barrier into consideration. This method is illustrated in a patient with zoster meningoencephalitis.

  10. New anti-GD2 monoclonal antibodies produced from gamma-interferon-treated neuroblastoma cells.

    PubMed

    Gross, N; Beck, D; Portoukalian, J; Favre, S; Carrel, S

    1989-04-15

    Three monoclonal antibodies (IgG2) have been produced from hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice hyperimmunized with gamma-interferon-treated neuroblastoma cells. The 3 MAbs, 7A4, 2A6 and IG8, detected an antigen present on neuroblastoma tumors and cell lines, but also on some neuro-ectoderm-derived tissues and cells. All 3 clones were shown to react with an epitope of the di-sialo-ganglioside GD2 molecules highly expressed by some neuro-ectoderm-derived tumors, mainly neuroblastoma. Whereas MAb IG8 specificity was restricted to GD2 and its o-acylated form, MAb 2A6 and 7A4 were also able to detect GD3 at high concentration of antibody as shown by TLC analysis and immunodetection. The 3 MAbs were able to lyse 100% neuroblastoma cells in the presence of rabbit or human complement. Direct binding assays with 125I-labelled MAbs showed that MAb 7A4 might be a good candidate for in vivo immunolocalization experiments. The high proportion of anti-GD2 MAbs obtained by our fusion and the increased binding of anti-GD2 MAbs on gamma-IFN-treated neuroblastoma cells suggests a modulation of the exposure and an increase in the immunogenicity of GD2 induced by gamma-IFN.

  11. Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens.

    PubMed

    Suyama, K; Goldstein, J

    1988-11-01

    Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. when isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.

  12. Rice-produced MSP142 of Plasmodium falciparum elicits antibodies that inhibit parasite growth in vitro.

    PubMed

    Chen, Q; Liang, W; Qian, F; Qian, B; Cao, J; Zhang, D; Xu, Y; Tang, L

    2016-10-01

    Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B-cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142 ) is such a kind of malaria antigen. In this study, we investigated the expression of MSP142 in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP142 gene was synthesized according to rice-preferred codons and transformed into rice plants via an Agrobacterium-mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP142 formulated with Freund's adjuvant. High antibody titres against MSP142 were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood-stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens. PMID:27493141

  13. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  14. Characterization of alpha-toxin hla gene variants, alpha-toxin expression levels, and levels of antibody to alpha-toxin in hemodialysis and postsurgical patients with Staphylococcus aureus bacteremia.

    PubMed

    Sharma-Kuinkel, Batu K; Wu, Yuling; Tabor, David E; Mok, Hoyin; Sellman, Bret R; Jenkins, Amy; Yu, Li; Jafri, Hasan S; Rude, Thomas H; Ruffin, Felicia; Schell, Wiley A; Park, Lawrence P; Yan, Qin; Thaden, Joshua T; Messina, Julia A; Fowler, Vance G; Esser, Mark T

    2015-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

  15. Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

    PubMed Central

    Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

    2014-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

  16. Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach.

    PubMed

    Zhang, Yuling; Bailey, Robert; Nightlinger, Nancy; Gillespie, Alison; Balland, Alain; Rogers, Richard

    2015-08-01

    In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.

  17. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  18. Human/BALB radiation chimera engrafted with splenocytes from patients with idiopathic thrombocytopenic purpura produce human platelet antibodies.

    PubMed Central

    Dekel, B; Marcus, H; Shenkman, B; Shimoni, A; Shechter, Y; Canaan, A; Berrebi, A; Varon, D; Reisner, Y

    1998-01-01

    We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post-transplant and declined after 6 weeks, while human anti-human platelet antibodies were detected 2-8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross-reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP. PMID:9767425

  19. Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

    PubMed

    Carey, Anne-Marie; Hacking, Kirsty; Picken, Nichola; Honkanen, Suvi; Kelly, Sharon; Niedzwiedzki, Dariusz M; Blankenship, Robert E; Shimizu, Yuuki; Wang-Otomo, Zheng-Yu; Cogdell, Richard J

    2014-11-01

    This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila. PMID:25111749

  20. Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

    PubMed

    Carey, Anne-Marie; Hacking, Kirsty; Picken, Nichola; Honkanen, Suvi; Kelly, Sharon; Niedzwiedzki, Dariusz M; Blankenship, Robert E; Shimizu, Yuuki; Wang-Otomo, Zheng-Yu; Cogdell, Richard J

    2014-11-01

    This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.

  1. Detection of the linalool-producing NES1 variant across diverse strawberry (Fragaria spp.) accessions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many volatile compounds have been shown to influence the flavor of strawberry (Fragaria spp.) fruit. A published study demonstrated that linalool, a critical flavor compound, is produced in cultivated F. xananassa varieties due to a truncated form of the NEROLIDOL SYNTHASE (NES) enzyme. The correspo...

  2. Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484.

    PubMed

    O'Connor, Paula M; O'Shea, Eileen F; Guinane, Caitriona M; O'Sullivan, Orla; Cotter, Paul D; Ross, R Paul; Hill, Colin

    2015-06-15

    Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions-Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys--and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin. PMID:25841003

  3. Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484.

    PubMed

    O'Connor, Paula M; O'Shea, Eileen F; Guinane, Caitriona M; O'Sullivan, Orla; Cotter, Paul D; Ross, R Paul; Hill, Colin

    2015-06-15

    Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions-Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys--and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.

  4. Directed selection of influenza virus produces antigenic variants that match circulating human virus isolates and escape from vaccine-mediated immune protection.

    PubMed

    DeDiego, Marta L; Anderson, Christopher S; Yang, Hongmei; Holden-Wiltse, Jeanne; Fitzgerald, Theresa; Treanor, John J; Topham, David J

    2016-06-01

    Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono-specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non-infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real-world drifted viruses, helping in annual vaccine re-formulation.

  5. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

    PubMed Central

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G.; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans. PMID:26437211

  6. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

    PubMed

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans. PMID:26437211

  7. Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

    PubMed

    Eisen, Herman N

    2014-05-01

    The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

  8. Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

    PubMed

    Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

    2014-02-01

    The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

  9. Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria.

    PubMed

    Pesarrodona, Mireia; Fernández, Yolanda; Foradada, Laia; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Unzueta, Ugutz; Xu, Zhikun; Roldán, Mónica; Villegas, Sandra; Ferrer-Miralles, Neus; Schwartz, Simó; Rinas, Ursula; Daura, Xavier; Abasolo, Ibane; Vázquez, Esther; Villaverde, Antonio

    2016-06-01

    Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source. PMID:27078873

  10. Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria.

    PubMed

    Pesarrodona, Mireia; Fernández, Yolanda; Foradada, Laia; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Unzueta, Ugutz; Xu, Zhikun; Roldán, Mónica; Villegas, Sandra; Ferrer-Miralles, Neus; Schwartz, Simó; Rinas, Ursula; Daura, Xavier; Abasolo, Ibane; Vázquez, Esther; Villaverde, Antonio

    2016-06-01

    Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source.

  11. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

    SciTech Connect

    Ye Ling; Lin Jianguo; Sun Yuliang; Bennouna, Soumaya; Lo, Michael; Wu Qingyang; Bu Zhigao; Pulendran, Bali; Compans, Richard W. . E-mail: compans@microbio.emory.edu; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.

  12. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    PubMed Central

    Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall’Acqua, William; Damschroder, Melissa; Hammond, Scott A.

    2012-01-01

    MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. PMID:22574157

  13. Exploiting highly ordered subnanoliter volume microcapillaries as microtools for the analysis of antibody producing cells.

    PubMed

    Fitzgerald, Valerie; Manning, Brian; O'Donnell, Barry; O'Reilly, Brian; O'Sullivan, Dermot; O'Kennedy, Richard; Leonard, Paul

    2015-01-20

    The interrogation of highly diverse repertoires of heterogeneous cell populations on a single cell basis increases the likelihood that a cell with unique characteristics will be identified. We have developed a new single cell analysis system comprising millions of bundled subnanoliter volume bioincubation chambers for the identification and recovery of target specific antibody secreting cells (ASCs). This platform integrates dual surface screening with dedicated user driven data analysis and automated cell recovery enabling multiple biophysical parameters to be tracked for millions of antibody leads in parallel. This direct clone analysis and selection technology is a clear deviation from current microfabricated well-based approaches and offers drastically enhanced screening throughput, simultaneous dual surface analysis, and rapid automated single cell recovery. The technology is also applicable to screening both bacterial and mammalian antibody secreting cells. We demonstrate the implementation and feasibility of this platform in identifying target specific antibodies from bacterial, hybridoma, and B cell libraries.

  14. Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease.

    PubMed

    Byun, Jae-Won; Jung, Byeong Yeal; Kim, Ha-Young; Fairbrother, John M; Lee, Myoung-Heon; Lee, Wan-Kyu

    2013-11-01

    One-step real-time PCR using one set of primers and four probes was developed for differentiation of F18 variants (F18 common, F18ab, F18ac, F18new variant) of enterotoxigenic (ETEC) and Shiga toxin-producing (STEC) Escherichia coli from piglets with diarrhoea and oedema disease. The limits of detection for F18common, F18ab, F18ac, and F18new variant were 10(7), 10(7), 10(5) and 10(7)colony forming units/g faeces, respectively. Of 94 Korean isolates of E. coli encoding F18, 70 were F18ac (43 STEC/ETEC, 4 STEC and 23 ETEC), 15 were F18ab (all STEC) and nine were F18new variant (1 STEC/ETEC, 7 STEC, 1 ETEC). PMID:23992871

  15. Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail.

    PubMed

    Kugelman, Jeffrey R; Kugelman-Tonos, Johanny; Ladner, Jason T; Pettit, James; Keeton, Carolyn M; Nagle, Elyse R; Garcia, Karla Y; Froude, Jeffrey W; Kuehne, Ana I; Kuhn, Jens H; Bavari, Sina; Zeitlin, Larry; Dye, John M; Olinger, Gene G; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

    2015-09-29

    MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape. PMID:26365189

  16. Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail.

    PubMed

    Kugelman, Jeffrey R; Kugelman-Tonos, Johanny; Ladner, Jason T; Pettit, James; Keeton, Carolyn M; Nagle, Elyse R; Garcia, Karla Y; Froude, Jeffrey W; Kuehne, Ana I; Kuhn, Jens H; Bavari, Sina; Zeitlin, Larry; Dye, John M; Olinger, Gene G; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

    2015-09-29

    MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.

  17. Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves

    PubMed Central

    Valgaeren, Bonnie R.; Pardon, Bart; Goossens, Evy; Verherstraeten, Stefanie; Roelandt, Sophie; Timbermont, Leen; Van Der Vekens, Nicky; Stuyvaert, Sabrina; Gille, Linde; Van Driessche, Laura; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip; Deprez, Piet

    2015-01-01

    Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin. PMID:26184311

  18. Mountain Yellow-legged Frogs (Rana muscosa) did not Produce Detectable Antibodies in Immunization Experiments with Batrachochytrium dendrobatidis.

    PubMed

    Poorten, Thomas J; Stice-Kishiyama, Mary J; Briggs, Cheryl J; Rosenblum, Erica Bree

    2016-01-01

    Chytridiomycosis is a devastating infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). A growing number of studies have examined the role of amphibian adaptive immunity in response to this pathogen, with varying degrees of immune activation reported. Here we present immunologic data for the mountain yellow-legged frog, Rana muscosa, and the Sierra Nevada yellow-legged frog, Rana sierrae, which are two endangered and ecologically important species experiencing Bd-inflicted declines. Previous studies on these species that examined transcriptional response during Bd infection, and the effective of immunization, provided little evidence of immune activation to Bd. However, the studies did not directly assay immune effectors in the frog hosts. We performed experiments to examine antibody production, which is a hallmark of systemic adaptive immune activation. We used controlled laboratory experiments and enzyme-linked immunosorbent assays to examine the antibody response to Bd immunization and live Bd exposure. Rana muscosa and R. sierrae individuals did not produce detectable antibodies with the capacity to bind to denatured Bd antigens under our experimental conditions. While we cannot rule out antibody response to Bd in these species, our results suggest weak, poor, or inefficient production of antibodies to denatured Bd antigens. Our findings are consistent with susceptibility to chytridiomycosis in these species and suggest additional work is needed to characterize the potential for adaptive immunity.

  19. Macrophages from chickens selected for high antibody response produced more nitric oxide and have greater phagocytic capacity.

    PubMed

    Guimarães, Marco Cesar Cunegundes; Guillermo, Landi Veivi Costilla; Matta, Marcos Fernando de Rezende; Soares, Sandro Gomes; DaMatta, Renato Augusto

    2011-04-15

    Macrophages are fundamental cells of the innate immune system, which, through phagocytosis and nitric oxide production, eliminate pathogens. The aim of the present study was to determine if macrophages from chicken families divergently selected to high and low antibodies response differ in nitric oxide production and phagocytic capacity. Blood monocytes derived macrophages were activated with lipopolysaccharide and supernatant from chicken spleen lymphocytes cultured with Concanavalin A (containing chicken interferon). Nitric oxide production was evaluated in culture supernatants. Phagocytic capacity of activated and non-activated macrophages was assayed using yeasts and IgY opsonized sheep red blood cells. Activated and non-activated macrophages from the high antibodies response family produced higher nitric oxide levels, internalized more yeast and significantly more opsonized sheep red blood cells than macrophages from the low antibodies response family. Moreover, activated macrophages became more elongated and widely spread. These findings indicate that macrophages from the high antibodies response family were more active suggesting that the differences in antibody response also depend on macrophage function.

  20. Vaccination of cattle with a methanogen protein produces specific antibodies in the saliva which are stable in the rumen.

    PubMed

    Subharat, Supatsak; Shu, Dairu; Zheng, Tao; Buddle, Bryce M; Janssen, Peter H; Luo, Dongwen; Wedlock, D Neil

    2015-04-15

    Methane is produced in the rumen of cattle by a group of archaea (single-celled organisms forming a domain distinct from bacteria and eucarya) called methanogens. Vaccination against methanogens has the potential to reduce methane emissions by inducing antibodies in saliva which are transferred to the rumen and diminish the ability of methanogens to produce methane. Since it is likely that an effective vaccination strategy will need to produce high levels of methanogen-specific antibody in the saliva; the choice of adjuvant, route of vaccination and stability of saliva-derived antibody in the rumen all need to be considered. In this study, stability of IgA and IgG in rumen fluid was determined using an in vitro assay. IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%. These results indicated that antibody is relatively stable in the bovine rumen. A trial was conducted in cattle to investigate induction of immune responses to a methanogen protein, recombinant glycosyl transferase protein (rGT2) from Methanobrevibacter ruminantium M1. Groups of cattle (n=6) were vaccinated subcutaneously with rGT2, formulated with Montanide ISA61 with or without the TLR4 agonist, monophosphoryl lipid A (MPL). A control group (n=6) was not vaccinated. Strong antigen-specific IgG and moderate IgA responses were measured in the serum and saliva of the vaccinated animals and antibody was also detected in the rumen.

  1. Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever

    PubMed Central

    Golden, Joseph W.; Maes, Piet; Kwilas, Steven A.; Ballantyne, John

    2016-01-01

    ABSTRACT Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT50), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can

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

    PubMed Central

    2012-01-01

    Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb) than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system. PMID:22587529

  3. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

    PubMed Central

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

  4. Do vitamin D levels affect antibody titers produced in response to HPV vaccine?

    PubMed Central

    Zimmerman, Richard K; Lin, Chyongchiou Jeng; Raviotta, Jonathan M; Nowalk, Mary Patricia

    2015-01-01

    In addition to its well-known effects on bone metabolism, vitamin D is an immunomodulating hormone. Serum vitamin D levels in males 18–25 years were measured at baseline, and HPV antibody titers were measured one month following the third quadrivalent HPV vaccine dose. Vitamin D levels were ≥30 ng/ml (normal) in 60 males and <30 ng/ml (low) in 113 males. Reverse cumulative distribution curves and scatter plots showed higher antibody titers with low vitamin D for all vaccine strains (P < 0.05). In linear regression analyses, antibody titers for all HPV strains were significantly higher among those with lower vitamin D levels and among younger participants (P < 0.05). These relationships add to the body of knowledge of the complex role of vitamin D in immunoregulation. PMID:26176493

  5. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

    PubMed

    Cigliola, Valentina; Populaire, Celine; Pierri, Ciro L; Deutsch, Samuel; Haefliger, Jacques-Antoine; Fadista, João; Lyssenko, Valeriya; Groop, Leif; Rueedi, Rico; Thorel, Fabrizio; Herrera, Pedro Luis; Meda, Paolo

    2016-01-01

    Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.

  6. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

    PubMed

    Voronina, E V; Seregin, Y A; Litvinova, N A; Shvets, V I; Shukurov, R R

    2016-01-01

    Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions. PMID:27652157

  7. In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris.

    PubMed

    Gomathinayagam, Sujatha; Laface, Drake; Houston-Cummings, Nga Rewa; Mangadu, Ruban; Moore, Renee; Shandil, Ishaan; Sharkey, Nathan; Li, Huijuan; Stadheim, Terrance A; Zha, Dongxing

    2015-08-20

    Monoclonal antibody (mAb) therapy has been successfully used for the treatment of B-cell lymphomas and is currently extended for the treatment of multiple myeloma (MM). New developments in MM therapeutics have achieved significant survival gains in patients but the disease still remains incurable. Elotuzumab (HuLuc63), an anti-CS1 monoclonal IgG1 antibody, is believed to induce anti-tumor activity and MM cytotoxicity through antibody dependent cellular cytotoxicity (ADCC) and inhibition of MM cell adhesion to bone marrow stromal cells (BMSCs). Modulations of the Fc glycan composition at the N297 site by selective mutations or afucosylation have been explored as strategies to develop bio-better therapeutics with enhanced ADCC activity. Afucosylated therapeutic antibodies with enhanced ADCC activity have been reported to possess greater efficacy in tumor growth inhibition at lower doses when compared to fucosylated therapeutic antibodies. The N-linked glycosylation pathway in Pichia pastoris has been engineered to produce human-like N-linked glycosylation with uniform afucosylated complex type glycans. The purpose of this study was to compare afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris with fucosylated anti-CS1 mAb expressed in mammalian HEK293 cells through in vitro ADCC and in vivo tumor inhibition models. Our results indicate that Fc glycosylation is critical for in vivo efficacy and afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris shows a better in vivo efficacy in tumor regression when compared to fucosylated anti-CS1 mAb expressed in HEK293 cells. Glycoengineered Pichia pastoris could provide an alternative platform for generating homogeneous afucosylated recombinant antibodies where Fc mediated immune effector function is important for efficacy. PMID:26015261

  8. Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

    PubMed

    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J

    2014-08-01

    Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication.

  9. Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

    PubMed Central

    2012-01-01

    Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule. PMID:22953695

  10. Murine hybridoma-derived antibodies producing circumoval precipitation (COP) reactions with eggs of Schistosoma japonicum.

    PubMed

    Cruise, K M; Mitchell, G F; Tapalés, F P; Garcia, E G; Huang, S R

    1981-08-01

    Of 7 hybridomas which secrete immunoglobulins binding to crude extracts of Schistosoma japonicum adult worms and/or eggs in solid-phase radioimmunoassays (RIAs), 3 gave positive precipitation reactions in the circumoval precipitin test (COPT). The COPT is a simple and inexpensive immunodiagnostic test for schistosomiasis japonica which involves the incubation of a selected batch of S. japonicum eggs with sera from patients and examination for precipitates one or more days later. Using a competitive RIA with an egg antigen extract and a labelled COPT-positive hybridoma ascites fluid, PwF.41-1-3, the surprising observation was made that only one anti-egg antibody specificity appeared to be represented in the series of 3 antibodies (as ascites fluids). Using sera as inhibitors in the competitive RIA, inhibitory activity (presumably antibodies to the target antigenic determinant of PwF.41-1-3) was readily detected in sera from egg-immunized mice and was of relatively high titre in a strain of mouse (C57BL/6) which can be readily sensitized for large granuloma formation around entrapped eggs in the lungs. Negligible inhibitory activity was found in the sera from S. japonicum-infected patients, even with sera from patients with prominent hepatosplenomegaly. The availability of COPT-positive hybridoma antibodies should facilitate isolation of at least one S. japonicum egg antigen involved in COP reactions and perhaps induction of immunopathological immune responses at least in mice.

  11. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses

    PubMed Central

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel

    2011-01-01

    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  12. A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin

    SciTech Connect

    Watkins, S.; Madison, J.; Davis, E.; Sakamoto, Yasushi; Putnam, F.W. ); Galliano, M.; Minchiotti, L. )

    1991-07-15

    At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5{prime} donor splice junction of intron 14 and the 3{prime} end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.

  13. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  14. Schistosoma japonicum soluble egg antigens activate naive B cells to produce antibodies: definition of parasite mechanisms of immune deviation.

    PubMed Central

    Yamashita, T; Watanabe, T; Saito, S; Araki, Y; Sendo, F

    1993-01-01

    This study analysed the effect of Schistosoma japonicum egg antigens (SEA) on the activation of lymphocytes from naive mice. T cells were found to be unaffected by SEA. B cells, however, were activated by SEA without participation of adherent cells such as macrophages. B-cell activating factor(s) in SEA were distributed into a fraction of M(r) 120,000 and a fraction of M(r) 650,000 by gel filtration. However, a fraction of M(r) 120,000 demonstrated the presence of a limited number of components by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. These activating factor(s) were destroyed by peroxidase oxidation, heat treatment, chymotrypsin and trypsin digestion. These results indicate that the B-cell activating factor(s) in SEA contain both carbohydrate and protein. IgM antibodies were detected in the culture supernatant of SEA-activated B cells after 48 hr in culture, but IgG antibodies were undetected in culture. These antibodies did not react with SEA but reacted with sheep, horse, mouse red blood cells, carbonic anhydrase and autoantigens in myelinated nerve fibres of cerebrum as well as luminal surface and parietal cells of the stomach of naive mice. Thus our data demonstrated that SEA directly stimulates naive B cells to produce antibodies against heterophile and autologous antigens. Images Figure 5 Figure 6 Figure 7 PMID:8344698

  15. Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India, after causing a cholera outbreak.

    PubMed

    Kumar, P; Mishra, D K; Deshmukh, D G; Jain, M; Zade, A M; Ingole, K V; Yadava, P K

    2014-05-01

    Vibrio cholerae O1 biotype El Tor producing Haitian variant Cholera Toxin (HCT) and showing reduced susceptibility to ciprofloxacin caused a cholera outbreak associated with a high case fatality rate (4.5) in India. HCT-secreting strains responsible for severe cholera epidemics in Orissa (India), Western Africa and Haiti were associated with increased mortality. There is a pressing need for an integrated multidisciplinary approach to combat further spread of newly emerging variant strains. The therapeutic effect of ciprofloxacin was diminished whereas use of doxycycline in moderate to severe cholera patients was found to be effective in outbreak management.

  16. Identification of a CD4 variant in Microminipigs not detectable with available anti-CD4 monoclonal antibodies.

    PubMed

    Matsubara, Tatsuya; Nishii, Naohito; Takashima, Satoshi; Takasu, Masaki; Imaeda, Noriaki; Aiki-Oshimo, Kayo; Yamazoe, Kazuaki; Kametani, Yoshie; Ando, Asako; Kitagawa, Hitoshi

    2015-12-15

    The Microminipig is an extra-small sized novel miniature pig developed in Japan. In the process of peripheral blood mononuclear cells analysis by flow cytometry, CD4+ cells could not be detected in some pigs with an anti-pig CD4 antibody (clone 74-12-4), or in some pigs with two other antibodies from different clones (MIL17 and PT90A). In a herd of 178 Microminipigs, 87 pigs (48.9%) were reactive with the anti-CD4 antibody (designated as CD4.A), and 91 pigs (51.1%) were non-reactive (designated as CD4.B). The CD4 types of piglets delivered from parents with CD4.A were CD4.A or CD4.B, and piglets delivered from parents with CD4.B were only CD4.B. This implies that the CD4.A pigs were homozygous for CD4.A or heterozygous for CD4.A and CD4.B, and the CD4.B pigs were homozygous for CD4.B. The CD4.B trait might be recessive. Significant differences could not be found in the percentage of CD3+ and CD8+ cells in whole lymphocytes between CD4.A and CD4.B animals. In the profile of CD4.B pigs, CD4+CD8+ T cells appeared to be detected in the CD4-CD8+ T cell region because the CD8 dull T cell population was observed. Thus, we considered that the CD4 molecules may be expressed on helper T cells, but the CD4 expressing cells could not be detected with the three anti-pig CD4 antibodies. Clinical abnormalities have not been observed in CD4.B pigs. Significant differences were not observed in immunoglobulin concentrations between CD4.A and CD4.B, though lower tendency was observed in plasma IgM concentrations from CD4.B pigs >36-months-old. These results imply that the CD4.B does not affect basic humoral immunity in vivo.

  17. Dose Range Evaluation of Mycograb C28Y Variant, a Human Recombinant Antibody Fragment to Heat Shock Protein 90, in Combination with Amphotericin B-Desoxycholate for Treatment of Murine Systemic Candidiasis ▿

    PubMed Central

    Louie, Arnold; Stein, Daniel S.; Zack, Julia Z.; Liu, Weiguo; Conde, Haley; Fregeau, Christine; VanScoy, Brian D.; Drusano, George L.

    2011-01-01

    Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t1/2α of ∼0.198 h and a t1/2β of ∼1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis. PMID:21502626

  18. Specific recognition pattern of IgM and IgG antibodies produced in the course of experimental paracoccidioidomycosis.

    PubMed Central

    Vaz, C A; Mackenzie, D W; Hearn, V M; Camargo, Z P; Singer-Vermes, L M; Burger, E; Calich, V L

    1992-01-01

    Specific IgM and IgG responses to Paracoccidioides brasiliensis produced in resistant and susceptible mice during experimental paracoccidioidomycosis were examined by the immunoblotting procedure. Sera from infected mice recognized 51 antigen bands with apparent molecular masses from 8 to 86 kD. Sixteen of these were defined as major antigen bands because of almost universal presence of antibodies to them, and their intense staining. All sera, including those from normal control mice, tested for both IgM and IgG antibody reacted with the major E antigen which appeared as a large diffuse band from 43 to 47 kD. Comparisons between resistant and susceptible mice showed some significant differences in IgM responses to many antigen bands. While IgG responses were quite similar for both strains, differences were apparent in the response to the antigens at 62 and 68 kD. Images Fig. 1 Fig. 2 Fig. 3 PMID:1563097

  19. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine.

