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Sample records for antibody fragments genetically

  1. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans

    2008-08-01

    Full Text Available Today’s proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

  2. Antibody Fragments and Their Purification by Protein L Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Gustav Rodrigo

    2015-09-01

    Full Text Available Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing.

  3. Recombinant fragment of an antibody tailored for direct radioiodination

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj; Fábry, Milan; Sieglová, Irena; Král, Vlastimil; Uhnáková, Bronislava; Mudra, M.; Kronrád, L.; Sawicka, A.; Mikolajczak, R.; Řezáčová, Pavlína

    2012-01-01

    Roč. 55, č. 1 (2012), s. 52-56 ISSN 0362-4803 R&D Projects: GA MPO 2A-2TP1/076; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50520514 Keywords : I125 labelling * single-chain antibody variable fragment * tyrosine-rich polypeptide segment * fusion protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.240, year: 2012

  4. Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

    Czech Academy of Sciences Publication Activity Database

    Ohradanova-Repic, A.; Nogueira, E.; Hartl, I.; Gomes, A.C.; Preto, A.; Steinhuber, E.; Muehlgrabner, V.; Repic, M.; Kuttke, M.; Zwirzitz, A.; Prouza, M.; Suchánek, M.; Wozniak-Knopp, G.; Hořejší, Václav; Schabbauer, G.; Cavaco-Paulo, A.; Stockinger, H.

    2018-01-01

    Roč. 14, č. 1 (2018), s. 123-130 ISSN 1549-9634 Institutional support: RVO:68378050 Keywords : Active targeting * Liposome functionalization * Immunoliposome * Antibody engineering * Recombinant Fab antibody fragment Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 5.720, year: 2016

  5. The paradox of forest fragmentation genetics

    Science.gov (United States)

    Andrea T. Kramer; Jennifer L. Ison; Mary V. Ashley; Henry F. Howe

    2008-01-01

    Theory predicts widespread loss of genetic diversity from drift and inbreeding in trees subjected to habitat fragmentation, yet empirical support of this theory is scarce. We argue that population genetics theory may be misapplied in light of ecological realities that, when recognized, require scrutiny of underlying evolutionary assumptions. One ecological reality is...

  6. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  7. Habitat fragmentation causes rapid genetic differentiation and ...

    African Journals Online (AJOL)

    ... city buildings. These results were supported by multiple statistical analyses including Mantel's test, PCOORDA and AMOVA. Genetic enrichment and epigenetic variation studies can be included in habitat fragmentation analysis and its implications in inducing homogenization and susceptibility in natural plant populations.

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

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do...

  9. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to fragments enabled the development of a sensitive (LoD=3.3ng/L) immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. Targeted cancer therapy through antibody fragments-decorated nanomedicines.

    Science.gov (United States)

    Alibakhshi, Abbas; Abarghooi Kahaki, Fatemeh; Ahangarzadeh, Shahrzad; Yaghoobi, Hajar; Yarian, Fatemeh; Arezumand, Roghaye; Ranjbari, Javad; Mokhtarzadeh, Ahad; de la Guardia, Miguel

    2017-12-28

    Active targeting in cancer nanomedicine, for improved delivery of agents and diagnose, has been reviewed as a successful way for facilitating active uptake of theranostic agents by the tumor cells. The application of a targeting moiety in the targeted carrier complexes can play an important role in differentiating between tumor and healthy tissues. The pharmaceutical carriers, as main part of complexes, can be polymeric nanoparticles, micelles, liposomes, nanogels and carbon nanotubes. The antibodies are among the natural ligands with highest affinity and specificity to target pharmaceutical nanoparticle conjugates. However, the limitations, such as size and long circulating half-lives, hinder reproducible manufacture in clinical studies. Therefore, novel approaches have moved towards minimizing and engineering conventional antibodies as fragments like scFv, Fab, nanobody, bispecific antibody, bifunctional antibody, diabody and minibody preserving their functional potential. Different formats of antibody fragments have been reviewed in this literature update, in terms of structure and function, as smart ligands in cancer diagnosis and therapy of tumor cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    Directory of Open Access Journals (Sweden)

    Katerina T. Xenaki

    2017-10-01

    Full Text Available The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

  12. Effects of genetic engineering on the pharmacokinetics of antibodies

    International Nuclear Information System (INIS)

    Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K.

    1999-01-01

    Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment

  13. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  14. Neutralisation and binding of VHS virus by monovalent antibody fragments

    DEFF Research Database (Denmark)

    Cupit, P.M.; Lorenzen, Niels; Strachan, G.

    2001-01-01

    single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using...... the 3F1H10 variable heavy (VH) chain and variable light (V kappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured...

  15. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama

  16. Habitat specialization predicts genetic response to fragmentation in tropical birds.

    Science.gov (United States)

    Khimoun, Aurélie; Eraud, Cyril; Ollivier, Anthony; Arnoux, Emilie; Rocheteau, Vincent; Bely, Marine; Lefol, Emilie; Delpuech, Martin; Carpentier, Marie-Laure; Leblond, Gilles; Levesque, Anthony; Charbonnel, Anaïs; Faivre, Bruno; Garnier, Stéphane

    2016-08-01

    Habitat fragmentation is one of the most severe threats to biodiversity as it may lead to changes in population genetic structure, with ultimate modifications of species evolutionary potential and local extinctions. Nonetheless, fragmentation does not equally affect all species and identifying which ecological traits are related to species sensitivity to habitat fragmentation could help prioritization of conservation efforts. Despite the theoretical link between species ecology and extinction proneness, comparative studies explicitly testing the hypothesis that particular ecological traits underlies species-specific population structure are rare. Here, we used a comparative approach on eight bird species, co-occurring across the same fragmented landscape. For each species, we quantified relative levels of forest specialization and genetic differentiation among populations. To test the link between forest specialization and susceptibility to forest fragmentation, we assessed species responses to fragmentation by comparing levels of genetic differentiation between continuous and fragmented forest landscapes. Our results revealed a significant and substantial population structure at a very small spatial scale for mobile organisms such as birds. More importantly, we found that specialist species are more affected by forest fragmentation than generalist ones. Finally, our results suggest that even a simple habitat specialization index can be a satisfying predictor of genetic and demographic consequences of habitat fragmentation, providing a reliable practical and quantitative tool for conservation biology. © 2016 John Wiley & Sons Ltd.

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  18. Genetic consequences of habitat fragmentation during a range expansion.

    Science.gov (United States)

    Mona, S; Ray, N; Arenas, M; Excoffier, L

    2014-03-01

    We investigate the effect of habitat fragmentation on the genetic diversity of a species experiencing a range expansion. These two evolutionary processes have not been studied yet, at the same time, owing to the difficulties of deriving analytic results for non-equilibrium models. Here we provide a description of their interaction by using extensive spatial and temporal coalescent simulations and we suggest guidelines for a proper genetic sampling to detect fragmentation. To model habitat fragmentation, we simulated a two-dimensional lattice of demes partitioned into groups (patches) by adding barriers to dispersal. After letting a population expand on this grid, we sampled lineages from the lattice at several scales and studied their coalescent history. We find that in order to detect fragmentation, one needs to extensively sample at a local level rather than at a landscape level. This is because the gene genealogy of a scattered sample is less sensitive to the presence of genetic barriers. Considering the effect of temporal changes of fragmentation intensities, we find that at least 10, but often >100, generations are needed to affect local genetic diversity and population structure. This result explains why recent habitat fragmentation does not always lead to detectable signatures in the genetic structure of populations. Finally, as expected, long-distance dispersal increases local genetic diversity and decreases levels of population differentiation, efficiently counteracting the effects of fragmentation.