    PubMed

    MacDermott, R P

    1985-01-01

    We have examined the ability of intestinal and peripheral blood mononuclear cells isolated from patients with inflammatory bowel disease to mediate killing against cell line targets in spontaneous, antibody-dependent, lectin-induced, and interferon-induced cell-mediated cytotoxicity assays, as well as responsiveness in the allogeneic mixed leukocyte reaction, and effector capabilities in cell-mediated lympholysis. IMC were poor mediators of spontaneous or antibody-dependent cellular cytotoxicity with cell line cells as targets (in comparison to normal PBMC, but were capable of killing antibody coated chicken red blood cells. Although IMC were capable of responding to allogeneic cell surface antigens in the mixed leukocyte reaction, they did not exhibit effector function in cell-mediated lympholysis. Mitogenic lectins induced cell-mediated cytotoxicity by isolated intestinal mononuclear cells from controls and patients. HFIF induces cytotoxicity by control but not inflammatory bowel disease intestinal cells. Pokeweed mitogen was the lectin which induced the greatest amount of killing against human cell line targets. We therefore speculate that exogenous agents, or endogenous factors released during viral infection, could play a role in inducing cell mediated cytotoxic damage to the intestine in inflammatory bowel disease patients. In addition, the functional differences between IMC and PBMC indicate that intestinal MNC may have unique cell capabilities which must be better understood prior to the delineation of immunopathologic events in solid organ tissues. We have also examined the secretion of IgA, IgM, and IgG by isolated human IMC, human bone marrow MNC from rib specimens, and PBMC from patients with CD, UC, SLE, or Henoch-Schoenlein purpura (HSP). Control IMC exhibited high spontaneous secretion of IgA, while intestinal MNC from UC and CD patients exhibited only modest increases in IgA secretion. PBMC from patients with CD, UC, SLE, or HSP exhibited markedly

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

    PubMed Central

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

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders. PMID:27006517

  1. Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles.

    PubMed

    Lin, Tao; Xianyu, Lingzhi; Lyu, Songya

    2015-06-01

    Periodic outbreaks of hand, foot and mouth disease (HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71 (EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice (6-8 weeks old) were immunized with virus-like particles (VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing mAbs (D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect >95% cells from 100 TCID50 EV71 infection at 25 µg/mL solution (lowest concentration). Those two neutralizing mAbs identified in the study may be promising candidates in development for mAbs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.

  2. Antibody-integrated and functionalized graphite-encapsulated magnetic beads, produced using ammonia gas plasma technology, for capturing Salmonella.

    PubMed

    Sakudo, Akikazu; Chou, Han; Nagatsu, Masaaki

    2015-03-01

    Salmonella spp. is the single and most important causative agent of foodborne infections, especially involving foods such as eggs, milk and meat. To prevent infection, a reliable surveillance system is required that can quickly and sensitively detect Salmonella. Here, we describe the development of antibody-integrated magnetic beads that are functionalized by a novel strategy using ammonia gas plasma. Ammonia plasma, produced by a radio frequency (RF) power supply, was allowed to react with the surface of graphite-encapsulated magnetic beads, resulting in the introduction of amino groups. An anti-Salmonella antibody was then anchored by sulfide groups present on the protein surface to the amino groups of the magnetic beads via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The potential usefulness of these magnetic beads for capturing Salmonella was examined as follows. The beads were incubated with Salmonella in liquid medium and then separated from the supernatant by applying a magnetic field. After thorough washing, adsorption of Salmonella to the beads was confirmed by immunochromatography, polymerase chain reaction and a direct culture assay. Our findings indicate that the capture and concentration of Salmonella using the antibody-integrated magnetic beads was more efficient than commercial Dynabeads® anti-Salmonella, which are conventionally used for concentrating Salmonella from liquid cultures. We believe this novel bead technology will contribute to the enhanced detection of Salmonella. PMID:25660257

  3. Antibody-integrated and functionalized graphite-encapsulated magnetic beads, produced using ammonia gas plasma technology, for capturing Salmonella.

    PubMed

    Sakudo, Akikazu; Chou, Han; Nagatsu, Masaaki

    2015-03-01

    Salmonella spp. is the single and most important causative agent of foodborne infections, especially involving foods such as eggs, milk and meat. To prevent infection, a reliable surveillance system is required that can quickly and sensitively detect Salmonella. Here, we describe the development of antibody-integrated magnetic beads that are functionalized by a novel strategy using ammonia gas plasma. Ammonia plasma, produced by a radio frequency (RF) power supply, was allowed to react with the surface of graphite-encapsulated magnetic beads, resulting in the introduction of amino groups. An anti-Salmonella antibody was then anchored by sulfide groups present on the protein surface to the amino groups of the magnetic beads via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The potential usefulness of these magnetic beads for capturing Salmonella was examined as follows. The beads were incubated with Salmonella in liquid medium and then separated from the supernatant by applying a magnetic field. After thorough washing, adsorption of Salmonella to the beads was confirmed by immunochromatography, polymerase chain reaction and a direct culture assay. Our findings indicate that the capture and concentration of Salmonella using the antibody-integrated magnetic beads was more efficient than commercial Dynabeads® anti-Salmonella, which are conventionally used for concentrating Salmonella from liquid cultures. We believe this novel bead technology will contribute to the enhanced detection of Salmonella.

  4. Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures

    USGS Publications Warehouse

    Harrahy, L.N.M.; Schreck, C.B.; Maule, A.G.

    2001-01-01

    The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21?? C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures. ?? 2001 Academic Press.

  5. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-01-01

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding. PMID:25828907

  6. Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes.

    PubMed

    Janda, Jozef; Plattet, Philippe; Torsteinsdottir, Sigurbjörg; Jonsdottir, Sigridur; Zurbriggen, Andreas; Marti, Eliane

    2012-06-30

    Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ∼13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with

  7. Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

    PubMed Central

    Verma, Harish; Sharma, Prashant; Yang, Jae Seung; Saletti, Giulietta; Ahmad, Mohammad; Bahl, Sunil K.; Wierzba, Thomas F.; Nandy, Ranjan K.; Deshpande, Jagadish M.; Sutter, Roland W.; Czerkinsky, Cecil

    2016-01-01

    Background The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. Methods 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. Results An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for

  8. [Transverse myelitis associated with toxocariasis and the importance of locally produced antibodies for diagnosis].

    PubMed

    Ural, Serap; Özer, Behiye; Gelal, Fazıl; Dirim Erdoğan, Derya; Sezak, Nurbanu; Balık, Recep; Demirdal, Tuna; Korkmaz, Metin

    2016-07-01

    Toxocariasis caused by Toxocara canis or less frequently by T.catis is a common parasitic infection worldwide. Clinical spectrum in humans can vary from asymptomatic infection to serious organ disfunction depending on the load of parasite, migration target of the larva and the inflammatory response of the host. Transverse myelitis (TM) due to toxocariasis is an uncommon illness identified mainly as case reports in literature. In this report, a case of TM who was diagnosed as neurotoxocariasis by serological findings has been presented. A 44-year-old male patient complained with backache was diagnosed as TM in a medical center in which he has admitted two years ago, and treated with pregabalin and nonsteroidal drugs for six months. Because of the progression of the lesions he readmitted to another center and treated with high dose steroid therapy for three months. After six months of follow up, improvement has been achieved, however, since his symptoms reccurred in the following year he was admitted to our hospital. Magnetic resonance imaging (MRI) examination revealed a TM in a lower segment of spinal cord. He was suffering with weakness and numbness in the left lower extremity. There was no history of rural life or contact with cats or dogs in his anamnesis. Physical examination revealed normal cranial nerve functions, sensory and motor functions. There has been no pathological reflexes, and deep tendon reflexes were also normal. Laboratory findings yielded normal hemogram and biochemical tests, negative PPD and parasitological examination of stool were negative for cysts and ova. Viral hepatitis markers, anti-HIV, toxoplasma-IgM, CMV-IgM, rubella-IgM, EBV-VCA-IgM, VDRL, Brucella tube agglutination, echinococcus antibody, autoantibody tests and neuromyelitis optica test were negative. Examination of CSF showed 20 cells/mm3 (mononuclear cells), 45 mg/dl protein and normal levels of glucose and chlorine. In both serum and CSF samples of the patient Toxocara

  9. Fermentanomics informed amino acid supplementation of an antibody producing mammalian cell culture.

    PubMed

    Read, Erik K; Bradley, Scott A; Smitka, Tim A; Agarabi, Cyrus D; Lute, Scott C; Brorson, Kurt A

    2013-01-01

    Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.

  10. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

    PubMed Central

    Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart

    2015-01-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  11. Selection of monoclonal antibodies for the identification of D variants: ability to detect weak D and to split epD2, epD5 and epD6/7.

    PubMed

    Jones, J; Filbey, D

    1996-01-01

    Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and RoHar cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 micrograms/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (Du) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with RoHarr cells, while only one IgG antibody was positive. PMID:8740011

  12. Induction of antihemagglutinin antibodies by polyclonal antiidiotype antibodies.

    PubMed

    Dinca, L; Neuwirth, S; Schulman, J; Bona, C

    1993-01-01

    Antiidiotypic antibodies can be envisioned as an alternative approach in the development of vaccines against influenza virus, which exhibits natural antigenic variations. In our work, we obtained two polyclonal cross-reactive anti-Id antibodies against PY102, VM113, and VM202 mAbs, which in turn are specific respectively for PR8 virus and laboratory-induced virus variants (PY102-V1 and VM113-V1). With these cross-reactive anti-Id antibodies, we were able to elicit anti-HA antibodies in mice. In comparing the anti-HA antibody response in animals injected with anti-Id antibodies to those immunized with PR8 influenza virus, we demonstrated that the HI titer was higher after virus immunization and that the PR8 virus boost was more efficient in this group. Our results showed that the polyclonal cross-reactive anti-Id antibodies were more efficient than the individual anti-Ids at eliciting responses. At the same time, we demonstrated that PR8-primed T cells, cultured with B cells from animals immunized with anti-Id antibodies, were able to produce anti-PR8 antibodies subsequent to stimulation with influenza virus. PMID:8476510

  13. Induction of antihemagglutinin antibodies by polyclonal antiidiotype antibodies.

    PubMed

    Dinca, L; Neuwirth, S; Schulman, J; Bona, C

    1993-01-01

    Antiidiotypic antibodies can be envisioned as an alternative approach in the development of vaccines against influenza virus, which exhibits natural antigenic variations. In our work, we obtained two polyclonal cross-reactive anti-Id antibodies against PY102, VM113, and VM202 mAbs, which in turn are specific respectively for PR8 virus and laboratory-induced virus variants (PY102-V1 and VM113-V1). With these cross-reactive anti-Id antibodies, we were able to elicit anti-HA antibodies in mice. In comparing the anti-HA antibody response in animals injected with anti-Id antibodies to those immunized with PR8 influenza virus, we demonstrated that the HI titer was higher after virus immunization and that the PR8 virus boost was more efficient in this group. Our results showed that the polyclonal cross-reactive anti-Id antibodies were more efficient than the individual anti-Ids at eliciting responses. At the same time, we demonstrated that PR8-primed T cells, cultured with B cells from animals immunized with anti-Id antibodies, were able to produce anti-PR8 antibodies subsequent to stimulation with influenza virus.

  14. Hemoglobin Abraham Lincoln, β32 (B14) Leucine → Proline AN UNSTABLE VARIANT PRODUCING SEVERE HEMOLYTIC DISEASE

    PubMed Central

    Honig, George R.; Green, David; Shamsuddin, Mir; Vida, Loyda N.; Mason, R. George; Gnarra, David J.; Maurer, Helen S.

    1973-01-01

    An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t½ using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[14C]leucine indicated a t½ of 7.2 days. When stroma-free hemolysates were heated at 50°C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37°C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant β-chain by precipitation with p-hydroxymercuribenzoate. Purification of the β-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for β32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at β31 to impair heme stabilization. Images PMID:4352462

  15. Hemoglobin Abraham Lincoln, beta32 (B14) leucine leads to proline. An unstable variant producing severe hemolytic disease.

    PubMed

    Honig, G R; Green, D; Shamsuddin, M; Vida, L N; Mason, R G; Gnarra, D J; Maurer, H S

    1973-07-01

    An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t(1/2) using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[(14)C]leucine indicated a t(1/2) of 7.2 days. When stroma-free hemolysates were heated at 50 degrees C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37 degrees C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant beta-chain by precipitation with p-hydroxymercuribenzoate. Purification of the beta-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for beta32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at beta31 to impair heme stabilization.

  16. A spontaneous hybridoma producing autoanti-idiotypic antibodies that recognize a V kappa-associated idiotope in mercury-induced autoimmunity.

    PubMed

    Guéry, J C; Druet, P

    1990-05-01

    Anti-idiotypic (Id) antibodies have been suggested to play a role in the self regulation process observed in Brown-Norway rats developing mercury-induced autoimmunity. However, the presence of such antibodies has not yet been directly demonstrated. For that purpose, spleen cells from a mercury-injected rat were fused and the resulting hybridomas tested for their anti-Id activity against monoclonal anti-glomerular basement membrane (GBM) antibodies produced in this model. A monoclonal antibody (mAb) was obtained that specifically reacted in an enzyme-linked immunosorbent assay with an anti-GBM mAb and to a much lesser extent with another one produced in the same fusion. In Western blot experiments this autoanti-Id mAb reacted under reducing conditions with the kappa L chains but not with the H chains of the two anti-GBM mAb. It did not react with the kappa L chains of eight other rat mAb. This mAb is therefore an autoanti-Id mAb that recognizes a V kappa-associated Id expressed on two anti-GBM mAb. These results demonstrate that anti-GBM antibodies and their corresponding autoanti-Id antibodies are simultaneously produced during this disease. Whether or not these autoanti-Id antibodies have a regulatory and/or a pathogenic role in this disease remains to be established.

  17. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  18. Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli.

    PubMed

    Bellini, Estela M; Elias, Waldir P; Gomes, Tânia A T; Tanaka, Tânia L; Taddei, Carla R; Huerta, Rocio; Navarro-Garcia, Fernando; Martinez, Marina B

    2005-02-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of pediatric and adult travellers diarrhea. The mechanism by which EAEC induce diarrhea is not completely known. Two serine protease autotransporter proteins, named Pet and Pic have been identified in EAEC strains. Pet has enterotoxic and cytotoxic activities, while the role of Pic in pathogenesis may lie on its mucinolytic activity. Little is known about Pet and Pic biological activities in vivo. In this study the antibody responses against these autotransporter proteins in convalescent children is investigated. Fifteen (83%) children showed specific antibodies against Pet or Pic in their sera. IgG and IgM antibodies were the main isotype found. Specific antibodies against Pic, but not against Pet, were detected in sera from age-matched control group. These data show that specific anti-Pet and anti-Pic antibodies are produced during the course of a natural EAEC infection in children.

  19. Changes of the immune reactivities of antibodies produced against gamma-irradiated antigen

    NASA Astrophysics Data System (ADS)

    Byun, Myung-Woo; Lee, Ju-Woon; Seo, Ji-Hyun; Kim, Jae-Hun; Jo, Cheorun; Kim, Dong-Ho; Chung, Hyung-Wook

    2004-09-01

    To observe the changes of immunogenicity and antigenicity of gamma-irradiated ovalbumin (OVA), an antigen (Ag) solution (2.0 mg/ml) was prepared and irradiated with the absorbed doses of 3 and 10 kGy. Immunoglobulin G (IgG) was produced for each Ag. 0, 3 and 10 kGy-IgG were individually reacted against 3 Ags in an ELISA cross reactivity test. Cross reactivity of each IgG was significantly different for each Ag. Especially the 10 kGy-irradiated OVA lost most antigenicity compared to the 0 kGy-IgG.

  20. Binding specificities of eight monoclonal antibodies to human glycophorin A - studies with M/sup c/M, and M/sub k/En(UK) variant human erythrocytes and M- and MN/sup V/-type chimpanzee erythrocytes

    SciTech Connect

    Bigbee, W.L.; Langlois, R.G.; Vanderlaan, M.; Jensen, R.H.

    1984-12-01

    Four newly derived mouse monoclonal antibodies to human glycophorin A are described. Three of these antibodies bind preferentially to the N form of glycophorin A; the fourth recognizes a shared determinant of the M and N forms. All four antibodies are directed toward the 39 amino acid, amino-terminal portion of the protein, and the N-specific antibodies require for binding the presence of N-acetyl-neuraminic acid on the glycosidically linked oligosaccharides. Cross-reaction of the N-specific antibodies to homozygous MM erythrocytes appears to result from binding to glycophorin B. In addition, these antibodies together with four previously reported glycophorin monoclonal antibodies, including two that specifically recognize the M form of glycophorin A, were tested for binding to M/sup c/M and M/sup k/En(UK) variant human erythrocytes. Results obtained for five of the six M- or N-specific monoclonal antibodies point to the general immunodominance of the amino-terminal serine-leucine polymorphism and the requirement for sialic acid. The epitopes for all three N-specific monoclonal antibodies include the amino terminal leucine that occurs in the N form of glycophorin A and may also include the glutamic acid that occurs at position five. Their studies support the proposed Lepore-type glycophorin A-B hybrid gene rearrangement for the En(UK) allele found in the English En(a-) family. The data also confirm the expression of the M-like glycoprotein on chimpanzee erythrocytes and the presence of a human glycophorin B-like antigen on the MN/sup V/-type cells.

  1. Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

    PubMed

    Zhou, Yu; Shen, Chaoyun; Zhang, Chao; Zhang, Wei; Wang, Lili; Lan, Ke; Liu, Qingwei; Huang, Zhong

    2016-08-01

    Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development. PMID:27315772

  2. Antithyroglobulin antibody

    MedlinePlus

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

  3. Serum anti-tissue transglutaminase antibodies detected during febrile illness may not be produced by the intestinal mucosa.

    PubMed

    De Leo, Luigina; Quaglia, Sara; Ziberna, Fabiana; Vatta, Serena; Martelossi, Stefano; Maschio, Massimo; Not, Tarcisio

    2015-03-01

    Anti-transglutaminase antibodies are the diagnostic marker of celiac disease, and are considered to be synthesized only by intestinal B-lymphocytes. During an infectious disease, these antibodies are transiently detected in serum. We show that these infection-triggered antibodies may not originate in the intestinal mucosa and are not an indication of celiac disease. PMID:25722272

  4. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  5. Cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, William L.; Fong, Stella S. N.; Jensen, Ronald H.; Vanderlaan, Martin; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  6. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level

    PubMed Central

    2012-01-01

    We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. PMID:22640862

  7. Growth factor(s) produced during infection with an adenovirus variant stimulates proliferation of nonestablished epithelial cells.

    PubMed

    Quinlan, M P; Sullivan, N; Grodzicker, T

    1987-05-01

    Infection of primary baby rat kidney cells with an adenovirus variant that encodes only the 12S gene of the E1A region, adenovirus type 5 (Ad5) 12S, results in the production of a growth factor that stimulates primary epithelial cells to proliferate. Increased epithelial cell DNA synthesis and proliferation is detectable between 24 and 36 hr after the addition of conditioned medium from Ad5 12S infected cells and not from cells infected with an E1A deletion mutant virus, Ad5 dl312. This mitogenic factor(s) is effective in the absence of serum and can override the inhibitory effect of serum on primary epithelial cells. Furthermore, there is a requirement for the continued presence of the growth factor(s) in the Ad5 12S conditioned medium to maintain epithelial cell proliferation, and the conditioned medium can maintain these cells in a proliferative state for at least 6 wk. The stimulatory activity in Ad5 12S conditioned medium is associated with large molecular weight complexes, from which it can be released by 4 M NaCl. Several characteristics of the growth factor(s) indicate that it is a unique mitogen for epithelial cells. PMID:2953026

  8. ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice.

    PubMed

    Phan, Hoang T; Pohl, Julia; Floss, Doreen M; Rabenstein, Frank; Veits, Jutta; Le, Binh T; Chu, Ha H; Hause, Gerd; Mettenleiter, Thomas; Conrad, Udo

    2013-06-01

    Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5-ELP. ELPylated trimers could be purified by a membrane-based inverse transition cycling procedure with the potential of successful scale-up. The trimeric form of AIV HA was found to enhance the HA-specific immune response compared with the monomeric form. Plant-derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus-like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale-up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.

  9. Application of an enzyme-labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes, in recurrent tonsillitis.

    PubMed

    Onouchi, Takanori; Mizutani, Yasuyoshi; Shiogama, Kazuya; Inada, Ken-ichi; Okada, Tatsuyoshi; Naito, Kensei; Tsutsumi, Yutaka

    2015-01-01

    Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme-labeled antigen method is a novel histochemical technique that visualizes specific antibody-producing cells in tissue sections by employing a biotin-labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde-fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme-labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR-detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti-Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A-reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.

  10. Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome.

    PubMed

    Ludwig, Kerstin; Sarkim, Volkan; Bitzan, Martin; Karmali, Mohamed A; Bobrowski, Christoph; Ruder, Hans; Laufs, Rainer; Sobottka, Ingo; Petric, Martin; Karch, Helge; Müller-Wiefel, Dirk E

    2002-05-01

    Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.

  11. Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients

    PubMed Central

    Ladenstein, Ruth; Weixler, Silke; Baykan, Bianca; Bleeke, Matthias; Kunert, Renate; Katinger, Dietmar; Pribill, Ingrid; Glander, Petra; Bauer, Steffen; Pistoia, Vito; Michon, Jean; Garaventa, Alberto; Lode, Holger N.

    2013-01-01

    Purpose: This study aimed to assess the safety, pharmacokinetic and activity profiles of the human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells (ch14.18/CHO). Methods: Sixteen children with recurrent/refractory neuroblastoma (median age 7.6 y) were enrolled in this Phase 1 dose-finding study. Patients received ch14.18/CHO courses of 10, 20 or 30 mg/m2/day as an eight-hour infusion over five consecutive days. Three courses at the same dose level were allowed unless disease progressed. Clearance and biodistribution of radiolabelled ch14.18/CHO in Balb/c and A/J mice were analyzed. Results: A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m2/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of 125I-ch14.18/CHO in mice with neuroblastoma was identical to 125I-ch14.18/SP2/0, indicating GD2 targeting activity in vivo. Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation. PMID:23924804

  12. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation

    PubMed Central

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-01-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies. PMID:26674415

  13. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation.

    PubMed

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-12-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action--proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide--were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate 'protective' antibodies.

  14. Anti-neurofilament antibodies in neuropathy with monoclonal gammopathy of undetermined significance produce experimental motor nerve conduction block.

    PubMed

    Stubbs, Evan B; Lawlor, Mike W; Richards, Michael P; Siddiqui, Kiran; Fisher, Morris A; Bhoopalam, Nirmala; Siegel, George J

    2003-02-01

    Elevated levels of serum antibodies to neurofilament proteins have been associated with a variety of neurological diseases, including autoimmune disorders such as neuropathy with monoclonal gammopathy of undetermined significance (MGUS). The pathological significance of anti-neurofilament antibodies in sera of affected patients, however, remains unclear. In this study, we report our findings of polyclonal antibodies in sera from 4 of 16 IgG MGUS neuropathy patients that react strongly on immunoblot with a high molecular weight neurofilament protein (NFH). The effect of anti-NFH polyclonal antibody on peripheral nerve function was tested in vivo by intraneural injection. Sera containing anti-NFH antibody, but not sera from age-matched control subjects, injected into the endoneurium of rat sciatic nerve significantly attenuated proximal-evoked motor nerve compound muscle action potential (CMAP) amplitudes in a complement-dependent manner. In contrast, ankle-evoked CMAP amplitudes were unaffected by intraneural injection of sera containing anti-NFH antibody. Anti-NFH serum-injected nerves showed changes in both axon caliber (shrinkage) and myelin ultrastructure (vesiculation and ovoid formation), suggestive of intramyelinic edema. Preincubation of sera containing anti-NFH antibody with purified NFH protein abolished immunoreactivity to NFH protein and neutralized the serum-mediated toxicity. The data suggest that anti-NFH polyclonal antibodies occurring in sera of some patients with IgG MGUS neuropathy may elicit peripheral nerve conduction block independent of the patients' IgG paraprotein. Anti-neural polyclonal antibodies in sera of IgG MGUS neuropathy patients may have a greater pathological significance than previously anticipated.

  15. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  16. Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors.

    PubMed

    van der Geest, Kornelis S M; Lorencetti, Pedro G; Abdulahad, Wayel H; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

    2016-03-01

    Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So far, few studies have directly assessed the effect of aging on the latter B cell function. Here, we determined if and how human aging influences the production of cytokines by B cells. In a cross-sectional study, we found that absolute numbers of circulating B cells were similar in 31 young (ages 19-39) and 73 old (age ≥ 60) individuals. Numbers of transitional B cells (CD19(+)CD27(-)CD38(High)CD24(High)) were decreased in old individuals, whereas numbers of naive and memory B cell subsets were comparable in young and old individuals. Short-term in vitro stimulation of whole blood samples revealed that numbers of B cells capable of producing TNF-α were similar in young and old individuals. In contrast, B cells capable of IL-10 production were decreased in old subjects. This decline of IL-10(+) B cells was observed in old individuals that were ANA positive, and in those that were negative for both ANAs and RFs. However, IL-10(+) B cells were remarkably well retained in the circulation of old subjects that were RF positive. Thus, pro-inflammatory TNF-α(+) B cells are retained in the elderly, whereas IL-10(+) B cells generally decline. In addition, our findings indicate that IL-10(+) B cells may differentially impact the development of ANAs and RFs in the elderly.

  17. Outbreak caused by an ertapenem-resistant, CTX-M-15-producing Klebsiella pneumoniae sequence type 101 clone carrying an OmpK36 porin variant.

    PubMed

    Poulou, Aggeliki; Voulgari, Evangelia; Vrioni, Georgia; Koumaki, Vasiliki; Xidopoulos, Grigorios; Chatzipantazi, Vasiliki; Markou, Fani; Tsakris, Athanassios

    2013-10-01

    Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.

  18. Outbreak Caused by an Ertapenem-Resistant, CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

    PubMed Central

    Poulou, Aggeliki; Voulgari, Evangelia; Vrioni, Georgia; Koumaki, Vasiliki; Xidopoulos, Grigorios; Chatzipantazi, Vasiliki; Markou, Fani

    2013-01-01

    Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak. PMID:23850951

  19. Hemoglobin Indianapolis (beta 112[G14] arginine). An unstable beta-chain variant producing the phenotype of severe beta-thalassemia.

    PubMed Central

    Adams, J G; Boxer, L A; Baehner, R L; Forget, B G; Tsistrakis, G A; Steinberg, M H

    1979-01-01

    Hemoglobin (Hb) Indianapolis is an extremely labile beta-chain variant, present in such small amounts that it was undetectable by usual techniques. Clinically, it produces the phenotype of severe beta-thalassemia. Biosynthetic studies showed a beta:alpha ratio of 0.5 in reticulocytes and about 1.0 in marrow after a 1-h incubation. These results, similar to those seen in typical heterozygous beta-thalassemia, suggested that betaIndianapolis was destroyed so rapidly that its net synthesis was essentially zero. To examine the kinetics of globin synthesis, reticulocyte incubations of 1.25--20 min were performed with [3H]leucine. The betaIndianapolis:beta A ratio at 1.25 min was 0.80 suggesting that beta Indianapolis was synthesized at a near normal rate. At 20 min, this ratio was 0.46 reflecting rapid turnover of beta Indianapolis. The erythrocyte ghosts from these incubations contained only betaIndianapolis and alpha-chains, and the proportion of betaIndianapolis decreased with time, indicating loss of betaIndianapolis. Pulse-chase studies showed little change in beta A:alpha ratio and decreasing betaIndianapolis:alpha and betaIndianapolis:beta A with time. The half-life of betaIndianapolis in the soluble hemoglobin was approximately equal to 7 min. There was also rapid loss of beta Indianapolis from the erythrocyte membrane. From these results, it may be inferred that betaIndianapolis is rapidly precipitated from the soluble cell phase to the membrane, where it is catabolized. Heterozygotes for beta 0-thalassemia usually have minimal hematologic abnormalities, whereas heterozygotes with betaIndianapolis, having a similar net content of beta-chain, have severe disease. The extremely rapid precipitation and catabolism of betaIndianapolis and the resulting excess of alpha-chains, both causing membrane damage, may be responsible for the severe clinical manifestations associated with this variant. It seems likely that other, similar disturbances in the primary sequence of

  20. Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3

    PubMed Central

    Tsekoa, Tsepo Lebiletsa; Lotter-Stark, Therese; Buthelezi, Sindisiwe; Chakauya, Ereck; Stoychev, Stoyan H.; Sabeta, Claude; Shumba, Wonderful; Phahladira, Baby; Hume, Steve; Morton, Josh; Rupprecht, Charles E.; Steinkellner, Herta; Pauly, Michael; Zeitlin, Larry; Whaley, Kevin; Chikwamba, Rachel

    2016-01-01

    Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG. PMID:27427976

  1. Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

    PubMed

    Tsekoa, Tsepo Lebiletsa; Lotter-Stark, Therese; Buthelezi, Sindisiwe; Chakauya, Ereck; Stoychev, Stoyan H; Sabeta, Claude; Shumba, Wonderful; Phahladira, Baby; Hume, Steve; Morton, Josh; Rupprecht, Charles E; Steinkellner, Herta; Pauly, Michael; Zeitlin, Larry; Whaley, Kevin; Chikwamba, Rachel

    2016-01-01

    Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG. PMID:27427976

  2. Comparative Analysis of Antibody Responses against HSP60, Invariant Surface Glycoprotein 70, and Variant Surface Glycoprotein Reveals a Complex Antigen-Specific Pattern of Immunoglobulin Isotype Switching during Infection by Trypanosoma brucei

    PubMed Central

    Radwanska, Magdalena; Magez, Stefan; Michel, Alain; Stijlemans, Benoît; Geuskens, Maurice; Pays, Etienne

    2000-01-01

    During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. brucei infection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching. PMID:10639455

  3. Anti-ICOS Monoclonal Antibody MEDI-570 in Treating Patients With Relapsed or Refractory Peripheral T-cell Lymphoma Follicular Variant or Angioimmunoblastic T-cell Lymphoma

    ClinicalTrials.gov

    2016-09-08

    Follicular Variant Peripheral T-Cell Lymphoma; Recurrent Angioimmunoblastic T-cell Lymphoma; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Refractory Angioimmunoblastic T-cell Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma

  4. Prevalence of toxic shock syndrome toxin 1-producing Staphylococcus aureus and the presence of antibodies to this superantigen in menstruating women.

    PubMed

    Parsonnet, Jeffrey; Hansmann, Melanie A; Delaney, Mary L; Modern, Paul A; Dubois, Andrea M; Wieland-Alter, Wendy; Wissemann, Kimberly W; Wild, John E; Jones, Michaelle B; Seymour, Jon L; Onderdonk, Andrew B

    2005-09-01

    Menstrual toxic shock syndrome (mTSS) is thought to be associated with colonization with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus aureus in women with insufficient antibody titers. mTSS has been associated with menstruation and tampon use, and although it is rare, the effects can be life threatening. It remains of interest because of the widespread use of tampons, reported to be about 70% of women in the United States, Canada, and much of Western Europe. This comprehensive study was designed to determine S. aureus colonization and TSST-1 serum antibody titers in 3,012 menstruating women in North America between the ages of 13 and 40, particularly among age and racial groups that could not be assessed reliably in previous small studies. One out of every four subjects was found to be colonized with S. aureus in at least one of three body sites (nose, vagina, or anus), with approximately 9% colonized vaginally. Eighty-five percent of subjects had antibody titers (> or =1:32) to TSST-1, and the vast majority (81%) of teenaged subjects (13 to 18 years) had already developed antibody titers. Among carriers of toxigenic S. aureus, a significantly lower percentage of black women than of white or Hispanic women were found to have antibody titers (> or =1:32) to TSST-1 (89% versus 98% and 100%). These findings demonstrate that the majority of teenagers have antibody titers (> or =1:32) to TSST-1 and are presumed to be protected from mTSS. These findings also suggest that black women may be more susceptible to mTSS than previously thought. PMID:16145118

  5. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    PubMed

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins.

  6. Immunization of healthy adults with a single recombinant pneumococcal surface protein A (PspA) variant stimulates broadly cross-reactive antibodies to heterologous PspA molecules.

    PubMed

    Nabors, G S; Braun, P A; Herrmann, D J; Heise, M L; Pyle, D J; Gravenstein, S; Schilling, M; Ferguson, L M; Hollingshead, S K; Briles, D E; Becker, R S

    2000-03-01

    Pneumococcal surface protein A (PspA) is a highly variable protein found on all strains of pneumococci. To be successful, a PspA-based vaccine for S. pneumoniae must induce antibodies that are broadly cross-reactive. To address whether cross-reactive antibodies could be induced in man, we evaluated serum from adults immunized with recombinant clade 2 PspA from strain Rx1. Immunization with 5-125 microg rPspA lead to a significant increase in circulating anti-PspA antibodies, as well as antibodies reactive to heterologous rPspA molecules. Increased binding of post-immune sera to 37 pneumococcal strains expressing a variety of PspA and capsule types was observed, versus pre-immune sera. The extent of cross-clade reactivity of human anti-rPspA followed roughly the amount of sequence homology to the non-clade 2 antigens. It is hypothesized that priming of humans by natural exposure to S. pneumoniae contributes to the breadth of the cross-reactivity of antibody to PspA.

  7. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology

    PubMed Central

    SAKUDO, AKIKAZU; VISWAN, ANCHU; CHOU, HAN; SASAKI, TADAHIRO; IKUTA, KAZUYOSHI; NAGATSU, MASAAKI

    2016-01-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito-borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave-excited inductively-coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody-integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV-infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1–4 onto the beads was confirmed using reverse transcription-polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody-integration, represents a significant improvement in the detection of DENV. PMID:27221214

  8. High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

    PubMed

    Lunha, Kamonwan; Chanawong, Aroonwadee; Lulitanond, Aroonlug; Wilailuckana, Chotechana; Charoensri, Nicha; Wonglakorn, Lumyai; Saenjamla, Pimjai; Chaimanee, Prajuab; Angkititrakul, Sunpetch; Chetchotisakd, Ploenchan

    2016-06-01

    Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future. PMID:27041106

  9. High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

    PubMed

    Lunha, Kamonwan; Chanawong, Aroonwadee; Lulitanond, Aroonlug; Wilailuckana, Chotechana; Charoensri, Nicha; Wonglakorn, Lumyai; Saenjamla, Pimjai; Chaimanee, Prajuab; Angkititrakul, Sunpetch; Chetchotisakd, Ploenchan

    2016-06-01

    Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future.

  10. Interaction between PLA2R1 and HLA-DQA1 variants associates with anti-PLA2R antibodies and membranous nephropathy.

    PubMed

    Lv, Jicheng; Hou, Wanyin; Zhou, Xujie; Liu, Gang; Zhou, Fude; Zhao, Na; Hou, Ping; Zhao, Minghui; Zhang, Hong

    2013-07-01

    Risk alleles at genome loci containing phospholipase A2 receptor 1 (PLA2R1) and HLA-DQA1 closely associate with idiopathic membranous nephropathy (IMN) in the European population, but it is unknown whether a similar association exists in the Chinese population and whether high-risk alleles promote the development of anti-PLA2R antibodies. Here, we genotyped 2132 Chinese individuals, including 1112 patients with IMN and 1020 healthy controls, for three single nucleotide polymorphisms (SNPs) within PLA2R1 and three SNPs within HLA genes. We also selected 71 patients, with varying genotypes, to assess for circulating anti-PLA2R antibody and for PLA2R expression in glomeruli. Three SNPs within PLA2R1 and one SNP within HLA-DQA1 strongly associated with IMN, and we noted gene-gene interactions involving these SNPs. Furthermore, these risk alleles strongly associated with the presence of anti-PLA2R antibodies and glomerular PLA2R expression. Among individuals who carried risk alleles for both genes, 73% had anti-PLA2R antibodies and 75% expressed PLA2R in glomeruli. In contrast, among individuals who carried protective genotypes of both genes, none had anti-PLA2R antibodies and glomerular expression of PLA2R was weak or absent. In conclusion, the interaction between PLA2R1 and HLA-DQA1 risk alleles associates with the development of IMN in the Chinese population. Individuals carrying risk alleles are predisposed to the generation of circulating anti-PLA2R autoantibodies, which may contribute to the development of IMN.

  11. A Phase I Study of a Combination of Yttrium-90 labeled Anti-CEA Antibody and Gemcitabine in Patients with CEA Producing Advanced Malignancies

    PubMed Central

    Shibata, Stephen; Raubitschek, Andrew; Leong, Lucille; Koczywas, Marianna; Williams, Lawrence; Zhan, Jiping; Wong, Jeffrey Y.C.

    2011-01-01

    Purpose To determine the maximum tolerated dose of combined therapy using an yttrium-90 labeled anti-CEA antibody with gemcitabine in patients with advanced CEA producing solid tumors. Experimental Design The chimeric human/murine cT84.66 is an anti-CEA intact IgG1, with high affinity and specificity to CEA. This was given at a fixed yttrium-90 labeled dose of 16.6 mCi/m2 to subjects who had and an elevated CEA in serum or in tumor by immunohistochemistry. Also required was a tumor that imaged with an 111In labeled cT84.66 antibody. Patients were treated with escalating doses of gemcitabine given intravenously over 30 minutes on day 1 and 3 after the infusion of the yttrium-90 labeled antibody. Patients were treated in cohorts of 3. The maximum tolerated dose was determined as the highest level at which no more than 1 of 6 patients experienced a dose limiting toxicity. Results A total of 36 patients were enrolled, and all but one had prior systemic therapy. The maximum tolerated dose of gemcitabine in this combination was 150mg/m2. Dose limiting toxicities at a gemcitabine dose of 165mg/m2 included a grade 3 rash and grade 4 neutropenia. One partial response was seen in a patient with colorectal cancer, and 4 patients had a > 50% decrease in baseline CEA levels associated with stable disease. Human antichimeric antibody responses were the primary reason for stopping treatment in 12 patients. Conclusions feasibility of combining gemcitabine with an yttrium-90 labeled anti-CEA antibody is demonstrated with preliminary evidence of clinical response. PMID:19351765

  12. Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.

    PubMed

    Lombardi, Raffaele; Villani, Maria Elena; Di Carli, Mariasole; Brunetti, Patrizia; Benvenuto, Eugenio; Donini, Marcello

    2010-12-01

    It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15-20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels

  13. A human-human hybridoma producing cytotoxic antibody to HLA-B15, cross-reacting with B17, B5, B35 and B18.

    PubMed

    Hansen, T; Kolstad, A; Thorsby, E; Hannestad, K

    1987-05-01

    Mononuclear blood cells from a multiparous woman were transformed with Epstein Barr virus, and a cell line (Tr2D8) producing anti-HLA antibody was obtained. This cell line was immortalized by hybridization to the human fusion partners KR4 and KR12. While the EBV line died after 7 months, the hybridomas have remained stable for 13 months. The EBV line supernatant (40 micrograms IgM/ml) lysed peripheral blood mononuclear cells (PBMC) bearing B15, B17, B5 and B35. Consistent lysis of B18 bearing cells was only observed with lymphoblastoid cell lines. The supernatant from the Tr2D8 (EBV line X KR4) hybridoma (2.7 micrograms IgM/ml) only lysed B15 bearing PBMC. At a concentration of 13.5 micrograms IgM/ml, the hybridoma antibody lysed lymphoblastoid cell lines bearing B15, B17, B5, B35 and B18.

  14. Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

    PubMed Central

    Tanyaratsrisakul, Sasipa; Jirapongsananuruk, Orathai; Kulwanich, Bhakkawarat; Hales, Belinda J.; Thomas, Wayne R.

    2016-01-01

    Purpose The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants. Methods The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured. Results The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants. Conclusions The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be

  15. Cavitary Pneumonia in an AIDS Patient Caused by an Unusual Bordetella bronchiseptica Variant Producing Reduced Amounts of Pertactin and Other Major Antigens

    PubMed Central

    Lorenzo-Pajuelo, Benito; Villanueva, José Luis; Rodríguez-Cuesta, Juan; Vergara-Irigaray, Nuria; Bernabeu-Wittel, Máximo; Garcia-Curiel, Andrés; Martínez de Tejada, Guillermo

    2002-01-01

    Although Bordetella bronchiseptica can infect and colonize immunocompromised humans, its role as a primary pathogen in pneumonia and other respiratory processes affecting those patients remains controversial. A case of cavitary pneumonia caused by B. bronchiseptica in an AIDS patient is presented, and the basis of the seemingly enhanced pathogenic potential of this isolate (designated 814) is investigated. B. bronchiseptica was the only microorganism recovered from sputum, bronchoalveolar lavage fluid, and samples taken through the protected brush catheter. Unlike previous work reporting the involvement of B. bronchiseptica in cases of pneumonia, antibiotic treatment selected on the basis of in vitro antibacterial activity resulted in clearance of the infection and resolution of the pulmonary infiltrate. Although isolate 814 produced reduced amounts of several major antigens including at least one Bvg-activated factor (pertactin), the molecular basis of this deficiency was found to be BvgAS independent since the defect persisted after the bvgAS locus of isolate 814 was replaced with a wild-type bvgAS allele. Despite its prominent phenotype, isolate 814 displayed only a modest yet a significant deficiency in its ability to colonize the respiratory tracts of immunocompetent rats at an early time point. Interestingly, the antibody response elicited by isolate 814 in these animals was almost undetectable. We propose that isolate 814 may be more virulent in immunocompromised patients due, at least in part, to its innate ability to produce low amounts of immunogenic factors which may be required at only normal levels for the interaction of this pathogen with its immunocompetent natural hosts. PMID:12202545

  16. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

    PubMed Central

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-01-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

  17. Crystal Structure of Serine Racemase that Produces Neurotransmitter variant:small-caps">d-Serine for Stimulation of the NMDA Receptor

    NASA Astrophysics Data System (ADS)

    Goto, Masaru

    variant:small-caps">d-Serine is an endogenous coagonist for the N-methyl-variant:small-caps">d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of variant:small-caps">l-serine to yield variant:small-caps">d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-variant:small-caps">d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-variant:small-caps">d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

  18. De Novo assembly of the complete genome of an enhanced electricity-producing variant of Geobacter sulfurreducens using only short reads.

    PubMed

    Nagarajan, Harish; Butler, Jessica E; Klimes, Anna; Qiu, Yu; Zengler, Karsten; Ward, Joy; Young, Nelson D; Methé, Barbara A; Palsson, Bernhard Ø; Lovley, Derek R; Barrett, Christian L

    2010-06-08

    State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.

  19. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

    PubMed

    Sakudo, Akikazu; Viswan, Anchu; Chou, Han; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2016-07-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito‑borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave‑excited inductively‑coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody‑integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV‑infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1‑4 onto the beads was confirmed using reverse transcription‑polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody‑integration, represents a significant improvement in the detection of DENV.

  20. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

    PubMed

    Sakudo, Akikazu; Viswan, Anchu; Chou, Han; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2016-07-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito‑borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave‑excited inductively‑coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody‑integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV‑infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1‑4 onto the beads was confirmed using reverse transcription‑polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody‑integration, represents a significant improvement in the detection of DENV. PMID:27221214

  1. Antibody Titer Has Positive Predictive Value for Vaccine Protection against Challenge with Natural Antigenic-Drift Variants of H5N1 High-Pathogenicity Avian Influenza Viruses from Indonesia

    PubMed Central

    Suarez, David L.; Spackman, Erica; Jadhao, Samadhan; Dauphin, Gwenaelle; Kim-Torchetti, Mia; McGrane, James; Weaver, John; Daniels, Peter; Wong, Frank; Selleck, Paul; Wiyono, Agus; Indriani, Risa; Yupiana, Yuni; Sawitri Siregar, Elly; Prajitno, Teguh; Smith, Derek; Fouchier, Ron

    2015-01-01

    antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses. IMPORTANCE H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear. PMID:25609805

  2. Determination of immunoglobulin M antibodies for hepatitis B core antigen with a capture enzyme immunoassay and biotin-labeled core antigen produced in Escherichia coli.

    PubMed Central

    Vilja, P; Turunen, H J; Leinikki, P O

    1985-01-01

    A new capture enzyme immunoassay for the determination of immunoglobulin M (IgM) antibodies against hepatitis B core antigen (HBcAg) is described. Core antigen produced in Escherichia coli was labeled with biotin and subsequently detected by an avidin-biotin-peroxidase complex. The biotin-labeled core antigen was effective at concentrations as low as 20 ng/ml. Of 561 serum samples from different groups of patients that were tested, 465 samples were negative for other hepatitis B virus markers and also for anti-HBcAg IgM. Sera from the early stages of hepatitis B infection had high levels of anti-HBcAg IgM, and a clear correlation with the acuteness of the disease was observed in 45 follow-up sera from 23 patients with acute or recent hepatitis B. Sera from 21 patients with past hepatitis B were all negative for anti-HBcAg IgM. Twenty serum samples from chronic carriers of hepatitis B surface antigen showed slightly elevated antibody levels for anti-HBcAg IgM. Ten sera which were positive for anti-HBcAg IgG antibodies and had high levels of rheumatoid factor were negative for anti-HBcAg IgM. PMID:3908476

  3. Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice.

    PubMed

    Zeng, Yan; Fest, Stefan; Kunert, Renate; Katinger, Hermann; Pistoia, Vito; Michon, Jean; Lewis, Gillan; Ladenstein, Ruth; Lode, Holger N

    2005-07-01

    Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

  4. Piperacillin, tazobactam, and gentamicin alone or combined in an endocarditis model of infection by a TEM-3-producing strain of Klebsiella pneumoniae or its susceptible variant.

    PubMed Central

    Mentec, H; Vallois, J M; Bure, A; Saleh-Mghir, A; Jehl, F; Carbon, C

    1992-01-01

    The efficacy of tazobactam, a beta-lactamase inhibitor, in combination with piperacillin, was studied in vitro and in rabbit experimental endocarditis due to a Klebsiella pneumoniae strain (KpR) producing an extended-spectrum beta-lactamase, TEM-3, or its nonproducing variant (KpS). In vitro, piperacillin was active against KpS (MIC = 4 micrograms/ml, MBC = 8 micrograms/ml with 10(7)-CFU/ml inoculum) but not against KpR (MIC = MBC = 256 micrograms/ml). Tazobactam (1 microgram/ml) restored the activity of piperacillin against KpR (MIC = 2 micrograms/ml, MBC = 4 micrograms/ml). Gentamicin was active against both strains (MIC = 0.25 and 0.5 micrograms/ml for KpS and KpR, respectively). The piperacillin-tazobactam-gentamicin combination was synergistic in vitro. The piperacillin/tazobactam ratio in plasma and in vegetations was always lower than the 4/1 injected dose ratio. In vivo, piperacillin (300 mg/kg of body weight four times a day [QID]) was active against KpS but not against KpR. Tazobactam (75 mg/kg QID) was able to restore the in vivo effect of piperacillin (300 mg/kg QID) against KpR (-3.0 log10 CFU/g of vegetation versus that of controls). Gentamicin (4 mg/kg twice a day [BID]) was active against both strains. Compared with controls, the combination of gentamicin plus piperacillin against KpS (-5.6 log10 CFU/g of vegetation), and the gentamicin-piperacillin-tazobactam combination against KpR (-4.4 log10 CFU/g of vegetation) achieved the greatest decrease in bacterial counts in vegetations and were the only regimens that significantly increased the proportion of sterile vegetations. It is concluded that (i) tazobactam was able to restore the effect of piperacillin against a TEM-3 extended-spectrum Beta-lactamase-producing strain of K. pneumoniae, both in vitro and in a severe experimental infection with high inoculum, when used in a 4/1 piperacillin/tazobactam dose ratio; (ii) gentamicin alone was effective because of the high peak/MBC ratio in plasma; (iii

  5. 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

  6. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  7. Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography.

    PubMed

    Joshi, Varsha; Kumar, Vijesh; Rathore, Anurag S

    2015-08-01

    A method is proposed for rapid development of a short, analytical cation exchange high performance liquid chromatography method for analysis of charge heterogeneity in monoclonal antibody products. The parameters investigated and optimized include pH, shape of elution gradient and length of the column. It is found that the most important parameter for development of a shorter method is the choice of the shape of elution gradient. In this paper, we propose a step by step approach to develop a non-linear sigmoidal shape gradient for analysis of charge heterogeneity for two different monoclonal antibody products. The use of this gradient not only decreases the run time of the method to 4min against the conventional method that takes more than 40min but also the resolution is retained. Superiority of the phosphate gradient over sodium chloride gradient for elution of mAbs is also observed. The method has been successfully evaluated for specificity, sensitivity, linearity, limit of detection, and limit of quantification. Application of this method as a potential at-line process analytical technology tool has been suggested.

  8. Cellulase variants

    DOEpatents

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  9. Versuche zur Gewinnung von katalytischen Antikörpern zur Hydrolyse von Arylcarbamaten und Arylharnstoffen. (English Title: Attempts to produce catalytic antibodies for hydrolysis of arylcarbamates and arylureas)

    NASA Astrophysics Data System (ADS)

    Werner, Deljana

    2002-05-01

    Umsatzgeschwindigkeit eine lineare Abhängigkeit festgestellt. Die thermodynamische Gleichtgewichtsdissoziationskonstante KD des Abzyms von 2,6 nM zeugt von einer sehr guten Affinität zum ÜZA. Hydrolytisch aktiv waren nur Antikörper, die gegen das Übergangszustandsanalogon Hei3 hergestellt worden waren. Es wird vermutet, dass die Hydrolyse der Benzylphenylcarbamate über einen Additions-Eliminierungsmechanismus unter Ausbildung eines tetraedrischen Übergangszustandes verläuft, dessen analoge Verbindung Hei3 ist. Im Rahmen der Generierung von Nachweisantikörpern zur Detektion der Substratabnahme bei der Hydrolyse wurden Anti-Diuron-Antikörper hergestellt. Einer der Antikörper (B91-CG5) ist spezifisch für das Herbizid Diuron und hat einen IC50-Wert von 0,19 µg/l und eine untere Nachweisgrenze von 0,04 µg/l. Ein anderer Antikörper (B91-KF5) reagiert kreuz mit einer Palette ähnlicher Herbizide. Mit diesen Antikörpern wurde ein empfindlicher Labortest, der ein Monitoring von Diuron auf Grundlage des durch die Trinkwasserverordnung festgeschriebenen Wertes für Pflanzenschutzmittel von 0,1 µg/l erlaubt, aufgebaut. Der Effekt der Anti-Diuron-Antikörper auf die Diuron-inhibierte Photosynthese wurde in vitro und in vivo untersucht. Es wurde nachgewiesen, dass sowohl in isolierten Thylakoiden, als auch in intakten Algen eine Vorinkubation der Anti-Diuron-Antikörper mit Diuron zur Inaktivierung seiner Photosynthese-hemmenden Wirkung führt. Wurde der Elektronentransport in den isolierten Thylakoiden oder in Algen durch Diuron unterbrochen, so führte die Zugabe der Anti-Diuron-Antikörper zur Reaktivierung der Elektronenübertragung. Attempts to produce catalytic antibodies for hydrolysis of arylcarbamates and arylureas: The aim of the investigations was to produce antibodies which are able to cleave herbicides resistant to naturally occuring enzymes. Structurally similar carbamate and urea derivatives were chosen for the experiments. Phosphonate derivatives were synthesized

  10. A novel β-globin gene mutation HBB.c.22 G>C produces a hemoglobin variant (Hb Vellore) mimicking HbS in HPLC.

    PubMed

    Edison, E S; Sathya, M; Rajkumar, S V; Nair, S C; Srivastava, A; Shaji, R V

    2012-10-01

    Hemoglobinopathies are highly prevalent in Indian population. DNA analysis to detect causative mutations is required for identifying rare hemoglobin variants or when hematological results are discordant with the clinical phenotype. In this report, we describe a novel hemoglobin variant caused by a mutation in beta-globin gene, Codon 7 GAG→CAG (Glu→Gln) that elutes in the position of sickle haemoglobin (HbS) in cation exchange high performance liquid chromatography. This report highlights possible diagnostic pitfalls in interpreting data solely based on haemoglobin analysis and usefulness of mutation screening in definitive diagnosis of hemoglobinopathies. PMID:22471768

  11. A new natural hGH variant--17.5 kd--produced by alternative splicing. An additional consensus sequence which might play a role in branchpoint selection.

    PubMed Central

    Lecomte, C M; Renard, A; Martial, J A

    1987-01-01

    From a human pituitary cDNA library, we have cloned 3 distinct human growth hormone (hGH) cDNAs, coding respectively for the 22 K hGH, the 20 K variant, and a yet unknown 17.5 K variant. S1 mapping analysis using human pituitary RNA confirms the existence of at least four distinct hGH mRNAs originating from alternative acceptor sites at the second intron of the primary transcript. We have analysed the hGH gene sequence to explain the high frequency of alternative splicings which occur only at this location. In this study we propose CTTGNNPyPyPy as an additional consensus sequence guiding the selection of the branched nucleotide. Images PMID:3627992

  12. Effects of cytotoxin-associated gene A (CagA) positive Helicobacter pylori infection on anti-platelet glycoprotein antibody producing B cells in patients with primary idiopathic thrombocytopenic purpura (ITP)

    PubMed Central

    Cheng, Yuan-Shan; Kuang, Li-Ping; Zhuang, Chun-Lan; Jiang, Jia-Dian; Shi, Man

    2015-01-01

    Objective: To explore the effects of cytotoxin-associated gene A (CagA) positive Helicobacter pylori (H. pylori or HP) infection on circulating B cells producing specific platelet glycoprotein antibodies and the association between therapeutic outcomes in primary idiopathic thrombocytopenic purpura (ITP) patients. Methods: A total of 76 newly diagnosed primary ITP patients were included in the study which was conducted at the first affiliated hospital of Shantou University Medical college, in Shantou city China, between January 2013 and January 2014. These patients were tested for H. pylori infection by 13C urea breath test and for anti-CagA antibody in H. pylori positive cases by enzyme-linked immunosorbent assay (ELISA) method. Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were measured using an enzyme-linked immunospot (ELISPOT) assay in all ITP patients and 30 controls. Anti-nuclear antibody (ANA) was also detected in ITP patients. Results: The numbers of anti-GPIIb/IIIa antibody-producing B cells in HP+CagA+ patients were higher than in HP+CagA- or HP- patients. However, anti-GPIb antibody-producing B cells were found higher in HP- patients. Analysis of treatment outcomes showed that a therapeutic response was more likely in patients presenting anti-GPIIb/IIIa B cells, but the poor response was found to be associated with anti-GPIb B cells and ANA presences. Conclusion: CagA antigen of H. pylori may induce anti-GPIIb/IIIa antibodies production by a molecular mimicry mechanism. Anti-GPIIb/IIIa and anti-GPIb antibody producing B Cells detection is useful for predicting treatment effects of primary ITP. PMID:25878627

  13. A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism*

    PubMed Central

    Liu, Zhi; Leng, Esther C.; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N.; Yan, Wei

    2015-01-01

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. PMID:25583986

  14. Antibody humanization by structure-based computational protein design

    PubMed Central

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH (“Computationally-Driven Antibody Humanization”) in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation. PMID:26252731

  15. The dermal-epidermal junction of human skin contains a novel laminin variant

    PubMed Central

    1992-01-01

    We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant. PMID:1383241

  16. Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation

    PubMed Central

    1992-01-01

    The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events. PMID:1370498

  17. Human Leukocyte Antigen Class II Alleles Influence Levels of Antibodies to the Plasmodium falciparum Asexual-Stage Apical Membrane Antigen 1 but Not to Merozoite Surface Antigen 2 and Merozoite Surface Protein 1

    PubMed Central

    Johnson, Armead H.; Leke, Rose G. F.; Mendell, Nancy R.; Shon, Dewon; Suh, Young Ju; Bomba-Nkolo, Dennis; Tchinda, Viviane; Kouontchou, Samuel; Thuita, Lucy W.; van der Wel, Anne Marie; Thomas, Alan; Stowers, Anthony; Saul, Allan; Zhou, Ainong; Taylor, Diane W.; Quakyi, Isabella A.