  19. Cultivation of Pichia pastoris carrying the scFv anti LDL (- antibody fragment. Effect of preculture carbon source

    Directory of Open Access Journals (Sweden)

    Cesar Andres Diaz Arias

    Full Text Available Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (- under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

  20. Stability of llama heavy chain antibody fragments under extreme conditions

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The

  1. Habitat fragmentation causes rapid genetic differentiation and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... inducing homogenization and susceptibility in natural plant populations. Key words: Leymus chinensis, AFLP, genetic ... Its habitat revegetation is influenced by artificial selection, habitat selection pressure ..... left below diagonal) and Nei's genetic distance (Nei, 1978, right above diagonal) between pairs.

  2. Genetic structure of fragmented November moth (Lepidoptera: Geometridae) populations in farmland

    DEFF Research Database (Denmark)

    Wynne, Ian Robert; Loxdale, Hugh D.; Brookes, Cliff P.

    2003-01-01

    allozymes, conservation genetics, Epirrita dilutata, Epirrita christyi, molecular markers, habitat fragmentation, population genetic structure......allozymes, conservation genetics, Epirrita dilutata, Epirrita christyi, molecular markers, habitat fragmentation, population genetic structure...

  3. Recombinant human antibody fragment against tetanus toxoid produced by phage display

    Science.gov (United States)

    Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

    2014-01-01

    Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

  4. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus.

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S; Jia, Letong; Lee, Peter P; Fouts, Timothy R; Parks, Thomas P; Lagenaur, Laurel A

    Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1 BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission.

  5.  Variable fragments of heavy chain antibodies (VHHs: a new magic bullet molecule of medicine?

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek

    2012-06-01

    Full Text Available  Serum of animals belonging to the Camelidae family (camels and llamas contains fully active antibodies that are naturally devoid of light chains. Variable domains derived from heavy chain antibodies (hcAb called VHHs or nanobodies™ can bind antigens as effectively as full-length antibodies and are easy to clone and express. Because of their potential, VHHs are being intensively studied as potential therapeutic, diagnostic and imaging tools. The paper reviews the molecular background of heavy chain antibodies and describes methods of obtaining recombinant fragments of heavy chain antibodies as well as their therapeutic, diagnostic and other applications.

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  7. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV

    Directory of Open Access Journals (Sweden)

    Hust Michael

    2008-09-01

    Full Text Available Abstract Background Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV and Eastern equine encephalitis virus (EEEV antigenic complex, nor did they react with Chikungunya virus (CHIKV, if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

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

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

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

  9. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    Science.gov (United States)

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

  10. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  11. Pharmacokinetics and biodistribution of genetically-engineered antibodies

    International Nuclear Information System (INIS)

    Colcher, D.; Pavlinkova, G.; Beresford, G.; Booth, B.J.M.; Choudhury, A.; Batra, S.K.; Omaha, Univ. of Nebraska Medical Center, NE

    1998-01-01

    Genetic manipulations of the immunoglobulin molecules are effective means of altering stability, functional affinity, pharmacokinetics, and biodistribution of the antibodies required for the generation of the 'magic bullet'

  12. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

    Science.gov (United States)

    Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

    2012-01-01

    Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

  13. Chemical design of radiolabeled antibody fragments for low renal radioactivity levels.

    Science.gov (United States)

    Arano, Y; Fujioka, Y; Akizawa, H; Ono, M; Uehara, T; Wakisaka, K; Nakayama, M; Sakahara, H; Konishi, J; Saji, H

    1999-01-01

    The renal uptake of radiolabeled antibody fragments presents a problem in targeted imaging and therapy. We hypothesized that the renal radioactivity levels of radiolabeled antibody fragments could be reduced if radiolabeled compounds of urinary excretion were released from glomerularly filtered antibody fragments before they were incorporated into renal cells by the action of brush border enzymes, present on the lumen of renal tubules. 3'-[131I]Iodohippuryl N(epsilon)-maleoyl-L-lysine ([131I]HML) was conjugated with a thiolated Fab fragment because the glycyl-lysine sequence in HML is a substrate for a brush border enzyme and metaiodohippuric acid is released by cleavage of the linkage. Fab fragments were also radiolabeled by direct radioiodination (125I-Fab) or by conjugation with meta-[125I]-iodohippuric acid via an amide bond [N-(5-maleimidopentyl) 3'-iodohippuric acid amide ([125I]MPH-Fab)] or an ester bond [maleimidoethy 3'-iodohippurate ([125I]MIH-Fab)] by procedures similar to those used for [131I]HML-Fab. In biodistribution experiments in mice, [131I]HML-Fab demonstrated markedly low renal radioactivity levels with kidney:blood ratios of radioactivity of 1 from 10 min to 1 h due to rapid release of meta-[131I]iodohippuric acid. [125]MIH-Fab and 1251-Fab reached their peak ratios of 3.8 and 7.3 at 1 h, respectively, and [125I]MPH-Fab showed the maximum ratio of 16.8 at 6 h. In subcellular distribution studies, both [125I]MIH-Fab and 125I-Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection, whereas the majority of the radioactivity was detected only in the membrane fraction after administration of [131I]HML-Fab at both time points. In nude mice, [131I]HML-Fab showed one-quarter of the renal radioactivity of simultaneously administered 125I-Fab without impairing the target radioactivity levels 3 h after injection. These findings indicated that HML is a useful reagent for targeted

  14. Computational identification of antigen-binding antibody fragments.

    Science.gov (United States)

    Burkovitz, Anat; Leiderman, Olga; Sela-Culang, Inbal; Byk, Gerardo; Ofran, Yanay

    2013-03-01

    Determining which parts of the Ab are essential for Ag recognition and binding is crucial for understanding B cell-mediated immunity. Identification of fragments of Abs that maintain specificity to the Ag will also allow for the development of improved Ab-based therapy and diagnostics. In this article, we show that structural analysis of Ab-Ag complexes reveals which fragments of the Ab may bind the Ag on their own. In particular, it is possible to predict whether a given CDR is likely to bind the Ag as a peptide by analyzing the energetic contribution of each CDR to Ag binding and by assessing to what extent the interaction between that CDR and the Ag depends on other CDRs. To demonstrate this, we analyzed five Ab-Ag complexes and predicted for each of them which of the CDRs may bind the Ag on its own as a peptide. We then show that these predictions are in agreement with our experimental analysis and with previously published experimental results. These findings promote our understanding of the modular nature of Ab-Ag interactions and lay the foundation for the rational design of active CDR-derived peptides.

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

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga

    2014-08-01

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

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

    Science.gov (United States)

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

    2004-09-01

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

  17. A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

    NARCIS (Netherlands)

    Ailor, E; Pathmanathan, J; Jongbloed, JDH; Betenbaugh, MJ

    1999-01-01

    The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the

  18. Optimization of the crystallizability of a single-chain antibody fragment

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Král, Vlastimil; Fábry, Milan; Sedláček, Juraj; Veverka, Václav; Řezáčová, Pavlína

    2014-01-01

    Roč. 70, č. 12 (2014), s. 1701-1706 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : single-chain antibody fragment * Thermofluor assay * differential scanning fluorimetry * crystallizability optimization * oligomerization * crystallization Subject RIV: CE - Biochemistry Impact factor: 0.527, year: 2014

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

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

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

  20. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  1. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  2. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    International Nuclear Information System (INIS)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-01-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection

  3. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    International Nuclear Information System (INIS)

    Page, R.L.; Garg, P.K.; Gard, S.