    2004-01-01

    The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP119) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP119 variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP119 but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP119 variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated. PMID:15102786

  18. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2011-05-31

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  19. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2008-12-02

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  20. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2012-08-07

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  1. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2011-08-16

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  2. Variant Humicola grisea CBH1.1

    SciTech Connect

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2014-03-18

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  3. Variant humicola grisea CBH1.1

    SciTech Connect

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Edmund, Larenas

    2014-09-09

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  4. Variant Humicola grisea CBH1.1

    SciTech Connect

    Goedegeburr, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2013-02-19

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  5. Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei.

    PubMed

    Colombi, Débora; Oliveira, Maria L S; Campos, Ivana B; Monedero, Vicente; Pérez-Martinez, Gaspar; Ho, Paulo L

    2006-12-01

    Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough. PMID:17106803

  6. Developing recombinant antibodies for biomarker detection

    SciTech Connect

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

    2010-10-01

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

  7. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines.

    PubMed

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B; Fontana, Juan; Steven, Alasdair C; King, Lisa R; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K; Golding, Hana

    2011-08-01

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

  8. Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

    SciTech Connect

    Crowell, R.L.; Field, A.K.; Schleif, W.A.; Long, W.L.; Colonno, R.J.; Mapoles, J.E.; Emini, E. A.

    1986-02-01

    BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: (i) protection against CB3 on HeLa, (ii) protection against CB3-RD on rhabdomyosarcoma (RD) cells, and (iii) protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of (/sup 35/S)methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-Rd virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and /sup 125/I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

  9. Staphylococcus aureus adapts to oxidative stress by producing H2O2-resistant small-colony variants via the SOS response.

    PubMed

    Painter, Kimberley L; Strange, Elizabeth; Parkhill, Julian; Bamford, Kathleen B; Armstrong-James, Darius; Edwards, Andrew M

    2015-05-01

    The development of chronic and recurrent Staphylococcus aureus infections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure of S. aureus to sublethal concentrations of H2O2 leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H2O2 exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within the S. aureus population. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H2O2 also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H2O2 than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2. Taken together, these data reveal a mechanism by which S. aureus adapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production.

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

    PubMed

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

    2009-09-01

    Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents. PMID:19758150

  11. Expression in systemic lupus erythematosus of an idiotype common to DNA-binding and nonbinding monoclonal antibodies produced by normal human lymphoid cells.

    PubMed Central

    Cairns, E; Massicotte, H; Bell, D A

    1989-01-01

    Rabbit antiserum raised against a normal-derived monoclonal anti-DNA antibody KIM 4.6.3 (IgM lambda) was used for idiotype analyses. This anti-serum (anti-4.6.3 ID) was rendered specific for KIM 4.6.3 idiotype (4.6.3 ID) by absorption with normal human IgM and IgG. The specificity of anti-4.6.3 was shown by its ability to bind to KIM 4.6.3 antibody but not to normal human IgM and IgG, by inhibition of anti-4.6.3 ID reactivity with KIM 4.6.3 antibody by the homologous monoclonal antibody and by the ability of anti-4.6.3 ID to inhibit the binding of single stranded DNA with KIM 4.6.3 antibody. The 4.6.3 ID was found to be commonly expressed since it was detected among 33% (10/30) DNA and 32% (23/72) non-DNA-reactive monoclonal antibodies that were obtained from five different unrelated normal individuals. The binding to ssDNA of the majority of idiotype positive anti-DNA antibodies however was not blocked by anti-4.6.3 ID suggesting that among these other monoclonal antibodies its expression is outside of the antigen binding site. The 4.6.3 ID, which was present among some normal-derived monoclonal IgM molecules was also found at a high frequency (90%) in the sera of patients with systemic lupus erythematosus (SLE) but only at a low frequency (24%) and concentration in normal sera. The level of 4.6.3 ID in SLE did not correlate with serum IgM and IgG nor with anti-DNA antibody concentrations. Idiotypic relatedness between SLE serum antibodies and monoclonal anti-DNA antibodies of normals implies the existence of a cross-reactive idiotype family and implies that a conserved common gene or closely related genes exist in the germ line encoding these 4.6.3 ID positive antibodies some of which are not exclusively associated with nucleic acid reactivity. The expression of these germ line genes in vivo thus distinguishes SLE from normals. PMID:2493481

  12. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  13. Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS-PSD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to severe outbreaks of foodborne illness throughout the world, e.g. Germany and France in 2011. STEC infections can result in bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. New analytical techniques are ne...

  14. Combination of low producer AA-genotypes in IFN-γ and IL-10 genes makes a high risk genetic variant for HIV disease progression.

    PubMed

    Singh, Sukhvinder; Sharma, Aman; Arora, Sunil K

    2016-01-01

    Rate of HIV disease progression varies considerably among individuals, host genetic makeup be one of the possible reasons. We aimed to determine association of functional single nucleotide polymorphism (SNPs), (-179G/T and +874T/A) in IFN-γ and (-1082A/G, -819C/T and -592C/A) in IL-10 genes, with the rate of disease progression or susceptibility to HIV infection. Therapy naïve HIV infected individuals from North India, categorized as slow progressors or fast progressors and HIV exposed seronegative individuals were recruited for this study. Genotyping results revealed significantly higher frequencies of low producer AA genotype at +874T/A in IFN-γ gene and -592C/A position in IL-10 gene in FPs (p<0.002). Multifactor dimensional reduction (MDR) analysis revealed this to be a high risk combination for faster disease progression in HIV-1 infected individuals. Low producer AA genotype carriers at +874T/A in IFN-γ gene produced significantly low amounts of cellular IFN-γ. Low producing haplotype 'ATA' at -1082, -819 and -592 loci in IL-10 gene was significantly over-represented in FPs as compared to SPs (p<0.01) and these individuals showed poor response to therapy in terms of CD4 count gains after one year of ART, compared to high producing haplotype (GCC) carriers. Thus, a combination of genetic variations in IFN-γ and IL-10 cytokine genes in a single host associate with HIV disease progression and may help clinicians to better manage the HIV disease if known earlier.

  15. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

    PubMed Central

    Desin, Taseen S.; Townsend, Hugh G.; Potter, Andrew A.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes. PMID:26451946

  16. Engineering antibody therapeutics.

    PubMed

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  17. Invasive Stratified Mucin-producing Carcinoma and Stratified Mucin-producing Intraepithelial Lesion (SMILE): 15 Cases Presenting a Spectrum of Cervical Neoplasia With Description of a Distinctive Variant of Invasive Adenocarcinoma.

    PubMed

    Lastra, Ricardo R; Park, Kay J; Schoolmeester, J Kenneth

    2016-02-01

    Stratified mucin-producing intraepithelial lesion (SMILE) is a cervical intraepithelial lesion, distinct from conventional squamous or glandular counterparts, believed to arise from embryonic cells at the transformation zone by transdifferentiation during high-risk HPV-associated carcinogenesis. It is characterized by stratified, immature epithelial cells displaying varying quantities of intracytoplasmic mucin throughout the majority of the lesional epithelium. We identified a distinct form of invasive cervical carcinoma with morphologic features identical to those in SMILE, which we have termed "invasive stratified mucin-producing carcinoma." Fifteen cases from 15 patients (mean 36 y; range, 22 to 64 y) were retrieved from the pathology archives of multiple institutions with a diagnosis of either SMILE or invasive cervical carcinoma with a description or comment about the invasive tumor's resemblance to SMILE. Seven cases had solely intraepithelial disease with a component of SMILE (mean 29 y; range, 22 to 40 y). The 8 other cases had invasive stratified mucin-producing carcinoma (mean 44; range, 34 to 64 y) in which SMILE was identified in 7. All cases of invasive stratified mucin-producing carcinoma demonstrated stratified, immature nuclei with intracytoplasmic mucin, which morphologically varied between cases from "mucin-rich" to "mucin-poor" in a similar manner to SMILE. All cases had mitotic figures and apoptotic debris, and an intralesional neutrophilic infiltrate was seen in the majority of cases. In cases of invasive carcinoma, the depth of invasion ranged from <1 to 19 mm. Follow-up information was available in 8 cases and ranged from 1 to 36 months (mean 11 mo). Three cases of invasive stratified mucin-producing carcinoma had biopsy or resection-proven metastatic carcinoma on follow-up. These 15 cases of cervical stratified mucin-producing lesions show a combination of intraepithelial and invasive growth patterns. Given that SMILE is well rooted as a

  18. Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry.

    PubMed

    Sanz, Miriam; Andreote, Ana Paula Dini; Fiore, Marli Fatima; Dörr, Felipe Augusto; Pinto, Ernani

    2015-06-18

    Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).

  19. Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

    PubMed Central

    Sanz, Miriam; Andreote, Ana Paula Dini; Fiore, Marli Fatima; Dörr, Felipe Augusto; Pinto, Ernani

    2015-01-01

    Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.). PMID:26096276

  20. Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

    PubMed Central

    Lehmacher, Anselm; Meier, Heidi; Aleksic, Stojanka; Bockemühl, Jochen

    1998-01-01

    The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC. PMID:9647814

  1. A human monoclonal antibody, produced following in vitro immunization, recognizing an epitope shared by HLA-A2 subtypes and HLA-A28.

    PubMed

    Mulder, A; Kardol, M; Blom, J; Jolley, W B; Melief, C J; Bruning, H

    1993-07-01

    In vitro immunization and subsequent immortalization of peripheral blood cells of a multiparous woman has resulted in the production of a stable human mouse heterohybridoma, 5C2A2, secreting an HLA-A2/A28-specific human monoclonal antibody. Although possibly exposed to HLA-A2 by transfusions, the cell donor showed no HLA-A2-specific serum antibodies. The present protocol for in vitro immunization includes the elimination of suppressor cells from the responder cell population, the presence of irradiated allogeneic lymphocytes as a source of antigen, as well as stimuli--recombinant interleukin-2 and a B-cell specific nucleoside analogue--causing the proliferation of B lymphocytes, prior to immortalization. The ability of the antibody 5C2A2 to detect all known HLA-A2 subtypes, except A2.3, and A28, allows identification of the serological epitope on the HLA-A2 molecule. Application of this in vitro immunization method allows the production of a set of HLA monoclonal antibody-secreting human hybridomas, independent of the existence of serum HLA antibodies in the lymphocyte donors. PMID:7504327

  2. Stiff-person syndrome with amphiphysin antibodies

    PubMed Central

    Murinson, Beth B.; Guarnaccia, Joseph B.

    2008-01-01

    Background: Stiff-person syndrome (SPS), formerly Stiff-man syndrome, is a rare autoimmune disease usually exhibiting severe spasms and thoracolumbar stiffness, with very elevated glutamic acid decarboxylase antibodies (GAD Ab). A paraneoplastic variant, less well characterized, is associated with amphiphysin antibodies (amphiphysin Ab). The objective of this study was to identify distinctive clinical features of amphiphysin Ab-associated SPS. Methods: Records associated with 845 sera tested in the Yale SPS project were examined, and 621 patients with clinically suspected SPS were included in the study. Clinical characteristics were assessed with correction for multiple comparisons. Results: In all, 116 patients had GAD antibodies and 11 patients had amphiphysin Ab; some clinical information was available for 112 and 11 of these patients, respectively. Patients with amphiphysin Ab-associated SPS were exclusively female; mean age was 60. All except one had breast cancer; none had diabetes. Compared to patients with GAD Ab-associated SPS, those with amphiphysin Ab were older (p = 0.02) and showed a dramatically different stiffness pattern (p < 0.0000001) with cervical involvement more likely, p ≤ 0.001. Electromyography showed continuous motor unit activity or was reported positive in eight. Benzodiazepines at high dose (average 50 mg/day diazepam) were partially effective. Four patients were steroid responsive and tumor excision with chemotherapy produced marked clinical improvement in three of five patients. Conclusions: Amphiphysin Ab-associated stiff-person syndrome is strongly associated with cervical region stiffness, female sex, breast cancer, advanced age, EMG abnormalities, and benzodiazepine responsiveness. The condition may respond to steroids and can dramatically improve with cancer treatment. GLOSSARY EAE = experimental autoimmune encephalitis; GAD Ab = glutamic acid decarboxylase antibodies; ICC = immunocytochemistry; PERM = progressive variant with

  3. Very High Plasma Concentrations of a Monoclonal Antibody against the Human Insulin Receptor Are Produced by Subcutaneous Injection in the Rhesus Monkey.

    PubMed

    Boado, Ruben J; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Pardridge, William M

    2016-09-01

    Brain penetration of recombinant protein drugs is possible following the re-engineering of the drug as an IgG fusion protein. The IgG domain is a monoclonal antibody (mAb) against an endogenous blood-brain barrier (BBB) receptor transporter, such as the insulin receptor. One such mAb targets the human insulin receptor (HIR) and is active in Rhesus monkeys. Prior work has measured the plasma pharmacokinetics of HIRMAb-derived fusion proteins following intravenous (IV) infusion. However, an alternative method of administration for chronic treatment of brain disease is the subcutaneous (SQ) route. The extent to which an antibody against the insulin receptor undergoes systemic distribution and clearance is unknown. Therefore, in the present study, the rate of plasma clearance of the HIRMAb is measured in Rhesus monkeys following IV or SQ administration of 3, 10, and 30 mg/kg doses of the antibody. The HIRMAb is readily absorbed into the systemic circulation following SQ injection with a 42% plasma bioavailability. The rate of plasma clearance of the antibody, 0.04-0.06 mL/min/kg, is the same following either IV or SQ administration. Owing to the slow rate of plasma clearance of the antibody, high concentrations of the HIRMAb are sustained in plasma for days after the SQ injection. The plasma concentration of the HIRMAb exceeds 0.8 mg/mL, which is 9% of the entire plasma IgG pool in the primate, after the SQ injection of the high dose, 30 mg/kg, of the antibody. In summary, the pharmacokinetics of plasma clearance of the HIRMAb are such that HIRMAb-derived fusion proteins can be developed as protein therapeutics for the brain with chronic SQ administration on a weekly or twice-weekly regimen. PMID:27513815

  4. Antibodies to both terminal and internal B-cell epitopes of Francisella tularensis O-polysaccharide produced by patients with tularemia.

    PubMed

    Lu, Zhaohua; Perkins, Hillary M; Sharon, Jacqueline

    2014-02-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals.

  5. The germinal center antibody response in health and disease

    PubMed Central

    DeFranco, Anthony L.

    2016-01-01

    The germinal center response is the delayed but sustained phase of the antibody response that is responsible for producing high-affinity antibodies of the IgG, IgA and/or IgE isotypes. B cells in the germinal center undergo re-iterative cycles of somatic hypermutation of immunoglobulin gene variable regions, clonal expansion, and Darwinian selection for cells expressing higher-affinity antibody variants. Alternatively, selected B cells can terminally differentiate into long-lived plasma cells or into a broad diversity of mutated memory B cells; the former secrete the improved antibodies to fight an infection and to provide continuing protection from re-infection, whereas the latter may jumpstart immune responses to subsequent infections with related but distinct infecting agents. Our understanding of the molecules involved in the germinal center reaction has been informed by studies of human immunodeficiency patients with selective defects in the production of antibodies. Recent studies have begun to reveal how innate immune recognition via Toll-like receptors can enhance the magnitude and selective properties of the germinal center, leading to more effective control of infection by a subset of viruses. Just as early insights into the nature of the germinal center found application in the development of the highly successful conjugate vaccines, more recent insights may find application in the current efforts to develop new generations of vaccines, including vaccines that can induce broadly protective neutralizing antibodies against influenza virus or HIV-1. PMID:27303636

  6. New engineered antibodies against prions

    PubMed Central

    Škrlj, Nives; Dolinar, Marko

    2014-01-01

    A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens. PMID:23941991

  7. Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

    PubMed Central

    Montefiori, David C.; Reimann, Keith A.; Wyand, Michael S.; Manson, Kelledy; Lewis, Mark G.; Collman, Ronald G.; Sodroski, Joseph G.; Bolognesi, Dani P.; Letvin, Norman L.

    1998-01-01

    The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1. PMID:9525675

  8. An application of capsid-specific artificial ankyrin repeat protein produced in E. coli for immunochromatographic assay as a surrogate for antibody.

    PubMed

    Nangola, Sawitree; Thongkum, Weeraya; Saoin, Somphot; Ansari, Aftab A; Tayapiwatana, Chatchai

    2014-07-01

    Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of antibodies and/or other proteins of interest. For the detection of target proteins, most of the commercial tests are assembled based on the conjugation of colloidal gold particles to monoclonal antibodies embedded within the conjugate pad of a strip test. In this study, we tested the novel concept of using an artificial non-antibody structure for generating a colloidal gold conjugate (CGC). We exploited the property of an ankyrin repeat protein that specifically binds to the HIV-1 capsid protein termed Ank(GAG)1D4. This construct was applied as a model structure to create Ank1D4-CGC and used as a new type of visible detector system and termed it ankyrin-based immunochromatographic strip (ABIS) test. The ABIS test was shown to be highly sensitive with a lower limit of detection of the target protein at 0.1 μg/ml. Moreover, the ABIS test was not only highly sensitive but also shared a level of specificity within the same range of the commercial test kit. The results of the studies presented herein therefore demonstrate the novel application of an artificial non-immunoglobulin structure (ankyrin repeat protein) as the new line of a visible detector using a rapid diagnostic test with characteristics that have the potential to be superior to those that utilize antibody-based tests.

  9. Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

    PubMed

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

    2015-11-01

    In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

  10. Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

    PubMed Central

    Campagnari, A A; Karalus, R; Apicella, M; Melaugh, W; Lesse, A J; Gibson, B W

    1994-01-01

    Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid. Images PMID:8188362

  11. Regulation of naturally occurring autoantibody-producing cells: detection of a suppressive substance which inhibits the secretion of antibodies directed against enzyme-treated isologous erythrocytes.

    PubMed

    Moticka, E J

    1983-10-01

    Normal mice possess spleen cells capable of forming hemolytic plaques against bromelain-treated autologous red cells (Br MRBC). There is present in the serum of these same mice a substance which can inhibit the formation of these plaques. This substance is inhibitory to the secretion of these antibodies following incubation of spleen cells in 20% serum at 4 degrees C for 5 min. This substance is not inhibitory to the formation of anti-sheep erythrocyte plaques from mice either immunized or nonimmunized with sheep erythrocytes. Characterization of the substance indicates that it is neither soluble antigen nor specific antibody. However, inclusion of nanogram amounts of soluble antigen from bromelain-treated red cells in the assay mixture effectively neutralized the suppression. In addition, passage of serum through a mouse anti-Br MRBC antibody immunoadsorbent effectively removed the suppressive activity of the serum while suppression could be recovered in the acid eluate from such a column. This suggests that the mechanism of suppression brought about by incubation in serum is due to the action of a molecule possessing anti-idiotypic activity directed against the cell surface receptors of anti-Br MRBC B cells. Attempts to isolate the molecule based on the postulate that it is immunoglobulin in nature have been unsuccessful.

  12. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  13. Antibody Production with Synthetic Peptides.

    PubMed

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column. PMID:27515072

  14. Association Energetics of Cross-reactive and Specific Antibodies

    PubMed Central

    Mohan, S.; Kourentzi, Katerina; Schick, Kari A.; Uehara, Christian; Lipschultz, Claudia A.; Acchione, Mauro; DeSantis, Morgan E.; Smith-Gill, Sandra J.; Willson, Richard C.

    2009-01-01

    HyHEL-8, HyHEL-10 and HyHEL-26 (HH8, HH10 and HH26, respectively) are murine monoclonal IgG1 antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally-characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more-extensive network of intramolecular salt links, and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition. PMID:19166328

  15. Antibody directed against GD(2) produces mechanical allodynia, but not thermal hyperalgesia when administered systemically or intrathecally despite its dependence on capsaicin sensitive afferents.

    PubMed

    Sorkin, L S; Yu, A L; Junger, H; Doom, C M

    2002-03-15

    Anti-GD(2) antibodies have been shown to be effective for immunotherapy of neuroblastoma and other GD(2) enriched malignancies. Infusion of anti-GD(2) antibodies frequently causes spontaneous pain and allodynia for the duration of the immunotherapy and occasionally longer lasting neuropathic pain. Bolus intravenous injection of anti-GD(2) in rats initiates mechanical allodynia as measured by withdrawal threshold of the hindpaws. In this study, thermal thresholds were measured prior to and for up to 6 h following systemic anti-GD(2) administration in adult rats. In addition, both thermal and mechanical thresholds were tested following intrathecal administration of anti-GD(2) and IgG(2a). Murine anti-GD(2) elicited mechanical allodynia when administered into either the vasculature or the intrathecal space. Effective systemic doses were 1--3 mg/kg as previously shown. Intrathecally, optimal doses ranged from 0.01 to 0.1 ng; a higher dose was ineffective. Thermal hyperalgesia was not observed via either route of administration. Intrathecal pretreatment 48--72 h prior to the experiment with capsaicin at doses sufficient to cause a 50% depletion of dorsal horn CGRP, caused a total blockade of the mechanical allodynia indicating an involvement of peptidergic fine afferent fibers. It is likely that the antibody reacts with an antigen on peripheral nerve and/or myelin to initiate its effect. The lack of observed thermal hyperalgesia is surprising especially in light of the capsaicin-associated blockade, however, it is consistent with several other immune system related models of pain.

  16. Cancer cells assemble and align gold nanorods conjugated to antibodies to produce highly enhanced, sharp, and polarized surface Raman spectra: a potential cancer diagnostic marker.

    PubMed

    Huang, Xiaohua; El-Sayed, Ivan H; Qian, Wei; El-Sayed, Mostafa A

    2007-06-01

    Human oral cancer cells are found to assemble and align gold nanorods conjugated to anti-epidermal growth factor receptor (anti-EGFR) antibodies. Immnoconjugated gold nanorods and nanospheres were shown previously to exhibit strong Rayleigh (Mie) scattering useful for imaging. In the present letter, molecules near the nanorods on the cancer cells are found to give a Raman spectrum that is greatly enhanced (due to the high surface plasmon field of the nanorod assembly in which their extended surface plasmon fields overlap), sharp (due to a homogeneous environment), and polarized (due to anisotropic alignments). These observed properties can be used as diagnostic signatures for cancer cells.

  17. Antithyroid microsomal antibody

    MedlinePlus

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

  18. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  19. Antimitochondrial antibody

    MedlinePlus

    ... antibodies (AMA) are substances ( antibodies ) that form against mitochondria. The mitochondria are an important part of cells. They are ... often, in people with other kinds of liver disease and some autoimmune diseases. Risks Risks for having ...

  20. 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. PMID:26850143

  1. 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.

  2. Humanized Antibodies for Antiviral Therapy

    NASA Astrophysics Data System (ADS)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  3. Antibody validation

    PubMed Central

    Bordeaux, Jennifer; Welsh, Allison W.; Agarwal, Seema; Killiam, Elizabeth; Baquero, Maria T.; Hanna, Jason A.; Anagnostou, Valsamo K.; Rimm, David L.

    2013-01-01

    Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence. PMID:20359301

  4. Monoclonal antibodies.

    PubMed

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  5. Restricted virus replication in the spinal cords of nude mice infected with a Theiler's virus variant.

    PubMed

    Zurbriggen, A; Yamada, M; Thomas, C; Fujinami, R S

    1991-02-01

    The Daniels strain of Theiler's murine encephalomyelitis produces a chronic disease which is an animal model for human demyelinating disorders. Previously, we selected a neutralization-resistant virus variant producing an altered and diminished central nervous system disease in immunocompetent mice which was evident during the later stage of infection (after 4 weeks) (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). The exact epitope determining neurovirulence was precisely mapped to a capsid protein, VP-1, and represents a neutralizing region (A. Zurbriggen, J. M. Hogle, and R. S. Fujinami, J. Exp. Med. 170:2037-2049, 1989). Here, we present experiments with immunoincompetent animals to determine viral replication, spread, and targeting to the central nervous system in the absence of detectable antibodies or functional T cells. Nude mice were infected orally, and the virus was monitored by plaque assay, immunohistochemistry, and in situ hybridization. Early during the infection (1 week), the variant virus induced an acute disease comparable to that induced by the wild-type virus in these nude mice. Alterations in tropism in the central nervous system were not apparent when wild-type parental Daniels strain virus was compared with the variant virus. Moreover, variant virus replicated in tissue culture (BHK-21 cells) to similarly high titers in a time course identical to that of the wild-type virus (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). However, replication of the variant virus versus the wild-type virus within the spinal cord of athymic nude mice infected per os was substantially restricted by 6 weeks postinfection. Therefore, the reduced neurovirulence in the later stage (6 weeks) of the disease is most likely due to a diminished growth rate or spread of the variant virus in the central nervous system rather than to marked differences in viral tropism.

  6. Restricted virus replication in the spinal cords of nude mice infected with a Theiler's virus variant.

    PubMed Central

    Zurbriggen, A; Yamada, M; Thomas, C; Fujinami, R S

    1991-01-01

    The Daniels strain of Theiler's murine encephalomyelitis produces a chronic disease which is an animal model for human demyelinating disorders. Previously, we selected a neutralization-resistant virus variant producing an altered and diminished central nervous system disease in immunocompetent mice which was evident during the later stage of infection (after 4 weeks) (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). The exact epitope determining neurovirulence was precisely mapped to a capsid protein, VP-1, and represents a neutralizing region (A. Zurbriggen, J. M. Hogle, and R. S. Fujinami, J. Exp. Med. 170:2037-2049, 1989). Here, we present experiments with immunoincompetent animals to determine viral replication, spread, and targeting to the central nervous system in the absence of detectable antibodies or functional T cells. Nude mice were infected orally, and the virus was monitored by plaque assay, immunohistochemistry, and in situ hybridization. Early during the infection (1 week), the variant virus induced an acute disease comparable to that induced by the wild-type virus in these nude mice. Alterations in tropism in the central nervous system were not apparent when wild-type parental Daniels strain virus was compared with the variant virus. Moreover, variant virus replicated in tissue culture (BHK-21 cells) to similarly high titers in a time course identical to that of the wild-type virus (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). However, replication of the variant virus versus the wild-type virus within the spinal cord of athymic nude mice infected per os was substantially restricted by 6 weeks postinfection. Therefore, the reduced neurovirulence in the later stage (6 weeks) of the disease is most likely due to a diminished growth rate or spread of the variant virus in the central nervous system rather than to marked differences in viral tropism. Images PMID:1987366

  7. Glycan modulation and sulfoengineering of anti–HIV-1 monoclonal antibody PG9 in plants

    PubMed Central

    Loos, Andreas; Gach, Johannes S.; Hackl, Thomas; Maresch, Daniel; Henkel, Theresa; Porodko, Andreas; Bui-Minh, Duc; Sommeregger, Wolfgang; Wozniak-Knopp, Gordana; Forthal, Donald N.; Altmann, Friedrich; Steinkellner, Herta; Mach, Lukas

    2015-01-01

    Broadly neutralizing anti–HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells (CHOPG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for CHOPG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti–HIV-1 antibodies with effector functions superior to PG9 made in CHO cells. PMID:26417081

  8. Lack of neutralizing antibodies to caprine arthritis-encephalitis lentivirus in persistently infected goats can be overcome by immunization with inactivated Mycobacterium tuberculosis.