    1994-01-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the 18 F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4'-( 18 F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T 1/2β = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of 18 F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10 -3 % injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of 18 F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs

  4. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Page, R.L.; Garg, P.K.; Gard, S. [North Carolina State Univ., Raleigh, NC (United States)]|[Duke Univ. Medical Center, Durham, NC (United States)]|[North Carolina and Norke Radium Hospital, Oslo (Norway)] [and others

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  5. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    Science.gov (United States)

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  6. Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155

    Directory of Open Access Journals (Sweden)

    J. Mala

    2017-06-01

    Full Text Available Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155 were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1 was confirmed by sequencing analysis. The complementarity determining regions (CDR I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

  7. Amplified restriction fragment length polymorphism in parasite genetics.

    Science.gov (United States)

    Masiga, D K; Tait, A; Turner, C M

    2000-08-01

    The amplified restriction fragment length polymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.

  8. Tumour targeting with monovalent fragments of anti-neuroblastoma antibody chCE7

    International Nuclear Information System (INIS)

    Carrel, F.; Novak-Hofer, I.; Ruch, C.; Zimmermann, K.; Amstutz, H.

    1997-01-01

    The in vitro and in vivo behaviour of the monovalent single chain (scFv) and Fab-fragments derived from anti-neuroblastoma antibody chCE7 is reported. When comparing tumour uptake and -retention of radioactivity of 67 Cu-labelled monovalent chCE7 with divalent chCE7 F(ab') 2 the advantage of the radiocopper label over the radioiodine label was more pronounced with the divalent (internalising) F(ab') 2 fragments. (author) 1 fig., 1 ref

  9. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.

    Science.gov (United States)

    Dada, Oluwatosin O; Rao, Romesh; Jones, Natalie; Jaya, Nomalie; Salas-Solano, Oscar

    2017-10-25

    Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Genetic effects of habitat fragmentation and population isolation on Etheostoma raneyi (Percidae)

    Science.gov (United States)

    Ken A. Sterling; David H. Reed; Brice P. Noonan; Melvin L. Warren

    2012-01-01

    The use of genetic methods to quantify the effects of anthropogenic habitat fragmentation on population structure has become increasingly common. However, in today’s highly fragmented habitats, researchers have sometimes concluded that populations are currently genetically isolated due to habitat fragmentation without testing the possibility that populations were...

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...... phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage....

  12. Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging

    Science.gov (United States)

    2016-10-01

    minibodies and cys-diabodies) can be labeled with radioisotopes for non-invasive PET imaging for use at multiple points in the prostate cancer treatment...1Crump Institute for Molecular Imaging , Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA...AWARD NUMBER: W81XWH-15-1-0725 TITLE: Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging PRINCIPAL

  13. Radioimmunoimaging using F(ab')2 fragment of monoclonal antibodies against human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Koizumi, Mitsuru; Ohta, Hitoya; Torizuka, Kanji; Okada, Kenichiro; Yoshida, Osamu; Nishi, Shinzo.

    1985-01-01

    Using monoclonal antibodies against human α-fetoprotein (AFP), radioiodinated F(ab') 2 fragments were compared with whole IgG as a radiotracer for radioimmunoimaging of cancer. F(ab') 2 fragments were obtained by pepsin digestion of whole IgG (IgGl). IgG and F(ab') 2 were labeled with 125 I or 131 I by the chloramine-T method with almost full retention of antibody activity. F(ab') 2 fragments were cleared more rapidly from the circulation in normal mice with a half life of 6.3 hours than whole IgG with a half life of 5.5 days. Radioactivity of F(ab') 2 in various organs also decreased faster than IgG. In nude mice transplanted with AFP-producing human testicular tumor, F(ab') 2 fragments demonstrated superior scintigrams to whole IgG at 2 days after the injection, because of the fast disappearance of background radioactivity. Although absolute accumulation of 131 I labeled F(ab') 2 in the tumor was less than that of 131 I labeled IgG, tumor to other organ ratios were much higher with F(ab') 2 than those of IgG. The tumor to blood ratio of 131 I labeled F(ab') 2 was 1.04 at day 2, whereas tumor to blood ratio of 131 I labeled IgG was 0.55 at day 2 and 0.92 at day 4, respectively. These results indicated that for the radiolabeling of monoclonal antibodies, F(ab') 2 fragments would be superior to whole IgG in the radioimmunoimaging of cancer. (author)

  14. Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

    International Nuclear Information System (INIS)

    Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

    1990-01-01

    Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

  15. Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S.J. (Oak Ridge National Lab., TN); Chen, J.P.; Lankford, P.K.; Foote, L.J.

    1981-01-01

    Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely wth Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 x 10/sup 9/ M/sup -1/ while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 x 10/sup 7/ and 4.7 x 10/sup 6/ M/sup -1/, respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.

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

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee

    2017-10-01

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

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

    Science.gov (United States)

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

    2017-10-27

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

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

    Science.gov (United States)

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

    2016-02-24

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

  19. Increased streptavidin uptake in tumors pretargeted with biotinylated antibody using a conjugate of streptavidin-Fab fragment

    International Nuclear Information System (INIS)

    Yao Zhengsheng; Zhang Meili; Sakahara, Harumi; Saga, Tsuneo; Kobayashi, Hisataka; Nakamoto, Yuji; Toyama, Sakuji; Konishi, Junji

    1998-01-01

    Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody

  20. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    Science.gov (United States)

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  1. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su

    2015-12-01

    Full Text Available Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM. Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF of the residues, as well as the MSF in the internal distance (MSFID between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

  2. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

    Science.gov (United States)

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio

    2013-01-01

    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  3. Evaluation of tumor targeting with radiolabeled F(ab2 fragment of a humanized monoclonal antibody

    Directory of Open Access Journals (Sweden)

    "Babaei MH

    2002-08-01

    Full Text Available Humanized monoclonal antibody U36 and its F(ab'2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F(ab'2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F(ab'2 showed highly specific localization in tumor tissue. The mean tumor uptake (n=3 is expressed as the percentage of the injected dose per gram of tumor tissue (%ID/g. %ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas %ID/g of F(ab'2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios (T/B at day 1 were 0.86 for IgG and 1.32 for F(ab'2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F(ab'2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting.

  4. Short-term genetic consequences of habitat loss and fragmentation for the neotropical palm Oenocarpus bataua.

    Science.gov (United States)

    Browne, L; Ottewell, K; Karubian, J

    2015-11-01

    Habitat loss and fragmentation may impact animal-mediated dispersal of seed and pollen, and a key question is how the genetic attributes of plant populations respond to these changes. Theory predicts that genetic diversity may be less sensitive to such disruptions in the short term, whereas inbreeding and genetic structure may respond more strongly. However, results from studies to date vary in relation to species, context and the parameter being assessed, triggering calls for more empirical studies, especially from the tropics, where plant-animal dispersal mutualisms are both disproportionately common and at risk. We compared the genetic characteristics of adults and recruits in a long-lived palm Oenocarpus bataua in a recently fragmented landscape (fragments and one nearby continuous forest. Our goal was to assess short-term consequences of fragmentation, with a focus on how well empirical data from this system follow theoretical expectations. Mostly congruent with predictions, we found stronger genetic differentiation and fine-scale spatial genetic structure among recruits in fragments compared with recruits in continuous forest, but we did not record differences in genetic diversity or inbreeding, nor did we record any differences between adults in fragments and adults in continuous forest. Our findings suggest that genetic characteristics of populations vary in their sensitivity to change in response to habitat loss and fragmentation, and that fine-scale spatial genetic structure may be a particularly useful indicator of genetic change in recently fragmented landscapes.

  5. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    Science.gov (United States)

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  7. Functional improvement of antibody fragments using a novel phage coat protein III fusion system

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Larsen, Martin; Pedersen, Jesper Søndergaard

    2002-01-01

    Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance...... of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria......(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity....

  8. Multifunctional PSCA antibody fragments for PET and optical prostate cancer imaging

    Science.gov (United States)

    2017-10-01

    radiolabeled antibody fragments specific for PSCA can be used for immunoPET imaging with high target-to-background at short imaging times post -injection2...1.3%ID/g) and 22Rv1 control tumor (1.2 ± 0.6%ID/g) uptake quantified by ex vivo biodistribution. The 124I-A11 cMb-Cy5.5 biodistribution was similar to...the singly labeled 124I-A11 cMb, resulting in positive-to-negative tumor ratios of 13:1 and 8:1, respectively. Fluorescent imaging post -mortem with

  9. Antibodies and genetically engineered related molecules: production and purification.

    Science.gov (United States)

    Roque, A Cecília A; Lowe, Christopher R; Taipa, M Angela

    2004-01-01

    Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification.

  10. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  11. Genetic consequences of forest fragmentation for a highly specialized arboreal mammal--the edible dormouse.

    Directory of Open Access Journals (Sweden)

    Joanna Fietz

    Full Text Available Habitat loss and fragmentation represent the most serious extinction threats for many species and have been demonstrated to be especially detrimental for mammals. Particularly, highly specialized species with low dispersal abilities will encounter a high risk of extinction in fragmented landscapes. Here we studied the edible dormouse (Glis glis, a small arboreal mammal that is distributed throughout Central Europe, where forests are mostly fragmented at different spatial scales. The aim of this study was to investigate the effect of habitat fragmentation on genetic population structures using the example of edible dormouse populations inhabiting forest fragments in south western Germany. We genotyped 380 adult individuals captured between 2001 and 2009 in four different forest fragments and one large continuous forest using 14 species-specific microsatellites. We hypothesised, that populations in small forest patches have a lower genetic diversity and are more isolated compared to populations living in continuous forests. In accordance with our expectations we found that dormice inhabiting forest fragments were isolated from each other. Furthermore, their genetic population structure was more unstable over the study period than in the large continuous forest. Even though we could not detect lower genetic variability within individuals inhabiting forest fragments, strong genetic isolation and an overall high risk to mate with close relatives might be precursors to a reduced genetic variability and the onset of inbreeding depression. Results of this study highlight that connectivity among habitat fragments can already be strongly hampered before genetic erosion within small and isolated populations becomes evident.

  12. Localization of tumors in vivo by scintigraphic identification of Clostridium butyricum using 131I-labelled antibodies and F(ab')2-antibody fragments

    International Nuclear Information System (INIS)

    Vogt, R.; Mehnert, W.H.; Schmidt, H.E.; Altenbrunn, H.J.; Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1979-01-01

    Tumor-bearing mice injected with clostridial spores show enrichment and germination of the spores within the tumor. 131 I-labelled anti-Clostridium-antibodies and anti-Clostridium-F(ab') 2 -fragments were used for a possible localization of tumors in vivo by scintiscanning. The application of the antibody revealed increased radioactivity in the tumors of mice pretreated with spores as well as in animals without pretreatment. In using F(ab') 2 -fragments instead of total antibody neither the apparently unspecific increase of radioactivity in not pretreated mice nor the specific fixation of labelled F(ab') 2 -fragments to clostridial rods in the tumors of pretreated animals could be demonstrated. The results are discussed with respect to further investigation

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

    Science.gov (United States)

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

    2015-12-24

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

  14. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    Science.gov (United States)

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. Copyright © 2015. Published by Elsevier Ltd.

  16. Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

    Directory of Open Access Journals (Sweden)

    Amram Torgeman

    2017-05-01

    Full Text Available The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs, which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50 of botulinum toxin, whereas the F(ab′2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

  17. ECT with /sup 123/I-labeled fragments of anti-CEA monoclonal antibodies in colo-rectal cancer

    International Nuclear Information System (INIS)

    Bischof-Delaloye, A.; Delaloye, B.

    1986-01-01

    The recent progress of tumor localization with labelled antibodies can be attributed to three techniques: 1) use of I-123 as a label; 2) fragmentation of antibodies; 3) tomographic recording and evaluation of patient radiation data. Under these conditions the method yields good sensitivity and specifity indexes (15/16 for primary tumors and local recurrences, 7/10 for metastasis). A strictly prospective study, however, remains mandatory in order to assess the clinical value of this method

  18. Packing motifs as predictors of the propensity of antibody fragments to crystallize

    Science.gov (United States)

    Edmundson, Allen B.; DeWitt, Christina R.; Goldsteen, Benjamin Z.; Ramsland, Paul A.

    1999-01-01

    A recurring theme in the crystallization of antibody fragments in our laboratory has been a packing pattern involving formation of intermolecular, antiparallel β-pleated sheets across two-fold axes. The most common motif is the antiparallel stacking of constant (C) domains of light (L) chain dimers or Fab molecules. Here, cross-molecule six-stranded sheets are produced by hydrogen-bonding interactions of three-residue polypeptide segments (triads), in the i, i+2 and i+4 positions of the final strands (designated 3-3) of the three-chain layers from two adjacent molecules. In the variable (V) domains the triads are supplied by the first strands (4-1) of the four-chain layers and the resulting cross-molecule sheets contain eight strands. The latter type of packing is more likely to be seen in crystals of Fv fragments (V domains only) than in those of L chain dimers or Fabs. Amongst the triads from either the V or C domains, there are on average four sets of backbone carbonyl and amide groups within hydrogen bonding distance (chain dimers, Fab and Fvs are likely to crystallize in these packing patterns.

  19. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage λ immunoexpression library

    International Nuclear Information System (INIS)

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A.; Hay, B.N.; Shopes, B.

    1990-01-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and κ light-chain variable and constant region domains, were inserted into modified bacteriophase λ expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen

  20. Meta-analysis of susceptibility of woody plants to loss of genetic diversity through habitat fragmentation.

    Science.gov (United States)

    Vranckx, Guy; Jacquemyn, Hans; Muys, Bart; Honnay, Olivier

    2012-04-01

    Shrubs and trees are assumed less likely to lose genetic variation in response to habitat fragmentation because they have certain life-history characteristics such as long lifespans and extensive pollen flow. To test this assumption, we conducted a meta-analysis with data on 97 woody plant species derived from 98 studies of habitat fragmentation. We measured the weighted response of four different measures of population-level genetic diversity to habitat fragmentation with Hedge's d and Spearman rank correlation. We tested whether the genetic response to habitat fragmentation was mediated by life-history traits (longevity, pollination mode, and seed dispersal vector) and study characteristics (genetic marker and plant material used). For both tests of effect size habitat fragmentation was associated with a substantial decrease in expected heterozygosity, number of alleles, and percentage of polymorphic loci, whereas the population inbreeding coefficient was not associated with these measures. The largest proportion of variation among effect sizes was explained by pollination mechanism and by the age of the tissue (progeny or adult) that was genotyped. Our primary finding was that wind-pollinated trees and shrubs appeared to be as likely to lose genetic variation as insect-pollinated species, indicating that severe habitat fragmentation may lead to pollen limitation and limited gene flow. In comparison with results of previous meta-analyses on mainly herbaceous species, we found trees and shrubs were as likely to have negative genetic responses to habitat fragmentation as herbaceous species. We also found that the genetic variation in offspring was generally less than that of adult trees, which is evidence of a genetic extinction debt and probably reflects the genetic diversity of the historical, less-fragmented landscape. ©2011 Society for Conservation Biology.