    PubMed Central

    Narayan, O; Sheffer, D; Griffin, D E; Clements, J; Hess, J

    1984-01-01

    The pathogenesis of the persistent progressive diseases of sheep (visna-maedi) and goats (arthritis-encephalitis) is dependent on continuous replication of the causative lentiviruses. One subgroup of these viruses, Icelandic visna virus, accomplishes this form of replication by undergoing antigenic mutation. Mutant viruses arising late in the infection escape neutralization by antibodies directed to the parental virus. In contrast, we show here that viruses obtained from persistently infected sheep and goats with natural disease in this country do not induce virus-neutralizing antibodies, although antibodies to virus core proteins were produced. The lack of neutralizing antibodies was not overcome by hyperimmunization of animals with concentrated preparations of live or inactivated virus. Thus, failure to produce these specific antibodies was not due to lack of sufficient antigen or interference with the immune response because of the ability of these viruses to infect macrophages. The hyporesponsive state, however, was overcome by immunization of animals with virus and large amounts of inactivated Mycobacterium tuberculosis. Induction of agglutinating and neutralizing antibodies by this method was probably due to a unique form of antigen processing by macrophages activated by M. tuberculosis. Neutralizing antibodies were produced for the first time against the caprine arthritis-encephalitis virus by this method. These antibodies have similar biological properties to those induced by Icelandic visna virus. They belong to the immunoglobulin G1 subclass, they are effective against a narrow range of caprine arthritis-encephalitis viruses, and they identify (for the first time) antigenic variants among these caprine agents. PMID:6319735

  9. Evaluation of options for harvest of a recombinant E. Coli fermentation producing a domain antibody using ultra scale‐down techniques and pilot‐scale verification

    PubMed Central

    Voulgaris, Ioannis; Chatel, Alex; Finka, Gary; Uden, Mark

    2016-01-01

    Ultra scale‐down (USD) methods operating at the millilitre scale were used to characterise full‐scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g., a centrifugation stage operating at 0.11 L/m2 equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m2/L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2016 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 32:382–392, 2016 PMID:26698375

  10. Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

    PubMed

    Hadiji-Abbes, Nadia; Mihoubi, Wafa; Martin, Marta; Karakasyan-Dia, Carole; Frikha, Fakher; Gergely, Csilla; Jouenne, Thierry; Gargouri, Ali; Mokdad-Gargouri, Raja

    2015-10-01

    Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

  11. Two-mAb cocktail protects macaques against the Makona variant of Ebola virus.

    PubMed

    Qiu, Xiangguo; Audet, Jonathan; Lv, Ming; He, Shihua; Wong, Gary; Wei, Haiyan; Luo, Longlong; Fernando, Lisa; Kroeker, Andrea; Fausther Bovendo, Hugues; Bello, Alexander; Li, Feng; Ye, Pei; Jacobs, Michael; Ippolito, Giuseppe; Saphire, Erica Ollmann; Bi, Shengli; Shen, Beifen; Gao, George F; Zeitlin, Larry; Feng, Jiannan; Zhang, Boyan; Kobinger, Gary P

    2016-03-01

    The 2014-2015 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. We show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV variant Makona, the virus responsible for the ongoing 2014-2015 outbreak, whereas a similar formulation of ZMapp protected two of three NHPs. The chimeric MIL77E mAb cocktail is produced in engineered Chinese hamster ovary cells and is based on mAbs c13C6 and c2G4 from ZMapp. The use of only two antibodies in MIL77E opens the door to a pan-ebolavirus cocktail.

  12. Two-mAb cocktail protects macaques against the Makona variant of Ebola virus.

    PubMed

    Qiu, Xiangguo; Audet, Jonathan; Lv, Ming; He, Shihua; Wong, Gary; Wei, Haiyan; Luo, Longlong; Fernando, Lisa; Kroeker, Andrea; Fausther Bovendo, Hugues; Bello, Alexander; Li, Feng; Ye, Pei; Jacobs, Michael; Ippolito, Giuseppe; Saphire, Erica Ollmann; Bi, Shengli; Shen, Beifen; Gao, George F; Zeitlin, Larry; Feng, Jiannan; Zhang, Boyan; Kobinger, Gary P

    2016-03-01

    The 2014-2015 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. We show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV variant Makona, the virus responsible for the ongoing 2014-2015 outbreak, whereas a similar formulation of ZMapp protected two of three NHPs. The chimeric MIL77E mAb cocktail is produced in engineered Chinese hamster ovary cells and is based on mAbs c13C6 and c2G4 from ZMapp. The use of only two antibodies in MIL77E opens the door to a pan-ebolavirus cocktail. PMID:26962157

  13. Engineering antibodies for therapy.

    PubMed

    Adair, J R

    1992-12-01

    Success in the generation of an antibody-based therapeutic requires careful consideration of the binding site, to achieve specificity and high affinity; of the effector, to produce the desired therapeutic effect; of the means of attachment of the effector to the binding site; production of the end product; and the response made by the patient to the administered compound. Each of these areas is receiving attention by antibody-engineering techniques. The number of potentially useful monoclonal antibodies developed over the last 10 years, and currently in clinical trials or preregistration, is now being increased by these engineered newcomers. It will be interesting to see over the next few years how many of these antibodies, and of which kind, emerge as products.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga tox...

  15. Comparison of antibody- versus pcr-based assays for serotyping shiga toxin-producing escherichia coli recovered from various cattle operations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotyping of Shiga toxin-producing Escherichia coli (STEC) strains is contingent upon the availability of quality antisera. In this study, the serogroup of 161 STEC strains typed by conventional antisera and isolated from the fecal samples of California cattle were compared to two newly developed ...

  16. New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin (Stx) is a major virulence factor for several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges in clinical diagnosis of infections caused by Shiga toxin-producing org...

  17. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies. PMID:20954327

  18. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies.

  19. Monoclonal antibody heterogeneity analysis and deamidation monitoring with high-performance cation-exchange chromatofocusing using simple, two component buffer systems.

    PubMed

    Kang, Xuezhen; Kutzko, Joseph P; Hayes, Michael L; Frey, Douglas D

    2013-03-29

    The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions.

  20. Glyco-variant library of the versatile enzyme horseradish peroxidase

    PubMed Central

    Capone, Simona; Pletzenauer, Robert; Maresch, Daniel; Metzger, Karl; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2014-01-01

    When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. PMID:24859724

  1. Glyco-variant library of the versatile enzyme horseradish peroxidase.

    PubMed

    Capone, Simona; Pletzenauer, Robert; Maresch, Daniel; Metzger, Karl; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2014-09-01

    When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. PMID:24859724

  2. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Daugharty, Harry; Hopper, John E.; MacDonald, A. Bruce; Nisonoff, Alfred

    1969-01-01

    Specifically purified anti-p-azobenzoate antibodies of the IgG class from individual rabbits were used to elicit anti-idiotypic antibodies in recipient rabbits. Allotypes of each donor and recipient were matched. When polymerized antibodies were used for immunization, more than 80% of the recipients responded with the formation of antibodies that precipitated the monomeric donor antibody. Percentages of precipitable molecules in the donor antibody population (D) varied from 4 to 56. As little as 4% was readily detectable by the Ouchterlony method or precipitin test. Specificity of the reaction was tested by double diffusion in agar gel against a panel of purified antibenzoate antibodies from 14 heterologous rabbits and, quantitatively, in three systems by measurement of the extent of coprecipitation of heterologous, radiolabeled antibenzoate antibodies. No cross-reactions were observed. Reactions were shown to be attributable to antibenzoate antibodies in the donor serum, and contributions of allotypic reactions were excluded. In three systems investigated quantitatively, and in one studied qualitatively, two recipients of the same donor antibody produced anti-antibody that reacted with essentially the same subfraction of the donor antibody population. The findings that only a portion of the D population is immunogenic, and that the same subfraction is frequently immunogenic in different recipients, suggest that the immunogenic population comprises a limited number of homogeneous groups of antibody molecules. This is supported by the small number of bands usually observed by the Ouchterlony technique. Quantitative methods of analysis should provide an approach to the study of cell populations producing antibodies of a particular idiotype. PMID:5347693

  3. Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.

    PubMed

    Omenn, Gilbert S

    2014-05-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

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

    PubMed

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

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. PMID:26654254

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

    PubMed

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

    2015-12-24

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

  6. Optimizing antibody expression by using the naturally occurring framework diversity in a live bacterial antibody display system

    PubMed Central

    Lombana, T. Noelle; Dillon, Michael; Bevers III, Jack; Spiess, Christoph

    2015-01-01

    Rapid identification of residues that influence antibody expression and thermostability is often needed to move promising therapeutics into the clinic. To establish a method that can assess small expression differences, we developed a Bacterial Antibody Display (BAD) system that overcomes previous limitations, enabling the use of full-length formats for antibody and antigen in a live cell setting. We designed a unique library of individual framework variants using natural diversity introduced by somatic hypermutation, and screened half-antibodies for increased expression using BAD. We successfully identify variants that dramatically improve expression yields and in vitro thermostability of two therapeutically relevant antibodies in E. coli and mammalian cells. While we study antibody expression, bacterial display can now be expanded to examine the processes of protein folding and translocation. Additionally, our natural library design strategy could be applied during antibody humanization and library design for in vitro display methods to maintain expression and formulation stability. PMID:26631978

  7. Hemoglobin variants in Cyprus.

    PubMed

    Kyrri, Andreani R; Felekis, Xenia; Kalogerou, Eleni; Wild, Barbara J; Kythreotis, Loukas; Phylactides, Marios; Kleanthous, Marina

    2009-01-01

    Cyprus, located at the eastern end of the Mediterranean region, has been a place of eastern and western civilizations, and the presence of various hemoglobin (Hb) variants can be considered a testimony to past colonizations of the island. In this study, we report the structural Hb variants identified in the Cypriot population (Greek Cypriots, Maronites, Armenians, and Latinos) during the thalassemia screening of 248,000 subjects carried out at the Thalassaemia Centre, Nicosia, Cyprus, over a period of 26 years. A sample population of 65,668 people was used to determine the frequency and localization of several of the variants identified in Cyprus. The localization of some of the variants in regions where the presence of foreign people was most prevalent provides important clues to the origin of the variants. Twelve structural variants have been identified by DNA sequencing, nine concerning the beta-globin gene and three concerning the alpha-globin gene. The most common beta-globin variants identified were Hb S (0.2%), Hb D-Punjab (0.02%), and Hb Lepore-Washington-Boston (Hb Lepore-WB) (0.03%); the most common alpha-globin variant was Hb Setif (0.1%). The presence of some of these variants is likely to be directly linked to the history of Cyprus, as archeological monuments have been found throughout the island which signify the presence for many years of the Greeks, Syrians, Persians, Arabs, Byzantines, Franks, Venetians, and Turks. PMID:19373583

  8. Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus.

    PubMed

    He, Junyun; Lai, Huafang; Engle, Michael; Gorlatov, Sergey; Gruber, Clemens; Steinkellner, Herta; Diamond, Michael S; Chen, Qiang

    2014-01-01

    Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH(1-3)) of human IgG (pHu-E16scFv-CH(1-3)) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH(1-3) with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH(1-3) and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components. Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH(1-3) and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens. PMID:24675995

  9. Antibody producing B lineage cells invade the central nervous system predominantly at the time of and triggered by acute Epstein-Barr virus infection: A hypothesis on the origin of intrathecal immunoglobulin synthesis in multiple sclerosis.

    PubMed

    Otto, Carolin; Hofmann, Jörg; Ruprecht, Klemens

    2016-06-01

    Patients with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), typically have an intrathecal synthesis of immunoglobulin (Ig)G. Intrathecal IgG is produced by B lineage cells that entered the CNS, but why and when these cells invade the CNS of patients with MS is unknown. The intrathecal IgG response in patients with MS is polyspecific and part of it is directed against different common viruses (e.g. measles virus, rubella virus, varicella zoster virus). Strong and consistent evidence suggests an association of MS and Epstein-Barr virus (EBV) infection and EBV seroprevalence in patients with MS is practically 100%. However, intriguingly, despite of the universal EBV seroprevalence, the frequency of intrathecally produced IgG to EBV in patients with MS is much lower than that of intrathecally produced IgG to other common viruses. The acute phase of primary EBV infection is characterized by a strong polyclonal B cell activation. As typical for humoral immune responses against viruses, EBV specific IgG is produced only with a temporal delay after acute EBV infection. Aiming to put the above facts into a logical structure, we here propose the hypothesis that in individuals going on to develop MS antibody producing B lineage cells invade the CNS predominantly at the time of and triggered by acute primary EBV infection. Because at the time of acute EBV infection EBV IgG producing B lineage cells have not yet occurred, the hypothesis could explain the universal EBV seroprevalence and the low frequency of intrathecally produced IgG to EBV in patients with MS. Evidence supporting the hypothesis could be provided by large prospective follow-up studies of individuals with symptomatic primary EBV infection (infectious mononucleosis). Furthermore, the clarification of the molecular mechanism underlying an EBV induced invasion of B lineage cells into the CNS of individuals going on to develop MS could corroborate it, too. If true, our

  10. Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

    PubMed

    Mallaney, Mary; Wang, Szu-Han; Sreedhara, Alavattam

    2014-01-01

    During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

  11. Molecular and biochemical characterization of CTX-M-131, a natural Asp240Gly variant derived from CTX-M-2, produced by a Providencia rettgeri clinical strain in São Paulo, Brazil.

    PubMed

    Dropa, Milena; Ghiglione, Barbara; Matté, Maria Helena; Balsalobre, Livia Carminato; Lincopan, Nilton; Matté, Glavur Rogério; Gutkind, Gabriel; Power, Pablo

    2015-03-01

    CTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in a Providencia rettgeri clinical strain from Brazil. Molecular analysis showed that blaCTX-M-131 was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime.

  12. Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin

    PubMed Central

    1990-01-01

    An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for

  13. Penicillinase Studies on L-Phase Variants, G-Phase Variants, and Reverted Strains of Staphylococcus aureus.

    PubMed

    Simon, H J; Yin, E J

    1970-11-01

    L-phase variants and small colony (G-phase) variants derived from penicillinase-producing Staphylococcus aureus strains were tested for penicillinase (beta lactamase) production. A refined variation of the modified Gots test for penicillinase was used to demonstrate penicillinase synthesis. Penicillinase synthesis was reduced in L-phase variants and G-phase variants when compared to parental strains. After reversion of variants to vegetative stages had been induced, revertants were tested for production of penicillinase, coagulase, and alpha hemolysin, mannitol fermentation, and pigment production, and comparisons were made between parent and reverted vegetative forms. All revertants of G-phase variants retained penicillinase activity. Most revertants of L-phase variants showed reduction or loss of penicillinase activity. Retention of coagulase activity, alpha hemolysin production, mannitol fermentation, pigmentation, and phage type varied among revertants.

  14. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    SciTech Connect

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  15. Metrics for antibody therapeutics development.

    PubMed

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  16. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

    SciTech Connect

    Miller, Keith D.; Feldhaus, Jane M.; Gray, Sean A.; Siegel, Robert W.; Feldhaus, Michael J.

    2005-08-01

    Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0-99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was also used to compare scFv production levels from the periplasm, inclusion bodies, and culture media. The E. coli production system was then used to produce variants of several scFv to determine structure function relationships.

  17. Mucopolysaccharidosis: A New Variant?

    ERIC Educational Resources Information Center

    Primrose, D. A.

    1972-01-01

    Described is a possibly new variant of mucopolysaccharidosis characterized by progressive mental and motor deficiency, bone abnormalities, a generalized skin lesion, and abnormal mucopolysaccharides in the urine as seen in a 20-year-old female. (DB)

  18. Normal Variants in Echocardiography.

    PubMed

    Sanchez, Daniel R; Bryg, Robert J

    2016-11-01

    Echocardiography is a powerful and convenient tool used routinely in the cardiac evaluation of many patients. Improved resolution and visualization of cardiac anatomy has led to the discovery of many normal variant structures that have no known pathologic consequence. Importantly, these findings may masquerade as pathology prompting unnecessary further evaluation at the expense of anxiety, cost, or potential harm. This review provides an updated and comprehensive collection of normal anatomic variants on both transthoracic and transesophageal imaging. PMID:27612473

  19. [Industrial production of monoclonal antibodies].

    PubMed

    Baron, D

    1990-10-01

    Murine monoclonal antibodies (mabs) are produced in either mouse ascites or bioreactors (spinner culture, stirred-tank reactor, airlift reactor, hollow-fiber reactor). Human mabs are produced solely in bioreactors. Encapsulation represents a special technology. Hybridoma cells have to be adapted prior to growth in bioreactors. Of crucial importance is the construction of over-producing cell lines by cell- and gene-technological methods. Manipulated cell lines often produce modified mabs.

  20. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ...

  1. Control of trypanodestructive antibody responses and parasitemia in mice infected with Trypanosoma (Duttonella) vivax.

    PubMed Central

    Mahan, S M; Hendershot, L; Black, S J

    1986-01-01

    After infection with a cloned population of Trypanosoma vivax, C57BL/6 mice controlled parasitemia during the exponential growth phase and survived, with intermittent parasitemia, for several weeks. In contrast, most mice of the C3H/He strain did not control the first wave of parasitemia and died within 9 to 13 days after infection. Control of parasitemia in C57BL/6 mice was mediated by the production of a variant surface glycoprotein-specific trypanodestructive antibody response which was accompanied by production of antibodies against antigens shared between procyclic and bloodstream T. vivax as well as antibodies against trinitrophenyl (TNP) and sheep erythrocytes. The infected C3H/He mice did not produce trypanodestructive antibodies or antibodies against procyclic antigens or TNP but did produce antibodies against sheep erythrocytes. Although infected C57BL/6 mice produced levels of serum immunoglobulin M four times higher than infected C3H/He mice, their parasite-induced B-cell DNA synthetic responses were similar, and both sets of mice developed similar numbers of spleen cells with cytoplasmic immunoglobulin M, a proportion of which could react with TNP. In vitro biosynthetic labeling studies accompanied by immunoglobulin precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the immunoglobulin-containing cells of infected C3H/He mice synthesized and secreted less immunoglobulin than similar cells from infected C57BL/6 mice. We concluded that some parasite-induced antibody-forming cells in C3H/He mice, perhaps including parasite-specific and certainly including TNP-specific cells, had an impaired capacity to make and release immunoglobulin. Within 24 h after Berenil-mediated elimination of T. vivax from infected C3H/He mice, a population of cyclophosphamide-sensitive spleen cells produced large amounts of parasite-specific and TNP-specific antibody. We concluded that the defect in terminal B-cell function leading to

  2. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  3. T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing.

    PubMed

    Kudo, Ko; Imai, Chihaya; Lorenzini, Paolo; Kamiya, Takahiro; Kono, Koji; Davidoff, Andrew M; Chng, Wee Joo; Campana, Dario

    2014-01-01

    To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-ζ T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

  4. New HIV plaque titration; application to the assay of neutralizing antibody.

    PubMed

    Tsunetsugu-Yokota, Y; Ohshima, M; Naito, A; Chermann, J C; Shih, J; Yoshikura, H

    1993-01-01

    A simple, sensitive and accurate plaque assay was developed using HPB-Ma, a variant of the human T-cell line HPB-ALL, which becomes adherent to the substratum after infection with an amphotropic murine sarcoma virus (MSVa). The simplicity of this novel plaque assay allowed us to examine a large number of serum samples from patients with HIV infection for neutralizing antibody activity against two human immunodeficiency virus type-1 (HIV-1) strains. During the progression of clinical disease, the neutralizing activity in the sera from two individual patients remained unchanged or increased. A patient with a known time of HIV infection produced cross-neutralizing antibody at 25-34 weeks. The neutralizing activity in the sera from 17 asymptomatic carriers, four patients with AIDS-related complex and four AIDS patients was also examined and was found to be unrelated to the clinical stage.

  5. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2008-11-18

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  6. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2011-05-31

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  7. Cellobiohydrolase I gene and improved variants

    DOEpatents

    Adney, William S.; Decker, Stephen R.; Mc Carter, Suzanne; Baker, John O.; Nieves, Raphael; Himmel, Michael E.; Vinzant, Todd B.

    2008-05-20

    The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

  8. Imaging with indium111-labeled anticarcinoembryonic antigen monoclonal antibody ZCE-025 of recurrent colorectal or carcinoembryonic antigen-producing cancer in patients with rising serum carcinoembryonic antigen levels and occult metastases

    SciTech Connect

    Patt, Y.Z.; Lamki, L.M.; Shanken, J.; Jessup, J.M.; Charnsangavej, C.; Ajani, J.A.; Levin, B.; Merchant, B.; Halverson, C.; Murray, J.L. )

    1990-07-01

    We tested whether nuclear imaging with indium111 (111In)-labeled murine monoclonal (MoAb) anticarcinoembryonic antigen (anti-CEA) ZCE-025 antibody could detect recurrent disease in patients with a rising serum CEA level but negative findings for computed tomographic (CT) scans of the abdomen and pelvis, chest radiograph, and colonoscopy or barium enema. Twenty patients with a history of completely resected CEA-producing adenocarcinoma and a rising serum CEA level were given an intravenous infusion of 2 mg of 111In-labeled ZCE-025 mixed with 38 mg of unlabeled ZCE-025. Planar and single-photon emission CT (SPECT) scans were acquired at 72 and 144 hours, and in 19 of the 20 patients these were positive. Of those 19, 13 underwent exploratory surgery, and cancer was found in 10, and two had a diagnostic biopsy, which confirmed cancer. Three patients who had negative laparotomies and all four patients who did not undergo surgery or biopsy were followed radiologically. In all seven, cancer was subsequently detected at the sites suggested by the ZCE-025 scan. Thus, tumor was confirmed in all 19 patients with positive scans. Five of 13 patients who were explored benefited from the study and the exploratory laparotomy, as disease was entirely resected in four or was subjected to definitive radiation therapy to the pelvis in the fifth. In two additional patients who were not explored, MoAb imaging resulted in definitive therapy to regionally confined recurrent disease. 111In-labeled anti-CEA MoAb ZCE-025 scanning in patients with rising CEA successfully imaged metastatic colorectal cancer that eluded detection by other methods and affected the care given to some. These results suggest an important role for 111In-labeled ZCE-025 scanning among patients with rising CEA and otherwise occult metastatic cancer.

  9. Charge variants of tubulin, tubulin S, membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells.

    PubMed

    Zambito, Anna Maria; Knipling, Leslie; Wolff, J

    2002-12-16

    Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested. PMID:12445483

  10. Antibody Engineering and Therapeutics

    PubMed Central

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

    2014-01-01

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

  11. Analyzing antibody-Fc-receptor interactions.

    PubMed

    Nimmerjahn, Falk; Ravetch, Jeffrey V

    2008-01-01

    Cellular receptors for immunoglobulins (Fc-receptors; FcR) are central mediators of antibody-triggered effector functions. Immune complex (IC) binding to FcRs results in a variety of reactions such as the release of inflammatory mediators, antibody dependent cellular cytotoxicity (ADCC) and phagocytosis of ICs. Analyzing antibody-FcR (Ab-FcR) interactions in vitro is essential to determine the effector mechanisms, binding characteristics and affinity parameters that will impact and predict antibody activity in vivo. The methods described in this chapter include the generation of ICs and soluble FcR variants, as well as ELISA and FACS-based assays to study Ab-FcR interactions.

  12. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

  13. A minimal cytomegalovirus intron A variant can improve transgene expression in different mammalian cell lines.

    PubMed

    Quilici, L S; Silva-Pereira, I; Andrade, A C; Albuquerque, F C; Brigido, M M; Maranhão, A Q

    2013-01-01

    The expression enhancement by cytomegalovirus promoter and different intron A (IA) variants were evaluated in CHO-K1, HepG2, HEK-293 and COS-7 cells by assessing the levels of luciferase activity. This data along with mRNA levels measurement indicated that the construct harboring an IA variant with a 200-nucleotide deletion (Δ200) had the greatest impact on increasing luciferase expression among all constructs evaluated. Based on these results, we redesigned pCMV-IA variants and cloned them into plasmids expressing a humanized antibody. These plasmids were then used to transfect CHO-K1 cells. Production of the antibody was not augmented with the Δ200 promoter variant. The 600-nucleotide deletion (Δ600) and whole IA promoter variants expressed similar levels of the recombinant protein. These data indicate that the IA-based enhanced expression of transgenes depends on a small region within the intron.

  14. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico

    PubMed Central

    Ramírez-Hernández, Dolores G.; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  15. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  16. Genetically engineered antibody molecules and their application.

    PubMed

    Morrison, S L; Wims, L; Wallick, S; Tan, L; Oi, V T

    1987-01-01

    Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease.

  17. Avian Diagnostic and Therapeutic Antibodies

    SciTech Connect

    Bradley, David Sherman

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  18. Hemoglobin Variants: Biochemical Properties and Clinical Correlates

    PubMed Central

    Thom, Christopher S.; Dickson, Claire F.; Gell, David A.; Weiss, Mitchell J.

    2013-01-01

    Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples. PMID:23388674

  19. Hemoglobin variants: biochemical properties and clinical correlates.

    PubMed

    Thom, Christopher S; Dickson, Claire F; Gell, David A; Weiss, Mitchell J

    2013-03-01

    Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

  20. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

    Baldwin, R.W.; Byers, V.S. )

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

  1. Antibodies and immunoassays.

    PubMed

    Madersbacher, S; Berger, P

    2000-05-01

    As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a single molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation variants, such as its free beta (hCGbeta) and alpha (hCGalpha) subunits and clipped, cleaved, terminally differently sialylated, and overglycosylated forms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is highly dependent on the selection of target molecules and the natural variability of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immunoassays are still the state-of-the-art technique for both clinical and research applications but a major problem is the different extents of recognition of hCG variants by mAbs used in different immunoassays. On the whole, construction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, cross-reactivities, epitope locations and compatibilities, overlap and overhang in specificities (pairs of mAbs), and, finally, overspecificity. Consequences of overhang and overlap in antigen recognition of coating and detection mAb specificities are nondesirable assay cross-reactions and competitive interference by antigenic variants. The general agreement on the most favorable assay design is contrasted by the variety of isotopic and nonisotopic detection systems in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity. By measuring substrate absorption values off the absorption maximum, the measuring range of any IEMA can be extended significantly, as shown for 3,3',5, 5'-tetramethylbenzidine (TMB), without jeopardizing assay characteristics. Sensitivity of the IEMA can be enhanced by modifying the horseradish peroxidase (HRPO) labeling technique by using highly purified m

  2. Utilization of monoclonal-antibody-based assay (HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies.

    PubMed

    Schultz, J C

    1995-01-01

    Sickle cell disease covers a group of conditions in which pathology may be attributed to the presence of sickle hemoglobin (HbS). The identification of HbS and other variants including those in combination with HbS is commonly achieved by cellulose acetate electrophoresis at alkaline pH. Because many hemoglobin variants with similar charges have similar electrophoretic migration patterns, they are difficult to differentiate by electrophoresis. The HemoCard assays address this concern through the use of monoclonal antibodies capable of specifically recognizing the unique amino acid substitution in the variant hemoglobin. The panel of HemoCard monoclonal antibodies confirms the absence and presence of HbA, HbC, HbE, HbS, and other sickling hemoglobin variants. The combination of alkaline cellulose acetate electrophoresis and HemoCard assays allows the technologist to reach a final conformation of both common and much less common sickle cell disease genotypes, combinations of HbS with other hemoglobins that ordinarily do not produce sickle cell disease, and other clinically important hemoglobinopathies including HbE/beta-thalassemia and hemoglobin C disease. PMID:8587004

  3. Antitubulin antibody in healthy adults and patients with infectious mononucleosis and its relationship to smooth muscle antibody (SMA).