  1. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-01-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80μCi of I-131-F(ab')/sub 2/ with a specific activity of 1.8μCi/μg. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice

  2. Inbreeding and gene flow : the population genetics of plant species in fragmented landscapes

    NARCIS (Netherlands)

    Mix, Carolin

    2006-01-01

    Habitat fragmentation has been recognized as one of the major threats to plant population persistence. Fragmented small and isolated populations are expected to be seriously affected by inbreeding and genetic drift. Gene flow through seed and pollen dispersal may counterbalance the negative

  3. Habitat Fragmentation Differentially Affects Genetic Variation, Phenotypic Plasticity and Survival in Populations of a Gypsum Endemic

    Directory of Open Access Journals (Sweden)

    Silvia Matesanz

    2017-05-01

    Full Text Available Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of Centaurea hyssopifolia, a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population’s similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations’ survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness

  4. Habitat Fragmentation Differentially Affects Genetic Variation, Phenotypic Plasticity and Survival in Populations of a Gypsum Endemic.

    Science.gov (United States)

    Matesanz, Silvia; Rubio Teso, María Luisa; García-Fernández, Alfredo; Escudero, Adrián

    2017-01-01

    Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of Centaurea hyssopifolia , a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population's similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations' survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness, our study highlights

  5. In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.; Rochester Univ., NY

    1986-01-01

    Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab') 2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radio-immunotherapy we think that Fab fragments are not useful because of their too short half-life the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab') 2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab') 2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab') 2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab') 2 fragments. (orig.) [de

  6. Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase.

    Science.gov (United States)

    Takazawa, Takeshi; Kamiya, Noriho; Ueda, Hiroshi; Nagamune, Teruyuki

    2004-05-20

    Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins. Copyright 2004 Wiley Periodicals, Inc.

  7. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Mapping of Monoclonal Antibody Binding Sites on CNBr Fragments of the S- Layer Protein Antigens of Rickettsia Typhi and Rickettsia Prowazekii

    Science.gov (United States)

    1992-01-01

    Security Clasification ) Mapping of monoclonal antibody binding sites on CNBr fragments o; the S-layer protein antigens of Rickettsia Typhi and...homology was found in all the viral polypeptides have long fatty acids attached to fragments which react with type I antibody (Fig. 4). A their N-termini

  9. Effects of landscape fragmentation on genetic diversity of Stipa ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... Stipa krylovii Roshev (Stipa L.) is one of the most important grass species for rangeland ecology and animal husbandry. But some populations of this species are under threat due to landscape fragmentation and habitat isolation resulting from the reclamation and cultivation in ecotone. To determine if and ...

  10. Genetic variation within and among fragmented populations of ...

    African Journals Online (AJOL)

    Our results showed contrary findings from the hypothesis for fragmented population. The findings showed higher diversity within population and lower diversity among population like larger unfragmented populations even though the population size was less than 100 in all populations. Percentage of polymorphic bands ...

  11. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille; van Diest, Eline; Schmidt, Florian I.; Schwartz, Thomas U.; Ploegh, Hidde L. (Whitehead); (MIT)

    2016-12-13

    Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

    IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and

  12. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment.

    Science.gov (United States)

    Hanke, Leo; Knockenhauer, Kevin E; Brewer, R Camille; van Diest, Eline; Schmidt, Florian I; Schwartz, Thomas U; Ploegh, Hidde L

    2016-12-13

    Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. Influenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and provide a well-characterized tool to

  13. Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

    Science.gov (United States)

    Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

    2015-06-01

    Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

  14. Species-genetic diversity correlations in habitat fragmentation can be biased by small sample sizes.

    Science.gov (United States)

    Nazareno, Alison G; Jump, Alistair S

    2012-06-01

    Predicted parallel impacts of habitat fragmentation on genes and species lie at the core of conservation biology, yet tests of this rule are rare. In a recent article in Ecology Letters, Struebig et al. (2011) report that declining genetic diversity accompanies declining species diversity in tropical forest fragments. However, this study estimates diversity in many populations through extrapolation from very small sample sizes. Using the data of this recent work, we show that results estimated from the smallest sample sizes drive the species-genetic diversity correlation (SGDC), owing to a false-positive association between habitat fragmentation and loss of genetic diversity. Small sample sizes are a persistent problem in habitat fragmentation studies, the results of which often do not fit simple theoretical models. It is essential, therefore, that data assessing the proposed SGDC are sufficient in order that conclusions be robust.

  15. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  16. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    Surinder Batra

    2006-01-01

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  17. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  18. Genetic population differentiation and connectivity among fragmented Moor frog (Rana arvalis) populations in The Netherlands

    NARCIS (Netherlands)

    Arens, P.F.P.; Sluis, van der T.; Westende, van 't W.P.C.; Vosman, B.; Vos, C.C.; Smulders, M.J.M.

    2007-01-01

    We studied the effects of landscape structure, habitat loss and fragmentation on genetic differentiation of Moor frog populations in two landscapes in The Netherlands (Drenthe and Noord-Brabant). Microsatellite data of eight loci showed small to moderate genetic differentiation among populations in

  19. Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth.

    Science.gov (United States)

    Boulding, N; Yim, S S S; Keshavarz-Moore, E; Ayazi Shamlou, P; Berry, M

    2002-08-20

    Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates. Copyright 2002 Wiley Periodicals, Inc.

  20. Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

    Directory of Open Access Journals (Sweden)

    Ch’ng Wei-Choong

    2012-08-01

    Full Text Available Abstract Enterovirus 71 (EV71 causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100 protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.

  1. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    Science.gov (United States)

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N SARS-CoV protein using a phage display approach

    Directory of Open Access Journals (Sweden)

    Grasso Felicia

    2005-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

  3. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach

    Science.gov (United States)

    Flego, Michela; Di Bonito, Paola; Ascione, Alessandro; Zamboni, Silvia; Carattoli, Alessandra; Grasso, Felicia; Cassone, Antonio; Cianfriglia, Maurizio

    2005-01-01

    Background Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells. PMID:16171519

  4. A 10-RESIDUE FRAGMENT OF AN ANTIBODY (MINI-ANTIBODY) DIRECTED AGAINST LYSOZYME AS LIGAND IN IMMUNOAFFINITY CHROMATOGRAPHY

    NARCIS (Netherlands)

    WELLING, GW; VANGORKUM, J; DAMHOF, RA; DRIJFHOUT, JW; BLOEMHOFF, W; WELLINGWESTER, S

    1991-01-01

    The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule

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

    Science.gov (United States)

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

    2018-04-01

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

  6. Effects of landscape fragmentation on genetic diversity of Stipa ...

    African Journals Online (AJOL)

    To determine if and how these disturbances have impacted on genetic structure of S. krylovii populations, an inter-simple sequence repeat (ISSR) markers was used to characterize them for the first time in China. S. krylovii populations from 10 isolated patches were compared with population from unbroken natural ...

  7. Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

    Science.gov (United States)

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function. PMID:18783590

  8. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina

    2008-09-01

    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  9. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc., La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  10. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  11. Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments.

    Science.gov (United States)

    Fagète, Séverine; Botas-Perez, Ledicia; Rossito-Borlat, Irène; Adea, Kenneth; Gueneau, Franck; Ravn, Ulla; Rousseau, François; Kosco-Vilbois, Marie; Fischer, Nicolas; Hartley, Oliver

    2017-09-01

    Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 105) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 105) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    OpenAIRE

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

    2017-01-01

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

  13. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

    Directory of Open Access Journals (Sweden)

    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  14. Isolation of populations of antipeptide antibodies directed against different epitopes of the same fragment.

    Science.gov (United States)

    Chersi, A; Greger, C; Houghten, R A

    1985-01-01

    Rabbit antibodies against small peptides may be composed by subpopulations recognizing different epitopes made likely by few amino acids. This explains the frequent crossreactivity of antipeptide antibodies with unrelated peptides. A suitable use of immunoadsorbents is suggested to obtain truly specific antibodies able to react with restricted amino acid sequences.

  15. Serum and urine analysis of the aminoterminal procollagen peptide type III by radioimmunoassay with antibody Fab fragments.

    Science.gov (United States)

    Rohde, H; Langer, I; Krieg, T; Timpl, R

    1983-09-01

    A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.

  16. Lack of Population Genetic Structuring in Ocelots (Leopardus pardalis in a Fragmented Landscape

    Directory of Open Access Journals (Sweden)

    Marina G. Figueiredo

    2015-07-01

    Full Text Available Habitat fragmentation can promote patches of small and isolated populations, gene flow disruption between those populations, and reduction of local and total genetic variation. As a consequence, these small populations may go extinct in the long-term. The ocelot (Leopardus pardalis, originally distributed from Texas to southern Brazil and northern Argentina, has been impacted by habitat fragmentation throughout much of its range. To test whether habitat fragmentation has already induced genetic differentiation in an area where this process has been documented for a larger felid (jaguars, we analyzed molecular variation in ocelots inhabiting two Atlantic Forest fragments, Morro do Diabo (MD and Iguaçu Region (IR. Analyses using nine microsatellites revealed mean observed and expected heterozygosity of 0.68 and 0.70, respectively. The MD sampled population showed evidence of a genetic bottleneck under two mutational models (TPM = 0.03711 and SMM = 0.04883. Estimates of genetic structure (FST = 0.027; best fit of k = 1 with STRUCTURE revealed no meaningful differentiation between these populations. Thus, our results indicate that the ocelot populations sampled in these fragments are still not significantly different genetically, a pattern that strongly contrasts with that previously observed in jaguars for the same comparisons. This observation is likely due to a combination of two factors: (i larger effective population size of ocelots (relative to jaguars in each fragment, implying a slower effect of drift-induced differentiation; and (ii potentially some remaining permeability of the anthropogenic matrix for ocelots, as opposed to the observed lack of permeability for jaguars. The persistence of ocelot gene flow between these areas must be prioritized in long-term conservation planning on behalf of these felids.

  17. Drifting to oblivion? Rapid genetic differentiation in an endangered lizard following habitat fragmentation and drought

    Science.gov (United States)

    Vandergast, Amy; Wood, Dustin A.; Thompson, Andrew R.; Fisher, Mark; Barrows, Cameron W.; Grant, Tyler J.

    2016-01-01

    Aim The frequency and severity of habitat alterations and disturbance are predicted to increase in upcoming decades, and understanding how disturbance affects population integrity is paramount for adaptive management. Although rarely is population genetic sampling conducted at multiple time points, pre- and post-disturbance comparisons may provide one of the clearest methods to measure these impacts. We examined how genetic properties of the federally threatened Coachella Valley fringe-toed lizard (Uma inornata) responded to severe drought and habitat fragmentation across its range. Location Coachella Valley, California, USA. Methods We used 11 microsatellites to examine population genetic structure and diversity in 1996 and 2008, before and after a historic drought. We used Bayesian assignment methods and F-statistics to estimate genetic structure. We compared allelic richness across years to measure loss of genetic diversity and employed approximate Bayesian computing methods and heterozygote excess tests to explore the recent demographic history of populations. Finally, we compared effective population size across years and to abundance estimates to determine whether diversity remained low despite post-drought recovery. Results Genetic structure increased between sampling periods, likely as a result of population declines during the historic drought of the late 1990s–early 2000s, and habitat loss and fragmentation that precluded post-drought genetic rescue. Simulations supported recent demographic declines in 3 of 4 main preserves, and in one preserve, we detected significant loss of allelic richness. Effective population sizes were generally low across the range, with estimates ≤100 in most sites. Main conclusions Fragmentation and drought appear to have acted synergistically to induce genetic change over a short time frame. Progressive deterioration of connectivity, low Ne and measurable loss of genetic diversity suggest that conservation efforts have

  18. Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product.

    Science.gov (United States)

    Havlik, David; Brandt, Ulrike; Bohle, Kathrin; Fleißner, André

    2017-07-25

    Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg1nr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the

  19. Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

    DEFF Research Database (Denmark)

    On, Stephen L.W.; Atabay, H.I.; Amisu, K.O.

    2004-01-01

    Aims: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...

  20. Differential diagnosis of genetic disease by DNA restriction fragment length polymorphisms

    NARCIS (Netherlands)

    Bolhuis, P. A.; Defesche, J. C.; van der Helm, H. J.

    1987-01-01

    DNA restriction fragment length polymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers

  1. Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

    Science.gov (United States)

    Säll, Anna; Sjöholm, Kristoffer; Waldemarson, Sofia; Happonen, Lotta; Karlsson, Christofer; Persson, Helena; Malmström, Johan

    2015-11-06

    Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

  2. Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

    International Nuclear Information System (INIS)

    Endo, K.; Kamma, H.; Ogata, T.

    1988-01-01

    Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells

  3. Delayed genetic effects of habitat fragmentation on the ecologically specialized Florida sand skink (Plestiodon reynoldsi)

    Science.gov (United States)

    Richmond, Jonathan Q.; Reid, Duncan T.; Ashton, Kyle G.; Zamudio, Kelly R.

    2009-01-01

    Populations rarely show immediate genetic responses to habitat fragmentation, even in taxa that possess suites of traits known to increase their vulnerability to extinction. Thus conservation geneticists must consider the time scale over which contemporary evolutionary processes operate to accurately portray the effects of habitat isolation. Here, we examine the genetic impacts of fragmentation on the Florida sand skink Plestiodon reynoldsi, a sand swimming lizard that is highly adapted to the upland scrub habitat of central Florida. We studied fragments located on the southern Lake Wales Ridge, where human activity in the latter half of the 20th century has modified the natural patchiness of the landscape. Based on a relaxed molecular clock method, we estimate that sand skinks have persisted in this region for approximately 1.5 million years and that the time frame of human disturbance is equivalent to fewer than 30 skink generations. Using genotypes from eight microsatellite loci, we screened for molecular signatures of this disturbance by assessing congruence between population structure, as inferred from spatially-informed Bayesian assignment tests, and the current geography of scrub fragments. We also tested for potential intrapopulation genetic effects of inbreeding in isolated populations by comparing the average pairwise relatedness of individuals within fragments of different areas and isolation. Our results indicate that although some patches show a higher degree of relatedness than expected under random mating, the genetic effects of recent isolation are not evident in this part of the species’ range. We argue that this result is an artefact of a time-lag in the response to disturbance, and that species-typical demographic features may explain the genetic inertia observed in these populations.