    PubMed Central

    Mead, G M; Cowin, P; Whitehouse, J M

    1980-01-01

    Antibody to tubulin in man has been studied using a specific radioimmunoassay, affinity chromatography radioimmunoassay but markedly increased levels were noted in patients with infectious mononucleosis where the antibody was predominantly IgM in type. This finding was confirmed on fluorescence microscopy. Affinity chromatography purified antibody produced characteristic microtubular staining of fixed 3T3 cells, but in addition, produced weak staining of cryostat sections of rat tissue, similar in distribution to that of smooth muscle antibody. Our studies indicate that the IgM smooth muscle antibody found in infectious mononucleosis by IF techniques is at least in part due to an antitubulin antibody. Images FIG. 1 PMID:6993069

  4. Analysis of the Mycoplasma genitalium MgpB Adhesin to Predict Membrane Topology, Investigate Antibody Accessibility, Characterize Amino Acid Diversity, and Identify Functional and Immunogenic Epitopes

    PubMed Central

    Iverson-Cabral, Stefanie L.; Wood, Gwendolyn E.; Totten, Patricia A.

    2015-01-01

    Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to

  5. Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.

    PubMed

    Ito, Taichi; Komiya-Ito, Akiyo; Okuda, Katsuji; Minaguchi, Kiyoshi; Saitoh, Eiichi; Yamada, Satoru; Kato, Tetsuo

    2005-06-01

    To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

  6. HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

    PubMed Central

    Williams, Katherine L.; Cortez, Valerie; Dingens, Adam S.; Gach, Johannes S.; Rainwater, Stephanie; Weis, Julie F.; Chen, Xuemin; Spearman, Paul; Forthal, Donald N.; Overbaugh, Julie

    2015-01-01

    HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the

  7. Heterologous flavivirus infection-enhancing antibodies in sera of Nigerians.

    PubMed

    Fagbami, A; Halstead, S B; Marchette, N; Larsen, K

    1988-01-01

    Human sera collected from Nigerians were examined for plaque reduction neutralizing and infection-enhancing antibodies against dengue 2, yellow fever, and West Nile viruses. Neutralization tests showed that 17 of 19 sera contained flavivirus neutralizing antibody; 11 were positive to all 3 viruses, 5 to dengue and yellow fever, and 1 to dengue virus only. Two sera had no detectable neutralizing antibody to any of the flaviviruses. Enhancement assays showed that 17 flavivirus neutralizing antibody-positive sera contained infection-enhancing antibodies to dengue 2, and 16 had antibody to yellow fever. Although 11 sera were positive for West Nile neutralizing antibody, 17 enhanced this virus. Heterologous infection-enhancing antibody titers were lower than the homologous ones. Broadly reacting sera and those with high neutralizing antibody titers produced the highest infection-enhancing antibody titers.

  8. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  9. Variants of glycoside hydrolases

    SciTech Connect

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  10. Paraneoplastic cerebellar degeneration with anti-Yo antibodies - a review.

    PubMed

    Venkatraman, Anand; Opal, Puneet

    2016-08-01

    The ataxic syndrome associated with Anti-Yo antibody, or Purkinje cell cytoplasmic antibody type 1 (PCA1), is the most common variant of paraneoplastic cerebellar degeneration (PCD). The typical presentation involves the subacute development of pancerebellar deficits with a clinical plateau within 6 months. The vast majority of cases have been reported in women with pelvic or breast tumors. Magnetic resonance imaging of the brain is often normal in the early stages, with cerebellar atrophy seen later. The underlying mechanism is believed to be an immunological reaction to cerebellar degeneration-related protein 2 (CDR2), a protein usually found in the cerebellum that is ectopically produced by tumor cells. Although both B- and T-cell abnormalities are seen, there is debate about the relative importance of the autoantibodies and cytotoxic T lymphocytes in the neuronal loss. Cerebrospinal fluid abnormalities, primarily elevated protein, lymphocytic pleocytosis, and oligoclonal bands, are common in the early stages. The low prevalence of this condition has not allowed for large-scale randomized controlled trials. Immunotherapies, such as steroids, intravenous immune globulins, and plasma exchange, have been extensively used in managing this condition, with limited success. Although some reports indicate benefit from antitumor therapies like surgery and chemotherapy, this has not been consistently observed. The prognosis for anti-Yo PCD is almost uniformly poor, with most patients left bedridden. Further studies are required to clarify the pathophysiology and provide evidence-based treatment options. PMID:27606347

  11. Paraneoplastic cerebellar degeneration with anti-Yo antibodies - a review.

    PubMed

    Venkatraman, Anand; Opal, Puneet

    2016-08-01

    The ataxic syndrome associated with Anti-Yo antibody, or Purkinje cell cytoplasmic antibody type 1 (PCA1), is the most common variant of paraneoplastic cerebellar degeneration (PCD). The typical presentation involves the subacute development of pancerebellar deficits with a clinical plateau within 6 months. The vast majority of cases have been reported in women with pelvic or breast tumors. Magnetic resonance imaging of the brain is often normal in the early stages, with cerebellar atrophy seen later. The underlying mechanism is believed to be an immunological reaction to cerebellar degeneration-related protein 2 (CDR2), a protein usually found in the cerebellum that is ectopically produced by tumor cells. Although both B- and T-cell abnormalities are seen, there is debate about the relative importance of the autoantibodies and cytotoxic T lymphocytes in the neuronal loss. Cerebrospinal fluid abnormalities, primarily elevated protein, lymphocytic pleocytosis, and oligoclonal bands, are common in the early stages. The low prevalence of this condition has not allowed for large-scale randomized controlled trials. Immunotherapies, such as steroids, intravenous immune globulins, and plasma exchange, have been extensively used in managing this condition, with limited success. Although some reports indicate benefit from antitumor therapies like surgery and chemotherapy, this has not been consistently observed. The prognosis for anti-Yo PCD is almost uniformly poor, with most patients left bedridden. Further studies are required to clarify the pathophysiology and provide evidence-based treatment options.

  12. Computationally driven antibody engineering enables simultaneous humanization and thermostabilization.

    PubMed

    Choi, Yoonjoo; Ndong, Christian; Griswold, Karl E; Bailey-Kellogg, Chris

    2016-10-01

    Humanization reduces the immunogenicity risk of therapeutic antibodies of non-human origin. Thermostabilization can be critical for clinical development and application of therapeutic antibodies. Here, we show that the computational antibody redesign method Computationally Driven Antibody Humanization (CoDAH) enables these two goals to be accomplished simultaneously and seamlessly. A panel of CoDAH designs for the murine parent of cetuximab, a chimeric anti-EGFR antibody, exhibited both substantially improved thermostabilities and substantially higher levels of humanness, while retaining binding activity near the parental level. The consistently high quality of the turnkey CoDAH designs, over a whole panel of variants, suggests that the computationally directed approach encapsulates key determinants of antibody structure and function.

  13. [Antinuclear antibodies].

    PubMed

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  14. Induction of adhesion-inhibitory antibodies against placental Plasmodium falciparum parasites by using single domains of VAR2CSA.

    PubMed

    Nielsen, Morten A; Pinto, Vera V; Resende, Mafalda; Dahlbäck, Madeleine; Ditlev, Sisse B; Theander, Thor G; Salanti, Ali

    2009-06-01

    In areas of endemicity pregnancy-associated malaria is an important cause of maternal anemia, stillbirth, and delivery of low-birth-weight children. The syndrome is precipitated by the accumulation of Plasmodium falciparum-infected erythrocytes in the placenta, mediated through an interaction between a parasite protein expressed on erythrocytes named variant surface antigen 2-chondroitin sulfate A (VAR2CSA) and CSA on syncytiotrophoblasts. VAR2CSA is a large polymorphic protein consisting of six Duffy binding-like (DBL), domains and with current constraints on recombinant protein production it is not possible to produce entire VAR2CSA recombinant proteins. Furthermore, the presence of polymorphisms has raised the question of whether it is feasible to define VAR2CSA antigens eliciting broadly protective antibodies. Thus, the challenge for vaccine development is to define smaller parts of the molecule which induce antibodies that inhibit CSA binding of different parasite strains. In this study, we produced a large panel of VAR2CSA proteins and raised antibodies against these antigens. We show that antibodies against the DBL4 domain effectively inhibit parasite binding. As the inhibition was not limited to homologous parasite strains, it seems feasible to base a protective malaria vaccine on a single VAR2CSA DBL domain. PMID:19307213

  15. Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function.

    PubMed

    Antes, Bernhard; Amon, Sabine; Rizzi, Andreas; Wiederkum, Susi; Kainer, Manuela; Szolar, Oliver; Fido, Markus; Kircheis, Ralf; Nechansky, Andreas

    2007-06-01

    During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

  16. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  17. Human AZU-1 gene, variants thereof and expressed gene products

    DOEpatents

    Chen, Huei-Mei; Bissell, Mina

    2004-06-22

    A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

  18. Stabilization of antibody fragments in adverse environments.

    PubMed

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  19. Peptide Antibodies: Past, Present, and Future.

    PubMed

    Houen, Gunnar

    2015-01-01

    Peptide antibodies recognize epitopes with amino acid residues adjacent in sequence ("linear" epitopes). Such antibodies can be made to virtually any sequence and have been immensely important in all areas of molecular biology and diagnostics due to their versatility and to the rapid growth in protein sequence information. Today, peptide antibodies can be routinely and rapidly made to large numbers of peptides, including peptides with posttranslationally modified residues, and are used for immunoblotting, immunocytochemistry, immunohistochemistry, and immunoassays. In the future, peptide antibodies will continue to be immensely important for molecular biology, TCR- and MHC-like peptide antibodies may be produced routinely, peptide antibodies with predetermined conformational specificities may be designed, and peptide-based vaccines may become part of vaccination programs.

  20. RNA-directed DNA polymerase from human leukemic blood cells and from primate type-C virus-producing cells: high- and low-molecular-weight forms with variant biochemical and immunological properties.

    PubMed Central

    Mondal, H; Gallagher, R E; Gallo, R C

    1975-01-01

    RNA-directed DNA polymerase (reverse transcriptase) from leukocytes of individual leukemic patients can be grouped by velocity gradient analyses into two distinct classes, a low-molecular-weight (LMW) class of approximately 70,000 and a high-molecular-weight (HMW) class of 130,000 to 140,000. The reverse transcriptases from mammalian type-C viruses have with one exception (see text) been isolated as enzymes with molecular weights of 70,000. In this study, the reverse transcriptase from extracellular gibbon ape leukemia virus was also isolated only as the LMW class. However, the enzyme from gibbon virus-producing cells was isolated partially in the HMW form; this form was converted completely to the LMW form by treatment with 0.5 M KC1 and 0.5% Triton X-100 and could be re-converted to the HMW form by lowering the KC1 and Triton X-100 concentrations. A similar conversion from a HMW form to a LMW form was demonstrated with enzyme from human leukemic cells. The LMW form of the human and gibbon ape cellular enzymes utilized synthetic primer-templates in a similar fashion to viral enzyme, and this form was strongly inhibited by antisera (IgG) to reverse transcriptase from simian (woolly monkey) type-C virus. The HMW form of both enzymes utilized synthetic primer-templates less efficiently than the LMW form, and was resistant to inhibition by antipolymerase IgG of simian type-C virus. The HMW form of the cellular reverse transcriptases transcribed viral 70S RNA in the absence of synthetic primer relatively more efficiently than did the extracellular viral form. These data suggest that the HMW form is due in part to aggregation of the LMW form and in part to a cellular factor(s) which may affect both the form and function of intracellular reverse transciptase. PMID:48250

  1. Induction and detection of antibodies to squalene.

    PubMed

    Matyas, G R; Wassef, N M; Rao, M; Alving, C R

    2000-11-01

    An enzyme-linked immunosorbent assay (ELISA) utilizing antigen coated on hydrophobic polyvinyldiene fluoride (PVDF) membranes is described for detecting antibodies that bind to squalene (SQE). Because of the prior lack of availability of validated antibodies to SQE, positive controls for the assay were made by immunization with formulations containing SQE to create monoclonal antibodies (mAbs) that reacted with SQE. Among eight immunogens tested, only two induced detectable murine antibodies to SQE: liposomes containing dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, 71% SQE, and lipid A [L(71% SQE+LA)], and, to a much lesser extent, an oil-in-water emulsion containing SQE, Tween 80, Span 85, and lipid A. In each case, lipid A served as an adjuvant, but neither SQE alone, SQE mixed with lipid A, liposomes containing 43% SQE and lipid A, nor several other emulsions containing both SQE and lipid A, induced antibodies that reacted with SQE. Monoclonal antibodies produced after immunizing mice with [L(71% SQE+LA)] served as positive controls for developing the ELISA. Monoclonal antibodies were produced that either recognized SQE alone but did not recognize squalane (SQA, the hydrogenated form of SQE), or that recognized both SQE and SQA. As found previously with other liposomal lipid antigens, liposomes containing lipid A also induced antibodies that reacted with the liposomal phospholipids. However, mAbs were also identified that reacted with SQE on PVDF membranes, but did not recognize either SQA or liposomal phospholipid. The polyclonal antiserum produced by immunizing mice with [L(71% SQE+LA)] therefore contained a mixed population of antibody specificities and, as expected, the ELISA of polyclonal antiserum with PVDF membranes detected antibodies both to SQE and SQA. We conclude that SQE is a weak antigen, but that antibodies that specifically bind to SQE can be readily induced by immunization with [L(71% SQE+LA)] and detected by ELISA with PVDF

  2. VARIANT: Command Line, Web service and Web interface for fast and accurate functional characterization of variants found by Next-Generation Sequencing

    PubMed Central

    Medina, Ignacio; De Maria, Alejandro; Bleda, Marta; Salavert, Francisco; Alonso, Roberto; Gonzalez, Cristina Y.; Dopazo, Joaquin

    2012-01-01

    The massive use of Next-Generation Sequencing (NGS) technologies is uncovering an unexpected amount of variability. The functional characterization of such variability, particularly in the most common form of variation found, the Single Nucleotide Variants (SNVs), has become a priority that needs to be addressed in a systematic way. VARIANT (VARIant ANalyis Tool) reports information on the variants found that include consequence type and annotations taken from different databases and repositories (SNPs and variants from dbSNP and 1000 genomes, and disease-related variants from the Genome-Wide Association Study (GWAS) catalog, Online Mendelian Inheritance in Man (OMIM), Catalog of Somatic Mutations in Cancer (COSMIC) mutations, etc). VARIANT also produces a rich variety of annotations that include information on the regulatory (transcription factor or miRNA-binding sites, etc.) or structural roles, or on the selective pressures on the sites affected by the variation. This information allows extending the conventional reports beyond the coding regions and expands the knowledge on the contribution of non-coding or synonymous variants to the phenotype studied. Contrarily to other tools, VARIANT uses a remote database and operates through efficient RESTful Web Services that optimize search and transaction operations. In this way, local problems of installation, update or disk size limitations are overcome without the need of sacrifice speed (thousands of variants are processed per minute). VARIANT is available at: http://variant.bioinfo.cipf.es. PMID:22693211

  3. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  4. Remembering antibodies coming of age.

    PubMed

    Melchers, Fritz

    2016-01-01

    Fifty years ago, Norbert Hilschmann discovered that antibodies have variable immunoglobulin domains to bind antigens, and constant domains to carry out effector functions in the immune system. Just as this happened, the author of this perspective entered the field of immunology. Ten years later, the genetic basis of antibody variability was discovered by Susumu Tonegawa and his colleagues at the Basel Institute for Immunology, where the author had become a scientific member. At the same time, Georges Köhler, a former graduate student of the author's at the Basel Institute, invented with Cesar Milstein at the Laboratory of Molecular Biology in Cambridge, England, the method to produce monoclonal antibodies. The author describes here his memories connected to these three monumental, paradigm-changing discoveries, which he observed in close proximity. PMID:27144253

  5. Remembering antibodies coming of age.

    PubMed

    Melchers, Fritz

    2016-01-01

    Fifty years ago, Norbert Hilschmann discovered that antibodies have variable immunoglobulin domains to bind antigens, and constant domains to carry out effector functions in the immune system. Just as this happened, the author of this perspective entered the field of immunology. Ten years later, the genetic basis of antibody variability was discovered by Susumu Tonegawa and his colleagues at the Basel Institute for Immunology, where the author had become a scientific member. At the same time, Georges Köhler, a former graduate student of the author's at the Basel Institute, invented with Cesar Milstein at the Laboratory of Molecular Biology in Cambridge, England, the method to produce monoclonal antibodies. The author describes here his memories connected to these three monumental, paradigm-changing discoveries, which he observed in close proximity.

  6. Molecular-specific urokinase antibodies

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  7. 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

  8. Back to the future: recombinant polyclonal antibody therapeutics.

    PubMed

    Wang, Xian-Zhe; Coljee, Vincent W; Maynard, Jennifer A

    2013-11-01

    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

  9. Primary Structural Variation in Anaplasma marginale Msp2 Efficiently Generates Immune Escape Variants

    PubMed Central

    Paradiso, Lydia; Broschat, Shira L.; Noh, Susan M.; Palmer, Guy H.

    2015-01-01

    Antigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection. Anaplasma marginale utilizes gene conversion of a repertoire of silent msp2 alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement of msp2 alleles alone is insufficient to generate the number of variants required for persistence, A. marginale uses segmental gene conversion, in which oligonucleotide segments from multiple alleles are recombined into the expression site to generate a novel msp2 mosaic not represented elsewhere in the genome. Whether these segmental changes are sufficient to evade a broad antibody response is unknown. We addressed this question by identifying Msp2 variants that differed in primary structure within the immunogenic hypervariable region microdomains and tested whether they represented true antigenic variants. The minimal primary structural difference between variants was a single amino acid resulting from a codon insertion, and overall, the amino acid identity among paired microdomains ranged from 18 to 92%. Collectively, 89% of the expressed structural variants were also antigenic variants across all biological replicates, independent of a specific host major histocompatibility complex haplotype. Biological relevance is supported by the following: (i) all structural variants were expressed during infection of a natural host, (ii) the structural variation observed in the microdomains corresponded to the mean length of variants generated by segmental gene conversion, and (iii) antigenic variants were identified using a broad antibody response that developed during infection of a natural host. The findings demonstrate that segmental gene conversion efficiently generates Msp2 antigenic variants. PMID:26259814

  10. Antibodies: an alternative for antibiotics?

    PubMed

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases. PMID:15844826

  11. The Polymorphic Aggregative Phenotype of Shiga Toxin-Producing Escherichia coli O111 Depends on RpoS and Curli

    PubMed Central

    Diodati, M. E.; Bates, A. H.; Miller, W. G.; Carter, M. Q.; Zhou, Y.

    2015-01-01

    Escherichia coli O111 is an emerging non-O157:H7 serotype of Shiga toxin-producing E. coli (STEC). We previously reported that outbreak and environmental, but not sporadic-case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and M. T. Brandl, Foodborne Pathog Dis 12:235−243, 2015, http://dx.doi.org/10.1089/fpd.2014.1887). We show here the natural occurrence of nonaggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae and lack RpoS function but synthesize cellulose. The deletion of csgBAC or rpoS in an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a nonaggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blotting with an anti-CsgA antibody. Immunomicroscopy revealed that curli were undetectable on the cells of the nonaggregative variant and the RpoS mutant but were present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis of rpoS in the aggregative strain and its variant showed a single substitution of threonine for asparagine at amino acid 124. Our results indicate that the multicellular behavior of STEC O111 is RpoS dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of nonaggregative variants may allow the cell population to adapt to conditions benefiting a planktonic lifestyle. PMID:26712542

  12. Admixture Mapping in Lupus Identifies Multiple Functional Variants within IFIH1 Associated with Apoptosis, Inflammation, and Autoantibody Production

    PubMed Central

    Looger, Loren L.; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A.; Niewold, Timothy B.; Gilkeson, Gary S.; Brown, Elizabeth E.; Alarcón, Graciela S.; Edberg, Jeffrey C.; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D.; Vilá, Luis M.; Freedman, Barry I.; Tsao, Betty P.; Criswell, Lindsey A.; Jacob, Chaim O.; Moore, Jason H.; Vyse, Timothy J.; Langefeld, Carl L.; Guthridge, Joel M.; Gaffney, Patrick M.; Moser, Kathy L.; Scofield, R. Hal; Alarcón-Riquelme, Marta E.; Williams, Scott M.; Merrill, Joan T.; James, Judith A.; Kaufman, Kenneth M.; Kimberly, Robert P.; Harley, John B.; Nath, Swapan K.

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID

  13. Admixture mapping in lupus identifies multiple functional variants within IFIH1 associated with apoptosis, inflammation, and autoantibody production.

    PubMed

    Molineros, Julio E; Maiti, Amit K; Sun, Celi; Looger, Loren L; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A; Niewold, Timothy B; Gilkeson, Gary S; Brown, Elizabeth E; Alarcón, Graciela S; Edberg, Jeffrey C; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D; Vilá, Luis M; Freedman, Barry I; Tsao, Betty P; Criswell, Lindsey A; Jacob, Chaim O; Moore, Jason H; Vyse, Timothy J; Langefeld, Carl L; Guthridge, Joel M; Gaffney, Patrick M; Moser, Kathy L; Scofield, R Hal; Alarcón-Riquelme, Marta E; Williams, Scott M; Merrill, Joan T; James, Judith A; Kaufman, Kenneth M; Kimberly, Robert P; Harley, John B; Nath, Swapan K

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22-24 (LOD=6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ~1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [P(meta) = 5.20×10(-14); odds ratio, 95% confidence interval = 0.82 (0.78-0.87)], and two missense variants, rs1990760 (Ala946Thr) [P(meta) = 3.08×10(-7); 0.88 (0.84-0.93)] and rs10930046 (Arg460His) [P(dom) = 1.16×10(-8); 0.70 (0.62-0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID:23441136

  14. Regulation of Mouse 4-1BB Expression: Multiple Promoter Usages and a Splice Variant

    PubMed Central

    Kim, Jung D.; Kim, Chang H.; Kwon, Byoung S.

    2011-01-01

    The expression of 4-1BB has been known to be dependent on T cell activation. Recent studies have, however, revealed that 4-1BB expression is not restricted to T cells. We sought to determine the molecular basis for the differential gene expression. Here we report the expression pattern of two mouse 4-1BB transcripts, type I and type II. Whereas the type I transcript was specifically expressed on immune organ as previously reported, the type II transcript was ubiquitously expressed in tissues and various cell lines. However, both type I and type II transcript were highly induced on activated T cells. Primer extension assay of the two 4-1BB transcripts suggested that mouse 4-1BB had more than two transcripts. Using luciferase assay we have identified three promoter regions (PI, PII and PIII), which located on upstream region of second exon 1, first exon 1, and exon 2, respectively. In particular, the type I transcript was preferentially induced when naïve T cells are stimulated by anti-CD3 monoclonal antibody (mAb) since NF-κB specifically binds to the putative NF-κB element of PI. We have also shown that a splice variant, in which the transmembrane domain was deleted, could inhibit 4-1BB signaling. The splicing variant was highly induced by TCR stimulation. Our results reveal 4-1BB also has a negative regulation system through soluble 4-1BB produced from a splice variant induced under activation conditions. PMID:21347708

  15. Variants of windmill nystagmus.

    PubMed

    Choi, Kwang-Dong; Shin, Hae Kyung; Kim, Ji-Soo; Kim, Sung-Hee; Choi, Jae-Hwan; Kim, Hyo-Jung; Zee, David S

    2016-07-01

    Windmill nystagmus is characterized by a clock-like rotation of the beating direction of a jerk nystagmus suggesting separate horizontal and vertical oscillators, usually 90° out of phase. We report oculographic characteristics in three patients with variants of windmill nystagmus in whom the common denominator was profound visual loss due to retinal diseases. Two patients showed a clock-like pattern, while in the third, the nystagmus was largely diagonal (in phase or 180° out of phase) but also periodically changed direction by 180°. We hypothesize that windmill nystagmus is a unique manifestation of "eye movements of the blind." It emerges when the central structures, including the cerebellum, that normally keep eye movements calibrated and gaze steady can no longer perform their task, because they are deprived of the retinal image motion that signals a need for adaptive recalibration.

  16. Variants of windmill nystagmus.

    PubMed

    Choi, Kwang-Dong; Shin, Hae Kyung; Kim, Ji-Soo; Kim, Sung-Hee; Choi, Jae-Hwan; Kim, Hyo-Jung; Zee, David S

    2016-07-01

    Windmill nystagmus is characterized by a clock-like rotation of the beating direction of a jerk nystagmus suggesting separate horizontal and vertical oscillators, usually 90° out of phase. We report oculographic characteristics in three patients with variants of windmill nystagmus in whom the common denominator was profound visual loss due to retinal diseases. Two patients showed a clock-like pattern, while in the third, the nystagmus was largely diagonal (in phase or 180° out of phase) but also periodically changed direction by 180°. We hypothesize that windmill nystagmus is a unique manifestation of "eye movements of the blind." It emerges when the central structures, including the cerebellum, that normally keep eye movements calibrated and gaze steady can no longer perform their task, because they are deprived of the retinal image motion that signals a need for adaptive recalibration. PMID:27159990

  17. Redesigned HIV antibodies exhibit enhanced neutralizing potency and breadth

    PubMed Central

    Willis, Jordan R.; Sapparapu, Gopal; Murrell, Sasha; Julien, Jean-Philippe; Singh, Vidisha; King, Hannah G.; Xia, Yan; Pickens, Jennifer A.; LaBranche, Celia C.; Slaughter, James C.; Montefiori, David C.; Wilson, Ian A.; Meiler, Jens; Crowe, James E.

    2015-01-01

    Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100FY, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100FY fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100FY, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies. PMID:25985274

  18. Production of monoclonal antibodies.

    PubMed

    Freysd'ottir, J

    2000-01-01

    The discovery of monoclonal antibodies (mAbs) produced by "hybridoma technology" by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recognized. It took around 10 years to appreciate the importance of using these mAbs in various fields of science other than immunology, such as cell biology, biochemistry, microbiology, virology, para-sitology, physiology, genetics, and molecular biology; and also in areas of clinical medicine, such as pathology, hematology, oncology, and infectious disease. The contribution of mAbs to science and clinical medicine was recognized in 1984 by the award of the Nobel Prize for Medicine to Köhler and Milstein.