  4. F(ab'2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

    Directory of Open Access Journals (Sweden)

    LORENA AGUILAR

    2005-01-01

    Full Text Available Trypanosoma cruzi calreticulin (TcCRT, described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host / parasite equilibrium. In an in vitro correlate of this situation, we show that the C1q / TcCRT interaction is inhibited by F(ab'2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion C1q, and this C1q is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate C1q could be inhibited by F(ab'2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process

  5. Population genetics of the olive-winged bulbul (Pycnonotus plumosus) in a tropical urban-fragmented landscape.

    Science.gov (United States)

    Tang, Grace S Y; Sadanandan, Keren R; Rheindt, Frank E

    2016-01-01

    With increasing urbanization, urban-fragmented landscapes are becoming more and more prevalent worldwide. Such fragmentation may lead to small, isolated populations that face great threats from genetic factors that affect even avian species with high dispersal propensities. Yet few studies have investigated the population genetics of species living within urban-fragmented landscapes in the Old World tropics, in spite of the high levels of deforestation and fragmentation within this region. We investigated the evolutionary history and population genetics of the olive-winged bulbul (Pycnonotus plumosus) in Singapore, a highly urbanized island which retains landscape.

  6. Genetic variation in an endemic salamander, Salamandra atra, using amplified fragment length polymorphism.

    Science.gov (United States)

    Riberon, Alexandre; Miaud, Claude; Guyetant, R; Taberlet, P

    2004-06-01

    The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.

  7. Genetic connectivity of the moth pollinated tree Glionnetia sericea in a highly fragmented habitat.

    Directory of Open Access Journals (Sweden)

    Aline Finger

    Full Text Available Long-distance gene flow is thought to be one prerequisite for the persistence of plant species in fragmented environments. Human influences have led to severe fragmentation of native habitats in the Seychelles islands, with many species surviving only in small and isolated populations. The endangered Seychelles endemic tree Glionnetia sericea is restricted to altitudes between 450 m and 900 m where the native forest vegetation has been largely lost and replaced with exotic invasives over the last 200 years. This study explores the genetic and ecological consequences of population fragmentation in this species by analysing patterns of genetic diversity in a sample of adults, juveniles and seeds, and by using controlled pollination experiments. Our results show no decrease in genetic diversity and no increase in genetic structuring from adult to juvenile cohorts. Despite significant inbreeding in some populations, there is no evidence of higher inbreeding in juvenile cohorts relative to adults. A Bayesian structure analysis and a tentative paternity analysis indicate extensive historical and contemporary gene flow among remnant populations. Pollination experiments and a paternity analysis show that Glionnetia sericea is self-compatible. Nevertheless, outcrossing is present with 7% of mating events resulting from pollen transfer between populations. Artificial pollination provided no evidence for pollen limitation in isolated populations. The highly mobile and specialized hawkmoth pollinators (Agrius convolvuli and Cenophodes tamsi; Sphingidae appear to promote extensive gene flow, thus mitigating the potential negative ecological and genetic effects of habitat fragmentation in this species. We conclude that contemporary gene flow is sufficient to maintain genetic connectivity in this rare and restricted Seychelles endemic, in contrast to other island endemic tree species with limited contemporary gene flow.

  8. Genetic variation of loci potentially under selection confounds species-genetic diversity correlations in a fragmented habitat.

    Science.gov (United States)

    Bertin, Angeline; Gouin, Nicolas; Baumel, Alex; Gianoli, Ernesto; Serratosa, Juan; Osorio, Rodomiro; Manel, Stephanie

    2017-01-01

    Positive species-genetic diversity correlations (SGDCs) are often thought to result from the parallel influence of neutral processes on genetic and species diversity. Yet, confounding effects of non-neutral mechanisms have not been explored. Here, we investigate the impact of non-neutral genetic diversity on SGDCs in high Andean wetlands. We compare correlations between plant species diversity and genetic diversity (GD) calculated with and without loci potentially under selection (outlier loci). The study system includes 2188 specimens from five species (three common aquatic macroinvertebrate and two dominant plant species) that were genotyped for 396 amplified fragment length polymorphism loci. We also appraise the importance of neutral processes on SGDCs by investigating the influence of habitat fragmentation features. Significant positive SGDCs were detected for all five species (mean SGDC = 0.52 ± 0.05). While only a few outlier loci were detected in each species, they resulted in significant decreases in GD and in SGDCs. This supports the hypothesis that neutral processes drive species-genetic diversity relationships in high Andean wetlands. Unexpectedly, the effects on genetic diversity GD of the habitat fragmentation characteristics in this study increased with the presence of outlier loci in two species. Overall, our results reveal pitfalls in using habitat features to infer processes driving SGDCs and show that a few loci potentially under selection are enough to cause a significant downward bias in SGDC. Investigating confounding effects of outlier loci thus represents a useful approach to evidence the contribution of neutral processes on species-genetic diversity relationships. © 2016 John Wiley & Sons Ltd.

  9. Understanding the genetic effects of recent habitat fragmentation in the context of evolutionary history: Phylogeography and landscape genetics of a southern California endemic Jerusalem cricket (Orthoptera: Stenopelmatidae: Stenopelmatus)

    Science.gov (United States)

    Vandergast, A.G.; Bohonak, A.J.; Weissman, D.B.; Fisher, R.N.

    2007-01-01

    Habitat loss and fragmentation due to urbanization are the most pervasive threats to biodiversity in southern California. Loss of habitat and fragmentation can lower migration rates and genetic connectivity among remaining populations of native species, reducing genetic variability and increasing extinction risk. However, it may be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric fragmentation due to previous natural geological and climatic changes. To address these challenges, we examined the phylogenetic and population genetic structure of a flightless insect endemic to cismontane southern California, Stenopelmatus 'mahogani' (Orthoptera: Stenopelmatidae). Analyses of mitochondrial DNA sequence data suggest that diversification across southern California began during the Pleistocene, with most haplotypes currently restricted to a single population. Patterns of genetic divergence correlate with contemporary urbanization, even after correcting for (geographical information system) GIS-based reconstructions of fragmentation during the Pleistocene. Theoretical simulations confirm that contemporary patterns of genetic structure could be produced by recent urban fragmentation using biologically reasonable assumptions about model parameters. Diversity within populations was positively correlated with current fragment size, but not prehistoric fragment size, suggesting that the effects of increased drift following anthropogenic fragmentation are already being seen. Loss of genetic connectivity and diversity can hinder a population's ability to adapt to ecological perturbations commonly associated with urbanization, such as habitat degradation, climatic changes and introduced species. Consequently, our results underscore the importance of preserving and restoring landscape connectivity for long-term persistence of low vagility native species. Journal compilation ?? 2006 Blackwell Publishing Ltd.