  19. Plasma Proteomics, The Human Proteome Project, and Cancer-Associated Alternative Splice Variant Proteins☆, ☆☆

    PubMed Central

    Omenn, Gilbert S.

    2016-01-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 “missing proteins” and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:24211518

  20. Plasmodium falciparum-Infected Erythrocyte Knob Density Is Linked to the PfEMP1 Variant Expressed

    PubMed Central

    Subramani, Ramesh; Quadt, Katharina; Jeppesen, Anine E.; Hempel, Casper; Petersen, Jens Emil Vang; Hassenkam, Tue; Hviid, Lars

    2015-01-01

    ABSTRACT Members of the clonally variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediate adhesion of infected erythrocytes (IEs) to vascular receptors. PfEMP1 expression is normally confined to nanoscale knob protrusions on the IE surface membrane. To investigate the relationship between the densities of these IE surface knobs and the PfEMP1 variant expressed, we used specific antibody panning to generate three sublines of the P. falciparum clone IT4, which expresses the PfEMP1 variants IT4VAR04, IT4VAR32b, and IT4VAR60. The knob density in each subline was then determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM) and compared to PfEMP1 and knob-associated histidine-rich protein (KAHRP) expression. Selection for uniform expression of IT4VAR04 produced little change in knob density, compared to unselected IEs. In contrast, selection for IT4VAR32b expression increased knob density approximately 3-fold, whereas IEs selected for IT4VAR60 expression were essentially knobless. When IT4VAR60+ IEs were subsequently selected to express IT4VAR04 or IT4VAR32b, they again displayed low and high knob densities, respectively. All sublines expressed KAHRP regardless of the PfEMP1 expressed. Our study documents for the first time that knob density is related to the PfEMP1 variant expressed. This may reflect topological requirements to ensure optimal adhesive properties of the IEs. PMID:26443460

  1. Alternatively spliced variants of the cell adhesion molecule CD44 and tumour progression in colorectal cancer.

    PubMed Central

    Gotley, D. C.; Fawcett, J.; Walsh, M. D.; Reeder, J. A.; Simmons, D. L.; Antalis, T. M.

    1996-01-01

    Increased expression of alternatively spliced variants of the CD44 family of cell adhesion molecules has been associated with tumour metastasis. In the present study, expression of alternatively spliced variants of CD44 and their cellular distribution have been investigated in human colonic tumours and in the corresponding normal mucosa, in addition to benign adenomatous polyps. The expression of CD44 alternatively spliced variants has been correlated with tumour progression according to Dukes' histological stage. CD44 variant expression was determined by immunohistochemisty using monoclonal antibodies directed against specific CD44 variant domains together with RT-PCR analysis of CD44 variant mRNA expression in the same tissue specimens. We demonstrate that as well as being expressed in colonic tumour cells, the full range of CD44 variants, CD44v2-v10, are widely expressed in normal colonic crypt epithelium, predominantly in the crypt base. CD44v6, the epitope which is most commonly associated with tumour progression and metastasis, was not only expressed by many benign colonic tumours, but was expressed as frequently in normal basal crypt epithelium as in malignant colonic tumour cells, and surprisingly, was even absent from some metastatic colorectal tumours. Expression of none of the CD44 variant epitopes was found to be positively correlated with tumour progression or with colorectal tumour metastasis to the liver, results which are inconsistent with a role for CD44 variants as indicators of colonic cancer progression. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:8695347

  2. Antibodies and antibody-derived analytical biosensors

    PubMed Central

    Sharma, Shikha; Byrne, Hannah

    2016-01-01

    The rapid diagnosis of many diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Biosensors are now being applied for rapid diagnostics due to their capacity for point-of-care use with minimum need for operator input. Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection of a plethora of analytes such as disease markers, food and environmental contaminants, biological warfare agents and illicit drugs. Antibodies are ideal biorecognition elements that provide sensors with high specificity and sensitivity. This review describes monoclonal and recombinant antibodies and different immobilization approaches crucial for antibody utilization in biosensors. Examples of applications of a variety of antibody-based sensor formats are also described. PMID:27365031

  3. Antibodies and antibody-derived analytical biosensors.

    PubMed

    Sharma, Shikha; Byrne, Hannah; O'Kennedy, Richard J

    2016-06-30

    The rapid diagnosis of many diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Biosensors are now being applied for rapid diagnostics due to their capacity for point-of-care use with minimum need for operator input. Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection of a plethora of analytes such as disease markers, food and environmental contaminants, biological warfare agents and illicit drugs. Antibodies are ideal biorecognition elements that provide sensors with high specificity and sensitivity. This review describes monoclonal and recombinant antibodies and different immobilization approaches crucial for antibody utilization in biosensors. Examples of applications of a variety of antibody-based sensor formats are also described. PMID:27365031

  4. Antibody Blood Tests

    MedlinePlus

    ... discovered that people with celiac disease who eat gluten have higher than normal levels of certain antibodies ... rye and barley that are generically known as “gluten.” Antibody Testing: Only A First Step To help ...

  5. RBC Antibody Screen

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Screen Share this page: Was this page ... Screen Related tests: Direct Antiglobulin Test ; Blood Typing ; RBC Antibody Identification ; Type and Screen; Crossmatch All content ...

  6. REGULATION OF CELLULAR ANTIBODY SYNTHESIS

    PubMed Central

    Möller, Göran

    1968-01-01

    Transfer of spleen cells from mice immunized against sheep red blood cells (SRBC) into irradiated (600 R) nonimmune, syngeneic mice in the presence of antigen resulted in excessive cellular 7S production 7 days later. The number of 7S plaque-forming cells usually exceeded 106 per spleen and the mean proportion varied between 1 and 70%. In occasional animals all spleen cells were producing antibodies to SRBC. Serum antibody synthesis was also excessively increased, the titers in agglutination after 2-ME treatment and in hemolysis varying between 215 and 225. The generation time of the 7S PFC was found to be 9.6 hr in the secondary hosts. It seemed possible that the excessive production of 7S PFC and antibodies in the irradiated nonimmune recipients was caused by the absence of feedback inhibition of the immune response by antibody, a mechanism which would normally function to restrict antibody synthesis. This conclusion was strengthened by the demonstration that transfer of antigen-stimulated immune cells into actively or passively immunized irradiated recipients resulted in a marked suppression of cellular 7S synthesis. Serial transfers of antigen-stimulated immune cell populations in irradiated hosts resulted in an equally high number of 7S PFC during the first four transfer generations. However, after the fifth to seventh transfer generation the number of 7S PFC rapidly declined and disappeared within one to three passages. Serum antibodies and 7S PFC declined in parallel during the last transfer generations. Further passages of antigen-stimulated spleen cells lacking 7S PFC did not lead to reappearance of PFC. Thus, antigen-sensitive cells have a limited lifespan and/or multiplication capacity. From the hypothesis that the 7S PFC developed by division from antigen-sensitive precursors it was calculated that 38–40 divisions occurred, Thus, one antigen-sensitive precursor has the potential to give rise to 1012 7S PFC. PMID:5635380

  7. Modeling Antibody Diversity.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy Ronstadt

    1998-01-01

    Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

  8. Cellobiohydrolase variants and polynucleotides encoding same

    SciTech Connect

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  9. Cellobiohydrolase variants and polynucleotides encoding the same

    SciTech Connect

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  10. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  11. Anti-insulin antibody test

    MedlinePlus

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

  12. Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from cultured isovaleric acidemia fibroblasts.

    PubMed Central

    Ikeda, Y; Keese, S M; Tanaka, K

    1985-01-01

    Variants of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10) in 15 isovaleric acidemia fibroblast lines were analyzed using [35S]methionine labeling, immunoprecipitation with anti-rat IVDHase antiserum, and NaDodSo4/polyacrylamide gel electrophoresis. Five distinct variants of IVDHase were detected. The molecular size of variant 1 (43 kDa) was indistinguishable from that of normal IVDHase (43 kDa), although the activity of this enzyme was as deficient (0-2.2% of normal control) as that of any other variant. It was synthesized as a precursor (45 kDa), which is the case for normal IVDHase. Variant 2 was synthesized as a 42-kDa precursor, but only a small portion of it was processed to the mature variant form (40 kDa). Variant 3 (41 kDa) was synthesized as a 43-kDa precursor. Variant 4 (40 kDa) was synthesized as a 42-kDa precursor that was readily processed to the mature form. In cells with variant 5, no material that crossreacted with the anti-rat IVDHase antibody was detected. These results suggest that variant 1 may be due to a point mutation, while variants 2-4 may be encoded by a different mutant IVDHase allele that causes the premature termination of translation, although other complex mechanisms are possible. A deletion, a nonsense mutation close to the NH2 terminus or an extremely labile mRNA may give rise to variant 5. Images PMID:3863140

  13. Gut Microbial Metabolites Fuel Host Antibody Responses.

    PubMed

    Kim, Myunghoo; Qie, Yaqing; Park, Jeongho; Kim, Chang H

    2016-08-10

    Antibody production is a metabolically demanding process that is regulated by gut microbiota, but the microbial products supporting B cell responses remain incompletely identified. We report that short-chain fatty acids (SCFAs), produced by gut microbiota as fermentation products of dietary fiber, support host antibody responses. In B cells, SCFAs increase acetyl-CoA and regulate metabolic sensors to increase oxidative phosphorylation, glycolysis, and fatty acid synthesis, which produce energy and building blocks supporting antibody production. In parallel, SCFAs control gene expression to express molecules necessary for plasma B cell differentiation. Mice with low SCFA production due to reduced dietary fiber consumption or microbial insufficiency are defective in homeostatic and pathogen-specific antibody responses, resulting in greater pathogen susceptibility. However, SCFA or dietary fiber intake restores this immune deficiency. This B cell-helping function of SCFAs is detected from the intestines to systemic tissues and conserved among mouse and human B cells, highlighting its importance. PMID:27476413

  14. Rare Copy Number Variants

    PubMed Central

    Grozeva, Detelina; Kirov, George; Ivanov, Dobril; Jones, Ian R.; Jones, Lisa; Green, Elaine K.; St Clair, David M.; Young, Allan H.; Ferrier, Nicol; Farmer, Anne E.; McGuffin, Peter; Holmans, Peter A.; Owen, Michael J.; O’Donovan, Michael C.; Craddock, Nick

    2015-01-01

    Context Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date. Objectives To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia. Design A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray. Setting The Wellcome Trust Case Control Consortium. Participants There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents. Main Outcome Measures Overall load of CNVs and presence of rare CNVs. Results The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder. Conclusions Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder. PMID:20368508

  15. Microscale purification of antigen-specific antibodies

    PubMed Central

    Brown, Eric P.; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E.; Chan, Ying N.; Lai, Jennifer I.; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E.

    2015-01-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain. PMID:26078040

  16. Microscale purification of antigen-specific antibodies.

    PubMed

    Brown, Eric P; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E; Chan, Ying N; Lai, Jennifer I; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E

    2015-10-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain.

  17. Heteromorphic variants of chromosome 9

    PubMed Central

    2013-01-01

    Background Heterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers. Results In this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes. Conclusions Based on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants. PMID:23547710

  18. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    PubMed

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  19. Analysis of monoclonal antibody oxidation by simple mixed mode chromatography.

    PubMed

    Pavon, Jorge Alexander; Li, Xiaojuan; Chico, Steven; Kishnani, Umesh; Soundararajan, Soundara; Cheung, Jason; Li, Huijuan; Richardson, Daisy; Shameem, Mohammed; Yang, Xiaoyu

    2016-01-29

    Analysis of oxidation of monoclonal antibodies (mAbs) in most cases relies on peptide mapping and LC-MS, which is time consuming and labor-intensive. A robust chromatography based method that is able to resolve and quantitate mAb oxidation variants due to oxidized methionine or tryptophan is highly desired. Here we developed a novel mixed mode chromatography method using the unique property of Sepax Zenix SEC-300MK column to analyze mAb oxidation levels. The separation of oxidized species relied upon the mixed mode of size exclusion and hydrophobic interaction between the resin and antibodies. The chromatography was performed in a regular SEC mobile phase, PBS, containing NaCl at a concentration (0-2.4M) specific for individual antibodies. This method was able to resolve and quantitate the oxidized antibodies as prepeaks, of either methionine-oxidized species induced by the common oxidants TBHP, tryptophan-oxidized species triggered by AAPH, or oxidized species by UV photo-irradiation. The prepeaks were further characterized by SEC-MALLS as monomers and confirmed by LC-MS as oxidized antibody variants with a mass increase of 16 or 32Da. This method has been successfully applied to monitor multiple monoclonal antibodies of IgG1, IgG2, and IgG4 subclasses. PMID:26774436

  20. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

    PubMed Central

    Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A.; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D.

    2015-01-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  1. Variants of beta-glucosidases

    SciTech Connect

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2014-10-07

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  2. Variants of beta-glucosidase

    SciTech Connect

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2015-07-14

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  3. Variants of beta-glucosidase

    SciTech Connect

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2009-12-29

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  4. Variants of beta-glucosidases

    DOEpatents

    Fidantsef, Ana; Lamsa, Michael; Clancy, Brian Gorre

    2008-08-19

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  5. Primary Aldosteronism and ARMC5 Variants

    PubMed Central

    Zilbermint, Mihail; Xekouki, Paraskevi; Faucz, Fabio R.; Berthon, Annabel; Gkourogianni, Alexandra; Schernthaner-Reiter, Marie Helene; Batsis, Maria; Sinaii, Ninet; Quezado, Martha M.; Merino, Maria; Hodes, Aaron; Abraham, Smita B.; Libé, Rossella; Assié, Guillaume; Espiard, Stéphanie; Drougat, Ludivine; Ragazzon, Bruno; Davis, Adam; Gebreab, Samson Y.; Neff, Ryan; Kebebew, Electron; Bertherat, Jérôme; Lodish, Maya B.

    2015-01-01

    Context: Primary aldosteronism is one of the leading causes of secondary hypertension, causing significant morbidity and mortality. A number of genetic defects have recently been identified in primary aldosteronism, whereas we identified mutations in ARMC5, a tumor-suppressor gene, in cortisol-producing primary macronodular adrenal hyperplasia. Objective: We investigated a cohort of 56 patients who were referred to the National Institutes of Health for evaluation of primary aldosteronism for ARMC5 defects. Methods: Patients underwent step-wise diagnosis, with measurement of serum aldosterone and plasma renin activity followed by imaging, saline suppression and/or oral salt loading tests, plus adrenal venous sampling. Cortisol secretion was also evaluated; unilateral or bilateral adrenalectomy was performed, if indicated. DNA, protein, and transfection studies in H295R cells were conducted by standard methods. Results: We identified 12 germline ARMC5 genetic alterations in 20 unrelated and two related individuals in our cohort (39.3%). ARMC5 sequence changes in 6 patients (10.7%) were predicted to be damaging by in silico analysis. All affected patients carrying a variant predicted to be damaging were African Americans (P = .0023). Conclusions: Germline ARMC5 variants may be associated with primary aldosteronism. Additional cohorts of patients with primary aldosteronism and metabolic syndrome, particularly African Americans, should be screened for ARMC5 sequence variants because these may underlie part of the known increased predisposition of African Americans to low renin hypertension. PMID:25822102

  6. [VGKC-complex antibodies].

    PubMed

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  7. Cloning and characterization of functional subtype A HIV-1 envelope variants transmitted through breastfeeding.

    PubMed

    Rainwater, Stephanie M J; Wu, Xueling; Nduati, Ruth; Nedellec, Rebecca; Mosier, Donald; John-Stewart, Grace; Mbori-Ngacha, Dorothy; Overbaugh, Julie

    2007-03-01

    Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.

  8. Humoral Immune Pressure Selects for HIV-1 CXC-chemokine Receptor 4-using Variants.

    PubMed

    Lin, Nina; Gonzalez, Oscar A; Registre, Ludy; Becerril, Carlos; Etemad, Behzad; Lu, Hong; Wu, Xueling; Lockman, Shahin; Essex, Myron; Moyo, Sikhulile; Kuritzkes, Daniel R; Sagar, Manish

    2016-06-01

    Although both C-C chemokine receptor 5 (CCR5)- and CXC chemokine receptor 4 (CXCR4)-using HIV-1 strains cause AIDS, the emergence of CXCR4-utilizing variants is associated with an accelerated decline in CD4+ T cells. It remains uncertain if CXCR4-using viruses hasten disease or if these variants only emerge after profound immunological damage. We show that exclusively CXCR4- as compared to cocirculating CCR5-utilizing variants are less sensitive to neutralization by both contemporaneous autologous plasma and plasma pools from individuals that harbor only CCR5-using HIV-1. The CXCR4-utilizing variants, however, do not have a global antigenic change because they remain equivalently susceptible to antibodies that do not target coreceptor binding domains. Studies with envelope V3 loop directed antibodies and chimeric envelopes suggest that the neutralization susceptibility differences are potentially influenced by the V3 loop. In vitro passage of a neutralization sensitive CCR5-using virus in the presence of autologous plasma and activated CD4+ T cells led to the emergence of a CXCR4-utilizing virus in 1 of 3 cases. These results suggest that in some but not necessarily all HIV-1 infected individuals humoral immune pressure against the autologous virus selects for CXCR4-using variants, which potentially accelerates disease progression. Our observations have implications for using antibodies for HIV-1 immune therapy. PMID:27428434

  9. Alternative Non-Antibody Protein Scaffolds for Molecular Imaging of Cancer

    PubMed Central

    Stern, Lawrence A.; Case, Brett A.; Hackel, Benjamin J.

    2013-01-01

    The development of improved methods for early detection and characterization of cancer presents a major clinical challenge. One approach that has shown excellent potential in preclinical and clinical evaluation is molecular imaging with small-scaffold, non-antibody based, engineered proteins. These novel diagnostic agents produce high contrast images due to their fast clearance from the bloodstream and healthy tissues, can be evolved to bind a multitude of cancer biomarkers, and are easily functionalized by site-specific bioconjugation methods. Several small protein scaffolds have been verified for in vivo molecular imaging including affibodies and their two-helix variants, knottins, fibronectins, DARPins, and several natural ligands. Further, the biodistribution of these engineered ligands can be optimized through rational mutation of the conserved regions, careful selection and placement of chelator, and modification of molecular size. PMID:24358455

  10. Advances in Antibody Design.

    PubMed

    Tiller, Kathryn E; Tessier, Peter M

    2015-01-01

    The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

  11. Anti-acetylcholine receptor antibodies.

    PubMed Central

    Vincent, A; Newsom Davis, J

    1980-01-01

    Early suggestions that a humoral factor might be implicated in the disorder of neuromuscular transmission in myasthenia gravis have been confirmed by the detection of anti-AChR antibody in 85-90% of the patients with generalised disease and in 75% of cases with restricted ocular myasthenia. Plasma exchange reveals that serum anti-AChR usually has an inverse relationship to muscle strength and present evidence indicates that patients responding to thymectomy and immunosuppressive durg treatment usually show a consistent decline in serum anti-AChR titres. The antibody is heterogeneous and can lead to a loss of muscle AChR by several mechanisms. Anti-AChR is produced in the thymus in relatively small amounts. Anti-AChR antibody synthesis by thymic lymphocytes and pokeweed stimulated peripheral lymphocytes in culture provides a means of studying the effect of different lymphocyte populations in vitro. Analysis of clinical, immunological and HLA antigen characteristics in MG suggest that more than one mechanism may underlie the breakdown in tolerance to AChR, leading to the production of anti-AChR antibodies. PMID:7400823

  12. Meta-analysis of gene-level associations for rare variants based on single-variant statistics.

    PubMed

    Hu, Yi-Juan; Berndt, Sonja I; Gustafsson, Stefan; Ganna, Andrea; Hirschhorn, Joel; North, Kari E; Ingelsson, Erik; Lin, Dan-Yu

    2013-08-01

    Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying "causal" rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available. PMID:23891470

  13. Meta-analysis of Gene-Level Associations for Rare Variants Based on Single-Variant Statistics

    PubMed Central

    Hu, Yi-Juan; Berndt, Sonja I.; Gustafsson, Stefan; Ganna, Andrea; Berndt, Sonja I.; Gustafsson, Stefan; Mägi, Reedik; Ganna, Andrea; Wheeler, Eleanor; Feitosa, Mary F.; Justice, Anne E.; Monda, Keri L.; Croteau-Chonka, Damien C.; Day, Felix R.; Esko, Tõnu; Fall, Tove; Ferreira, Teresa; Gentilini, Davide; Jackson, Anne U.; Luan, Jian’an; Randall, Joshua C.; Vedantam, Sailaja; Willer, Cristen J.; Winkler, Thomas W.; Wood, Andrew R.; Workalemahu, Tsegaselassie; Hu, Yi-Juan; Lee, Sang Hong; Liang, Liming; Lin, Dan-Yu; Min, Josine L.; Neale, Benjamin M.; Thorleifsson, Gudmar; Yang, Jian; Albrecht, Eva; Amin, Najaf; Bragg-Gresham, Jennifer L.; Cadby, Gemma; den Heijer, Martin; Eklund, Niina; Fischer, Krista; Goel, Anuj; Hottenga, Jouke-Jan; Huffman, Jennifer E.; Jarick, Ivonne; Johansson, Åsa; Johnson, Toby; Kanoni, Stavroula; Kleber, Marcus E.; König, Inke R.; Kristiansson, Kati; Kutalik, Zoltán; Lamina, Claudia; Lecoeur, Cecile; Li, Guo; Mangino, Massimo; McArdle, Wendy L.; Medina-Gomez, Carolina; Müller-Nurasyid, Martina; Ngwa, Julius S.; Nolte, Ilja M.; Paternoster, Lavinia; Pechlivanis, Sonali; Perola, Markus; Peters, Marjolein J.; Preuss, Michael; Rose, Lynda M.; Shi, Jianxin; Shungin, Dmitry; Smith, Albert Vernon; Strawbridge, Rona J.; Surakka, Ida; Teumer, Alexander; Trip, Mieke D.; Tyrer, Jonathan; Van Vliet-Ostaptchouk, Jana V.; Vandenput, Liesbeth; Waite, Lindsay L.; Zhao, Jing Hua; Absher, Devin; Asselbergs, Folkert W.; Atalay, Mustafa; Attwood, Antony P.; Balmforth, Anthony J.; Basart, Hanneke; Beilby, John; Bonnycastle, Lori L.; Brambilla, Paolo; Bruinenberg, Marcel; Campbell, Harry; Chasman, Daniel I.; Chines, Peter S.; Collins, Francis S.; Connell, John M.; Cookson, William; de Faire, Ulf; de Vegt, Femmie; Dei, Mariano; Dimitriou, Maria; Edkins, Sarah; Estrada, Karol; Evans, David M.; Farrall, Martin; Ferrario, Marco M.; Ferrières, Jean; Franke, Lude; Frau, Francesca; Gejman, Pablo V.; Grallert, Harald; Grönberg, Henrik; Gudnason, Vilmundur; Hall, Alistair S.; Hall, Per; Hartikainen, Anna-Liisa; Hayward, Caroline; Heard-Costa, Nancy L.; Heath, Andrew C.; Hebebrand, Johannes; Homuth, Georg; Hu, Frank B.; Hunt, Sarah E.; Hyppönen, Elina; Iribarren, Carlos; Jacobs, Kevin B.; Jansson, John-Olov; Jula, Antti; Kähönen, Mika; Kathiresan, Sekar; Kee, Frank; Khaw, Kay-Tee; Kivimaki, Mika; Koenig, Wolfgang; Kraja, Aldi T.; Kumari, Meena; Kuulasmaa, Kari; Kuusisto, Johanna; Laitinen, Jaana H.; Lakka, Timo A.; Langenberg, Claudia; Launer, Lenore J.; Lind, Lars; Lindström, Jaana; Liu, Jianjun; Liuzzi, Antonio; Lokki, Marja-Liisa; Lorentzon, Mattias; Madden, Pamela A.; Magnusson, Patrik K.; Manunta, Paolo; Marek, Diana; März, Winfried; Leach, Irene Mateo; McKnight, Barbara; Medland, Sarah E.; Mihailov, Evelin; Milani, Lili; Montgomery, Grant W.; Mooser, Vincent; Mühleisen, Thomas W.; Munroe, Patricia B.; Musk, Arthur W.; Narisu, Narisu; Navis, Gerjan; Nicholson, George; Nohr, Ellen A.; Ong, Ken K.; Oostra, Ben A.; Palmer, Colin N.A.; Palotie, Aarno; Peden, John F.; Pedersen, Nancy; Peters, Annette; Polasek, Ozren; Pouta, Anneli; Pramstaller, Peter P.; Prokopenko, Inga; Pütter, Carolin; Radhakrishnan, Aparna; Raitakari, Olli; Rendon, Augusto; Rivadeneira, Fernando; Rudan, Igor; Saaristo, Timo E.; Sambrook, Jennifer G.; Sanders, Alan R.; Sanna, Serena; Saramies, Jouko; Schipf, Sabine; Schreiber, Stefan; Schunkert, Heribert; Shin, So-Youn; Signorini, Stefano; Sinisalo, Juha; Skrobek, Boris; Soranzo, Nicole; Stančáková, Alena; Stark, Klaus; Stephens, Jonathan C.; Stirrups, Kathleen; Stolk, Ronald P.; Stumvoll, Michael; Swift, Amy J.; Theodoraki, Eirini V.; Thorand, Barbara; Tregouet, David-Alexandre; Tremoli, Elena; Van der Klauw, Melanie M.; van Meurs, Joyce B.J.; Vermeulen, Sita H.; Viikari, Jorma; Virtamo, Jarmo; Vitart, Veronique; Waeber, Gérard; Wang, Zhaoming; Widén, Elisabeth; Wild, Sarah H.; Willemsen, Gonneke; Winkelmann, Bernhard R.; Witteman, Jacqueline C.M.; Wolffenbuttel, Bruce H.R.; Wong, Andrew; Wright, Alan F.

    2013-01-01

    Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying “causal” rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available. PMID:23891470

  14. Spread of Plasmids Carrying Multiple GES Variants.

    PubMed

    Cuzon, Gaelle; Bogaerts, Pierre; Bauraing, Caroline; Huang, Te-Din; Bonnin, Rémy A; Glupczynski, Youri; Naas, Thierry

    2016-08-01

    Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems. PMID:27216071

  15. Gene Variants Reduce Opioid Risks

    MedlinePlus

    ... Charts Emerging Trends and Alerts Alcohol Club Drugs Cocaine Hallucinogens Heroin Inhalants Marijuana MDMA (Ecstasy/Molly) Methamphetamine ... a decreased risk for addiction to heroin or cocaine. The other linked variants in two genes— OPRM1 , ...

  16. Oligomeric Recombinant H5 HA1 Vaccine Produced in Bacteria Protects Ferrets from Homologous and Heterologous Wild-Type H5N1 Influenza Challenge and Controls Viral Loads Better than Subunit H5N1 Vaccine by Eliciting High-Affinity Antibodies

    PubMed Central

    Verma, Swati; Dimitrova, Milena; Munjal, Ashok; Fontana, Juan; Crevar, Corey J.; Carter, Donald M.; Ross, Ted M.

    2012-01-01

    Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission. PMID:22951833

  17. Antibody Therapeutics in Oncology

    PubMed Central

    Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

    2016-01-01

    One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment. PMID:27081677

  18. [Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

    PubMed

    Dzagurova, T K; Solopova, O N; Sveshnikov, P G; Korotina, N A; Balovneva, M V; Leonovich, O A; Varlamov, N E; Malkin, G A; Sotskova, S E; Tkachenko, E A

    2013-01-01

    The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

  19. Monoclonal antibodies as blood grouping reagents.

    PubMed

    Voak, D

    1990-04-01

    The large volume requirements for high quality ABO and Rh(D) typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents can be prepared, from blends of at least two antibodies, to optimize the intensity of agglutination for slide tests and the potency for the detection of the weaker sub-groups, including Ax and Bw, by tube techniques. New quality control steps have been described for some highly sensitive anti-A/anti-B antibodies to avoid the detection of traces of A on B cells or traces of B on A1 cells, which results from the non-specific activity of A and B transferases. Excellent anti-A,B reagents may also be made by blends of at least two antibodies to optimize both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline Rh(D) typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants, e.g. the epitopes on D category VI cells. However, this can be achieved by blending an IgM anti-D with IgG (polyclonal) anti-D which can detect these types after conversion of negative saline tests to an antiglobulin phase. In addition, high grade Du, D categories and variants can be reliably detected (for typing donors) by selected monoclonal IgM and IgG anti-Ds by use of suitably enhanced tests without the use of an antiglobulin test.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Entamoeba histolytica: expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain.

    PubMed

    López-Vancell, R; Arreguín Espinosa, R; González-Canto, A; Néquiz Avendaño, M; García de León, M C; Olivos-García, A; López-Vancell, D; Pérez-Tamayo, R

    2010-07-01

    We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG's anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.

  1. Immune responses to borrelial VlsE IR6 peptide variants.

    PubMed

    Sillanpää, Heidi; Lahdenne, Pekka; Sarvas, Heikki; Arnez, Maja; Steere, Allen; Peltomaa, Miikka; Seppälä, Ilkka

    2007-02-01

    Laboratory confirmation of Lyme borreliosis (LB) relies mainly on the demonstration of anti-borrelial antibodies. In recent studies, a novel VlsE protein IR(6) peptide-based assay has been introduced. Our aim was to evaluate the IR(6) peptides from three Borrelia burgdorferi sensu lato genospecies in the serodiagnosis of European and North American patients. Five VlsE protein IR(6) peptide variants representing sequences from B. burgdorferi sensu stricto, B. garinii, and B. afzelii were used as antigens in both IgG and IgM enzyme-linked immunosorbent assays (ELISA). Serum antibodies of 187 patients at different stages of LB from Europe and the United States were evaluated for serodiagnosis. For comparison samples were tested with one of the commercial IR(6) ELISAs. Three B. afzelii IR(6) variant peptides revealed antibodies that were concordant with each other. B. burgdorferi sensu stricto peptide antibodies mostly paralleled B. afzelii peptide antibodies, and positive values were also obtained in the majority of European sera. For several sera, B. garinii IR(6) peptide antibodies were discordant to B. afzelii peptide antibodies. The commercial IR(6) peptide antibody assay (C6 ELISA) results correlated better with B. burgdorferi sensu stricto IR(6) than with B. garinii IR(6) peptide IgG results, especially in sera from patients with facial palsy. Thus, antibody specificity to IR(6) peptides may vary according to the infecting Borrelia species. In some manifestations of the disease, C6 ELISA may not cover all LB cases. Evidently, the methodological aspects in ELISA design for peptide antibody measurements are important as well as the amino acids sequence of the antigen.

  2. Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant.

    PubMed

    Braga, Catarina J M; Rodrigues, Juliana F; Medina-Armenteros, Yordanka; Farinha-Arcieri, Luís E; Ventura, Armando M; Boscardin, Silvia B; Sbrogio-Almeida, Maria E; Ferreira, Luís C S

    2014-01-01

    Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.

  3. Antibodies to prothrombin.

    PubMed

    Bertolaccini, M L

    2012-06-01

    Research on antiphospholipid antibodies (aPL) and the thrombotic manifestations associated with these antibodies has grown since the description of anticardiolipin antibodies (aCL) by Harris and colleagues in the early 1980s. Antiprothrombin (aPT) antibodies are commonly detected by ELISA, using irradiated plates (aPT) or prothrombin in complex with phosphatidylserine (aPS/PT). Although aPT and/or aPS/PT are associated with antiphospholipid syndrome (APS) -related clinical features and these antibodies correlate with each other, aPT and aPS/PT belong to different populations of autoantibodies even though they can both be present in the same patient. Early studies suggested that these antibodies might be the antigenic target of lupus anticoagulant (LA) and their correlation and clinical significance is being investigated. PMID:22635215

  4. Antibodies in Transplantation

    PubMed Central

    Platt, Jeffrey L.

    2011-01-01

    Transplantation of cells, tissues, and organs from one individual to another can incite the production of antibodies specific for foreign antigens, especially major histocompatibility antigens, in the graft. Antibodies specific for a graft provide an index of immunity and a potential trigger for injury and rejection. However, the index of immunity can sometimes miss antibody-mediated rejection and besides causing injury the antibodies against a graft can also protect a graft from injury by blocking immune recognition, called enhancement, regulating activation of complement, and inducing changes in the graft that resist damage. Reviewed here are potential limitations in the use of antibodies as an index of immunity and the ways antibodies cause and/or prevent injury. PMID:20807473

  5. Synthetic approach to the generation of antibody diversity

    PubMed Central

    Shim, Hyunbo

    2015-01-01

    The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494] PMID:26129672

  6. Donor non-specific MICA antibodies in renal transplant recipients.

    PubMed

    Sapák, Michal; Chreňová, Silvia; Tirpáková, Jana; Žilinská, Zuzana; Ďurmanová, Vladimíra; Shawkatová, Ivana; Jakuš, Vladimír; Kuba, Daniel; Buc, Milan

    2014-02-01

    Despite recent advances in solid organ transplantations, an antibody mediated rejection caused by donor specific antibodies is still a major problem in kidney graft survival. Besides HLA-induced humoral response, antibodies against MICA antigens have recently attracted attention because of their possible role in graft rejection. The aim of our study was to establish whether renal recipients produce antibodies against MICA molecules due to the transplantation and if they are specific for MICA antigens of the donors. MICA antibody screening was performed in 124 kidney recipient sera. 22 sera, that were found to be MICA antibody positive, were further examined for MICA antibody profiles and compared with donor MICA alleles. The analysis of MICA antibody positive sera showed mostly more complex reactivity patterns. A significant fraction of patient sera (59%) reacted not only with the donor MICA antigens, but also with other MICA patterns. A match between antibody specificities and MICA antigens was observed in 41% of renal recipients only. On the other hand, as much as in 36% of recipient sera were detected antibodies against their own MICA molecules. We did not prove a complete correlation between the recipient MICA antibody specificities and MICA antigens of the donor. We assume that MICA antibody induction occurs not only due to the allogeneic stimulation itself but also due to other factors that need to be elucidated.

  7. A general method for greatly improving the affinity of antibodies by using combinatorial libraries

    PubMed Central

    Rajpal, Arvind; Beyaz, Nurten; Haber, Lauric; Cappuccilli, Guido; Yee, Helena; Bhatt, Ramesh R.; Takeuchi, Toshihiko; Lerner, Richard A.; Crea, Roberto

    2005-01-01

    Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-α antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each VH and VL domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-α. These beneficial mutations in both VH and VL were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-α neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins. PMID:15939870

  8. High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC

    PubMed Central

    Gribenko, Alexey V.; Parris, Kevin; Mosyak, Lidia; Li, Sheng; Handke, Luke; Hawkins, Julio C.; Severina, Elena; Matsuka, Yury V.; Anderson, Annaliesa S.

    2016-01-01

    The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 – group 1, mAB 305-78-7 – group 2, and mAB 305-101-8 – group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity. PMID:27689696

  9. Antibodies to cholesterol.

    PubMed Central

    Swartz, G M; Gentry, M K; Amende, L M; Blanchette-Mackie, E J; Alving, C R

    1988-01-01

    Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained. Images PMID:3162316

  10. Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

    PubMed

    Okeley, Nicole M; Toki, Brian E; Zhang, Xinqun; Jeffrey, Scott C; Burke, Patrick J; Alley, Stephen C; Senter, Peter D

    2013-10-16

    The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

  11. Purification and properties of non-precipitating rabbit antibodies

    PubMed Central

    Margni, R.; Binaghi, R.

    1972-01-01

    Precipitating and non-precipitating anti-egg albumin and anti-dinitrophenyl rabbit antibodies were specifically purified from hyperimmunized sera. Both populations of antibody were similar with regard to electrophoretic mobility and molecular size. Non-precipitating antibodies brought about passive haemagglutination and PCA, although with less efficiency than precipitating antibodies. On the other hand, only precipitating antibodies fixed complement and produced a reverse Arthus reaction. The F(ab′)2 fragment obtained from non-precipitating antibody did not precipitate with antigen. These results are compatible with the hypothesis that non-precipitability is due to a particular configuration of the molecule that makes it impossible for one molecule of antibody to combine with two different molecules of antigen simultaneously. PMID:4259530

  12. Clear Speech Variants: An Acoustic Study in Parkinson's Disease

    ERIC Educational Resources Information Center

    Lam, Jennifer; Tjaden, Kris

    2016-01-01

    Purpose: The authors investigated how different variants of clear speech affect segmental and suprasegmental acoustic measures of speech in speakers with Parkinson's disease and a healthy control group. Method: A total of 14 participants with Parkinson's disease and 14 control participants served as speakers. Each speaker produced 18 different…

  13. Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice

    SciTech Connect

    Selden, R.F.; Wagner, T.E.; Blethen, S.; Yun, J.S.; Rowe, M.E.; Goodman, H.M. )

    1988-11-01

    The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, the authors have expressed a mouse methallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties.

  14. Monoclonal antibodies: their importance to surgeons.

    PubMed

    Estabrook, A; Mesa-Tejada, R

    1989-01-01

    A tremendous technological advance occurred in 1975 when a method was developed to fuse two cells producing a "hybridoma" which secretes a single clone of antibody, having one immunoglobulin (Ig) class, one structure, one affinity, and one specificity for an antigenic determinant. Because monoclonal antibodies are more precise reagents than conventional antisera they open new doors to diagnosis and therapy of disease, and they are useful tools in research. The pathologist uses monoclonals in immunocytochemistry to determine tumor type; the surgeon uses monoclonals for immunosuppression in renal transplantation; the immunologist uses monoclonals to decipher cellular and humoral interactions that could not be appreciated with polyclonal reagents. This review outlines the background of monoclonal antibodies and some of their clinically important uses, both in vitro and in vivo. We also project into the future and describe chimeric antibodies and their possible uses.

  15. Goat serums for fluorescent antibody conjugates to chlamydial antigens.

    PubMed Central

    Tessler, J

    1984-01-01

    Serums from goats hyperimmunized with Chlamydia psittaci consistently produce antichlamydial fluorescent antibody conjugate of high titer. The titer of the fluorescent antibody conjugate prepared from a given serum correlated well with the titer obtained by agar gel precipitin, but not with the complement fixation. The agar gel precipitin test can be used to predict whether a given serum is satisfactory for use in production of a conjugate for direct fluorescent antibody tests. Serums with an agar gel precipitin titer of 1/8 or higher generally produce a usable fluorescent antibody conjugate. Labeling gamma globulins with fluorescein isothiocyanate at a ratio of 1/150 resulted in satisfactory fluorescent antibody conjugates. Cultures of Vero cells infected with chlamydiae were found to be suitable for titration of the fluorescent antibody conjugates. PMID:6372973

  16. Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

    PubMed

    Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

    2011-12-01

    The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

  17. Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

    PubMed Central

    Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

    2014-01-01

    While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs. PMID:24492301

  18. Molecular data mining to improve antibody-based detection of Grapevine leafroll-associated virus 1 (GLRaV-1).

    PubMed

    Esteves, Filipa; Teixeira Santos, Margarida; Eiras-Dias, José Eduardo; Fonseca, Filomena

    2013-12-01

    Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

  19. Computerized design and generation of space-variant holographic filters. II - Applications of space-variant filters to optical computing

    NASA Technical Reports Server (NTRS)

    Ambs, P.; Fainman, Y.; Esener, S.; Lee, S. H.

    1988-01-01

    Holographic optical elements (HOEs) of space-variant impulse response have been designed and generated using a computerized optical system. HOEs made of dichromated gelatin have been produced and used for spatial light modulator defect removal and optical interconnects. Experimental performance and characteristics are presented.

  20. Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining

    PubMed Central

    Wang, Bo; Kluwe, Christien A.; Lungu, Oana I.; DeKosky, Brandon J.; Kerr, Scott A.; Johnson, Erik L.; Jung, Jiwon; Rezigh, Alec B.; Carroll, Sean M.; Reyes, Ann N.; Bentz, Janelle R.; Villanueva, Itamar; Altman, Amy L.; Davey, Robert A.; Ellington, Andrew D.; Georgiou, George

    2015-01-01

    The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138+ antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported. PMID:26355042

  1. Antibodies Against Three Forms of Urokinase

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Atassi, M. Zouhair

    2007-01-01

    Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

  2. Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

    PubMed

    Brindle, R J; Bryant, T N; Draper, P W

    1989-03-01

    A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1. PMID:2654183

  3. Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

    PubMed

    Kaloyanova, D; Kyurkchiev, S; Xu, J; Abouhaidar, M; Ivanov, I

    1999-08-01

    Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

  4. Monoclonal antibody to an integral membrane protein, the lactose permease.

    PubMed

    Eash, J; Villarejo, M R

    1983-02-01

    A monoclonal IgG antibody directed against the lactose permease was produced from animals inoculated with membranes of a lac Y plasmid strain. The appropriate antibody was selected by a series of ELISA assays in which membranes, purified permease, or a lac Y-Z chimeric protein was the immobilized antigen. The antibody recognizes a portion of the permease exposed on the surface of membrane vesicles but does not inhibit lactose transport.

  5. Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin a production

    SciTech Connect

    Chelack, W.S.; Borsa, J.; Szekely, J.G. ); Marquardt, R.R.; Frohlich, A.A. )

    1991-09-01

    The present studies, using Asperigillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28C and 25% moisture. After 10 days of incubation, two colony types, ocher (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ocher (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.

  6. Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

    PubMed Central

    Chelack, W S; Borsa, J; Szekely, J G; Marquardt, R R; Frohlich, A A

    1991-01-01

    The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants. Images PMID:1768122

  7. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  8. Antibodies as natural adjuvants.

    PubMed

    Heyman, Birgitta

    2014-01-01

    Antibodies in complex with specific antigen can dramatically change the antibody response to this antigen. Depending on antibody class and type of antigen, >99 % suppression or >100-fold enhancement of the response can take place. IgM and IgG3 are efficient enhancers and operate via the complement system. In contrast, IgG1, IgG2a, and IgG2b enhance antibody and CD4(+) T cell responses to protein antigens via activating Fcγ-receptors. IgE also enhances antibody and CD4(+) T cell responses to small proteins but uses the low-affinity receptor for IgE, CD23. Most likely, IgM and IgG3 work by increasing the effective concentration of antigen on follicular dendritic cells in splenic follicles. IgG1, IgG2a, IgG2b, and IgE probably enhance antibody responses by increasing antigen presentation by dendritic cells to T helper cells. IgG antibodies of all subclasses have a dual effect, and suppress antibody responses to particulate antigens such as erythrocytes. This capacity is used in the clinic to prevent immunization of Rhesus-negative women to Rhesus-positive fetal erythrocytes acquired via transplacental hemorrage. IgG-mediated suppression in mouse models can take place in the absence of Fcγ-receptors and complement and to date no knock-out mouse strain has been found where suppression is abrogated.

  9. 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.

  10. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  11. 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...

  12. Immunization of mice with a newly identified thyroid-stimulating hormone receptor splice variant induces Graves'-like disease.

    PubMed

    Endo, Toyoshi; Kobayashi, Teturo

    2013-06-01

    We have cloned a thyroid-stimulating hormone receptor (TSHR) cDNA from mouse thyroid glands. The sequence of this cDNA indicated that it encoded a 739 amino acid TSHR splice variant that lacked exon 5 (TSHR739). In thyroid gland samples from adult mice, the amount of TSHR739 mRNA was about 10% of the amount of full-length TSHR (TSHR764) mRNA. A eCFP-tagged TSHR739 integrated into plasma membrane, but lacked TSH binding activity and it did not produce cAMP in response to TSH. However, thyroid-stimulating antibodies from patients with Graves' disease stimulated cAMP production in HEK293 cells that expressed TSHR739. Quantitative PCR revealed that TSHR739 transcript levels were low in the fetal mouse thyroid samples, but TSHR739 transcript levels increased after birth and as the mice grew. We used plasmid injection combined with electroporation into skeletal muscles to immunize BALB/c mice with TSHR739, TSHR764,, or control plasmid; TSHR739 caused goiters, high (125)I uptake activity, thyrotoxicosis, and production of thyroid-stimulating antibodies, but TSHR764, or control did not. These results indicated that immunization with an autologous TSHR antigen, TSHR739, induced Graves'-like disease in mice, and that TSHR739 is a candidate autoantigen in autoimmune thyroid disease. PMID:23538203

  13. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include 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 facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  14. Common Variants for Heart Failure

    PubMed Central

    Shen, Shutong; Tao, Lichan; Wang, Xiuzhi; Kong, Xiangqing; Li, Xinli

    2015-01-01

    Heart failure (HF) is a common disease with high morbidity and mortality; however, none of the drugs available are now entirely optimal for the treatment of HF. In addition to various clinical diseases and environment influences, genetic factors also contribute to the development and progression of HF. Identifying the common variants for HF by genome-wide association studies will facilitate the understanding of pathophysiological mechanisms underlying HF. This review summarizes the recently identified common variants for HF risk and outcome and discusses their implications for the clinic therapy. PMID:26085806

  15. Anti-DNA antibody mediated catalysis is isotype dependent.

    PubMed

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies. PMID:26655427

  16. Anti-DNA antibody mediated catalysis is isotype dependent.

    PubMed

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.

  17. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen.

    PubMed

    Kucinskaite-Kodze, Indre; Pleckaityte, Milda; Bremer, Corinna M; Seiz, Pia L; Zilnyte, Milda; Bulavaite, Aiste; Mickiene, Gitana; Zvirblis, Gintautas; Sasnauskas, Kestutis; Glebe, Dieter; Zvirbliene, Aurelija

    2016-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic "a" determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV

  18. Fingerprint-like Analysis of “Nanoantibody” Selection by Phage Display Using Two Helper Phage Variants

    PubMed Central

    Tillib, S.V.; Ivanova, T.I.; Vasilev, L.A.

    2010-01-01

    This paper discusses the selection of mini-antibody (nanoantibody, nanobody® or single domain antibody) sequences of desired specificity by phage display-based method using a generated library of antigen-binding domains of special heavy-chain only antibodies (single-stranded antibodies) of immunized camel. A comprehensive comparison of the efficiency of parallel selection procedures was performed by using the traditional (M13KO7) and modified (with N-terminal deletion in the surface gIII protein) helper phages. These two methods are partly complementary, and by using them in parallel one can significantly improve the selection efficiency. Parallel restriction analysis (fingerprinting) of PCR-amplified cloned sequences coding for mini-antibodies (HMR-analysis) is proposed for identifying individual clones, as a replacement to sequencing (to a certain extent). Using this method, unique data were collected on the selection of mini-antibody variants with the required specificity at various stages of a multi-stage selection procedure. It has been shown that different sequences coding for mini-antibodies are selected in different ways, and that, if this feature is not taken into account, some mini-antibody variants may be lost. PMID:22649655

  19. A monoclonal antibody to the rat nuclear triiodothyronine receptor: production and characterization.

    PubMed

    Luo, M; Faure, R; Ruel, J; Dussault, J H

    1988-07-01

    The nuclear T3 receptor (NTR) was affinity-labeled with bromoacetyl-[125I]T3, purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and used to immunize BALB/c mice. Spleen cells from one strongly immunoreactive mouse were fused with Sp2 mouse myeloma cells, and 328 hybridomas were screened by a dot-blot immunoassay using as antigen, a preparation of NTR partially purified by diethylaminoethyl-Sephadex chromatography. Four positive cultures were thus found; three of which were confirmed by comparing Western blotting patterns with the electrophoretic mobility of the affinity-labeled NTR. One of these 3 hybridomas was further subcloned by limiting dilution and gave rise to the 2B3 clone, which produces an immunoglobulin of the immunoglobulin G1 subclass. Several lines of evidence indicated that the 2B3 monoclonal antibody was indeed directed against the NTR. The antibody recognized a protein with the same electrophoretic mobility as the affinity-labeled receptor. Thus, Western blotting revealed a predominant protein with a mol wt of 57,000 and a less abundant 45,000 component on sodium dodecyl sulfate gels, and multiple isoelectric variants of the 57,000 protein, with a predominant form at pI 6.2, were detected on two-dimensional gels. Incubation of the 2B3 antibody with the NTR labeled with [125I]T3 resulted in the formation of an antibody-receptor complex, as indicated by a shift of the radioactivity peak upon gel filtration on Sephacryl S-300. In contrast, control ascitic fluid did not change the elution profile of the labeled NTR. The 2B3 antibody is able to remove the T3-binding activity from rat liver nuclear extracts. Finally, in accordance with previous T3-binding experiments, expected amounts of NTR were found in pituitary, liver, brain, kidney, spleen, and testis with the use of the Western blotting technique and immunohistochemistry on frozen tissue sections. This antibody should prove useful in the characterization and

  20. Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

    PubMed Central

    Hugenholtz, Jeroen; Veldkamp, Hans; Konings, Wil N.

    1987-01-01

    Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically. Images PMID:16347256

  1. Comparison of nonhuman primate antibodies against Haemophilus influenzae type b polysaccharide with human antibodies in oligoclonality and in vivo protective potency.

    PubMed

    Kim, K H; Park, M K; Peeters, C C; Poolman, J T; Shearer, M H; Kennedy, R C; Nahm, M H

    1994-06-01

    Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

  2. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  3. NMDA receptor antibodies

    PubMed Central

    Ramberger, Melanie; Bsteh, Gabriel; Schanda, Kathrin; Höftberger, Romana; Rostásy, Kevin; Baumann, Matthias; Aboulenein-Djamshidian, Fahmy; Lutterotti, Andreas; Deisenhammer, Florian; Berger, Thomas

    2015-01-01

    Objectives: To analyze the frequency of NMDA receptor (NMDAR) antibodies in patients with various inflammatory demyelinating diseases of the CNS and to determine their clinical correlates. Methods: Retrospective case-control study from 2005 to 2014 with the detection of serum IgG antibodies to NMDAR, aquaporin-4, and myelin oligodendrocyte glycoprotein by recombinant live cell-based immunofluorescence assays. Fifty-one patients with acute disseminated encephalomyelitis, 41 with neuromyelitis optica spectrum disorders, 34 with clinically isolated syndrome, and 89 with multiple sclerosis (MS) were included. Due to a known association of NMDAR antibodies with seizures and behavioral symptoms, patients with those clinical manifestations were preferentially included and are therefore overrepresented in our cohort. Nine patients with NMDAR encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were used as controls. Results: NMDAR antibodies were found in all 9 patients with NMDAR encephalitis but in only 1 of 215 (0.5%) patients with inflammatory demyelination and in none of the controls. This patient had relapsing-remitting MS with NMDAR antibodies present at disease onset, with an increase in NMDAR antibody titer with the onset of psychiatric symptoms and cognitive deficits. Conclusion: In demyelinating disorders, NMDAR antibodies are uncommon, even in those with symptoms seen in NMDAR encephalitis. PMID:26309901

  4. Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies?

    PubMed Central

    Kuus-Reichel, K; Grauer, L S; Karavodin, L M; Knott, C; Krusemeier, M; Kay, N E

    1994-01-01

    While monoclonal antibodies show promise for use in the treatment of a variety of disease states, including cancer, autoimmune disease, and allograft rejection, generation of anti-antibody responses still remains a problem. For example, 50% of the patients who receive OKT3 produce blocking antibodies that interfere with its binding to T cells, thus decreasing the therapeutic effect (51). HAMA responses have also interfered with tumor imaging (39,40) and radioimmunotherapy (56). The generation of an anti-antibody response is dependent on many factors. These include the dose of antibody, the number of injections of antibody, the immunogenicity of the antibody, the form of the antibody, and the immunocompetence of the recipient. Predictably, both the number of injections of antibody and the dosage are influential in the generation of an anti-antibody response. It is apparent that human antibodies, chimeric antibodies, and mouse Fab fragments are much less likely to induce anti-antibody responses than intact mouse monoclonal antibodies or mouse F(ab')2 fragments when one injection is administered. Injections of human or chimeric antibodies appears to reduce immunogenicity, but the probability that anti-antibody responses can still be induced on multiple injections must be considered and appropriately evaluated. Several areas demand extensive investigation to enhance the clinical utility of monoclonal antibodies. First, results of thorough clinical trials with human or chimeric antibodies need to be evaluated for the induction of anti-antibodies after multiple injections of antibodies. Second, less immunogenic forms of antibodies (Fab, Fv) need to be studied for their clinical efficacies and for their abilities to induce anti-antibody responses. PMID:8556470

  5. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  6. Studies on a Smooth Phage-resistant Variant of Brucella abortus

    PubMed Central

    Corbel, M. J.; Morris, J. A.

    1974-01-01

    The immunological properties of a phage-resistant variant of Brucella abortus were examined. This variant, which was smooth and identical with the phage-susceptible parent strain in all cultural, biochemical and serological properties studied, was strongly antigenic in rabbits and guineapigs, inducing high titres of antibodies and delayed hypersensitivity to Br. abortus antigens. It was also fully virulent for mice and guinea-pigs. Contrary to other reports on the properties of phage-resistant Brucella strains, the in vivo properties of this strain showed no evidence of attenuation of virulence. PMID:4209104

  7. Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H.

    PubMed Central

    Hutchins, J T; Kull, F C; Bynum, J; Knick, V C; Thurmond, L M; Ray, P

    1995-01-01

    Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy. PMID:8618827

  8. Impedance measurements for detecting pathogens attached to antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2004-12-28

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  9. Method for detecting pathogens attached to specific antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2005-01-25

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  10. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  11. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    PubMed

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  12. Variant (Swine Origin) Influenza Viruses in Humans

    MedlinePlus

    ... What's this? Submit Button Past Newsletters Variant Influenza Viruses: Background and CDC Risk Assessment and Reporting Language: ... Background CDC Assessment Reporting Background On Variant Influenza Viruses Swine flu viruses do not normally infect humans. ...

  13. Swine Influenza/Variant Influenza Viruses

    MedlinePlus

    ... Humans Key Facts about Human Infections with Variant Viruses Interim Guidance for Clinicians on Human Infections Background, Risk Assessment & Reporting Reported Infections with Variant Influenza Viruses in the United States since 2005 Prevention Treatment ...

  14. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  15. Anti-cartilage antibody.

    PubMed

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  16. Anti-cartilage antibody.

    PubMed Central

    Greenbury, C L; Skingle, J

    1979-01-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change. Images Fig. 1 PMID:389957

  17. HIV Antibody Test

    MedlinePlus

    ... despite the fact that the person is infected ( false negative ). If an HIV antibody test is negative ... infection (around 28 days) and may give a false-negative result. ^ Back to top Is there anything ...