  10. The Power to Detect Recent Fragmentation Events Using Genetic Differentiation Methods

    Science.gov (United States)

    Lloyd, Michael W.; Campbell, Lesley; Neel, Maile C.

    2013-01-01

    Habitat loss and fragmentation are imminent threats to biological diversity worldwide and thus are fundamental issues in conservation biology. Increased isolation alone has been implicated as a driver of negative impacts in populations associated with fragmented landscapes. Genetic monitoring and the use of measures of genetic divergence have been proposed as means to detect changes in landscape connectivity. Our goal was to evaluate the sensitivity of Wright’s Fst, Hedrick’ G’st, Sherwin’s MI, and Jost’s D to recent fragmentation events across a range of population sizes and sampling regimes. We constructed an individual-based model, which used a factorial design to compare effects of varying population size, presence or absence of overlapping generations, and presence or absence of population sub-structuring. Increases in population size, overlapping generations, and population sub-structuring each reduced Fst, G’st, MI, and D. The signal of fragmentation was detected within two generations for all metrics. However, the magnitude of the change in each was small in all cases, and when Ne was >100 individuals it was extremely small. Multi-generational sampling and population estimates are required to differentiate the signal of background divergence from changes in Fst, G’st, MI, and D associated with fragmentation. Finally, the window during which rapid change in Fst, G’st, MI, and D between generations occurs can be small, and if missed would lead to inconclusive results. For these reasons, use of Fst, G’st, MI, or D for detecting and monitoring changes in connectivity is likely to prove difficult in real-world scenarios. We advocate use of genetic monitoring only in conjunction with estimates of actual movement among patches such that one could compare current movement with the genetic signature of past movement to determine there has been a change. PMID:23704965

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

    Directory of Open Access Journals (Sweden)

    Laura Frigotto

    2015-05-01

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

  12. Radioimmunodetection (RID) with OC-125 antibody F(ab)2 fragments in the follow-up of ovarian carcinoma

    International Nuclear Information System (INIS)

    Bockisch, A.; Broeckelmann, J.; Briele, B.; Vogel, J.; Oehr, P.; Osterthun, B.; Rheinsberg, J.; Hotze, A.; Biersack, H.J.; Krebs, D.

    1990-01-01

    Radioimmunodetection (RID) using 131 I-labelled OC-125 antibody F(ab') 2 fragments proved to be sensitive and specific for detecting ovarian carcinoma metastases and recurrences. 40 investigations in patients suffering from advanced ovarian carcinoma or carcinoma of unknown origin but with elevated CA-125 serum levels were investigated. The sensitivity, specificity, and accuracy were found to be 84%, 73%, and 80%, respectively. Analysing the 37 investigations in patients with ovarian carcinomas separately, the numbers were 91%, 71%, and 84%. This imaging modality may therefore be used as an alternative to exploratory surgery. No side-effects were observed clinically. After RID the serum levels of CA-125 were unexpected high in some patients when the measurement was performed in a routine procedure using the same anti-CA-125 antibody in the in vitro test as was used for imaging. (author)

  13. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

    DEFF Research Database (Denmark)

    Encinas, P.; Gomez-Casado, E.; Grandes, Fregeneda

    2011-01-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using...... refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV...

  14. Highly Specific PET Imaging of Prostate Tumors in Mice with an Iodine-124-Labeled Antibody Fragment That Targets Phosphatidylserine

    OpenAIRE

    Stafford, Jason H.; Hao, Guiyang; Best, Anne M.; Sun, Xiankai; Thorpe, Philip E.

    2013-01-01

    Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab')2 was...

  15. Radioimmunolocalization and selective delivery of radiation in a rat model system: comparison of intact and fragmented antibody

    International Nuclear Information System (INIS)

    Walker, K.Z.; Seymour-Munn, K.; Axiak, S.M.; Raison, R.L.; Basten, A.; Towson, J.E.; Bautovitch, G.J.; Morris, J.

    1988-01-01

    Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab') 2 and Fab fragments using a normal rat model system. Two days after injection of 131 I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab') 2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 ± 3.9 for Fab, 7.9 ± 1.8 for F(ab') 2 and 2.0 ± 0.3 for intact K-1-21. The improvement associated with the use of 131 I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab') 2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab') 2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates. (author)

  16. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    Science.gov (United States)

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

  17. Population fragmentation leads to spatial and temporal genetic structure in the endangered Spanish imperial eagle.

    Science.gov (United States)

    Martínez-Cruz, B; Godoy, J A; Negro, J J

    2007-02-01

    The fragmentation of a population may have important consequences for population genetic diversity and structure due to the effects of genetic drift and reduced gene flow. We studied the genetic consequences of the fragmentation of the Spanish imperial eagle (Aquila adalberti) population into small patches through a temporal analysis. Thirty-four museum individuals representing the population predating the fragmentation were analysed for a 345-bp segment of the mitochondrial control region and a set of 10 nuclear microsatellite loci. Data from a previous study on the current population (N = 79) were re-analysed for this subset of 10 microsatellite markers and results compared to those obtained from the historical sample. Three shared mitochondrial haplotypes were found in both populations, although fluctuations in haplotype frequencies and the occurrence of a fourth haplotype in the historical population resulted in lower current levels of haplotype and nucleotide diversity. However, microsatellite markers revealed undiminished levels of nuclear diversity. No evidence for genetic structure was observed for the historical Spanish imperial eagle population, suggesting that the current pattern of structure is the direct consequence of population fragmentation. Temporal fluctuations in mitochondrial and microsatellite allelic frequencies were found between the historical and the current population as well as for each pairwise comparison between historical and current Centro and historical and current Parque Nacional de Doñana nuclei. Our results indicate an ancestral panmictic situation for the species that management policies should aim to restore. A historical analysis like the one taken here provides the baseline upon which the relative role of recent drift in shaping current genetic patterns in endangered species can be evaluated and this knowledge is used to guide conservation actions.

  18. Habitat fragmentation in coastal southern California disrupts genetic connectivity in the cactus wren (Campylorhynchus brunneicapillus).

    Science.gov (United States)

    Barr, Kelly R; Kus, Barbara E; Preston, Kristine L; Howell, Scarlett; Perkins, Emily; Vandergast, Amy G

    2015-05-01

    Achieving long-term persistence of species in urbanized landscapes requires characterizing population genetic structure to understand and manage the effects of anthropogenic disturbance on connectivity. Urbanization over the past century in coastal southern California has caused both precipitous loss of coastal sage scrub habitat and declines in populations of the cactus wren (Campylorhynchus brunneicapillus). Using 22 microsatellite loci, we found that remnant cactus wren aggregations in coastal southern California comprised 20 populations based on strict exact tests for population differentiation, and 12 genetic clusters with hierarchical Bayesian clustering analyses. Genetic structure patterns largely mirrored underlying habitat availability, with cluster and population boundaries coinciding with fragmentation caused primarily by urbanization. Using a habitat model we developed, we detected stronger associations between habitat-based distances and genetic distances than Euclidean geographic distance. Within populations, we detected a positive association between available local habitat and allelic richness and a negative association with relatedness. Isolation-by-distance patterns varied over the study area, which we attribute to temporal differences in anthropogenic landscape development. We also found that genetic bottleneck signals were associated with wildfire frequency. These results indicate that habitat fragmentation and alterations have reduced genetic connectivity and diversity of cactus wren populations in coastal southern California. Management efforts focused on improving connectivity among remaining populations may help to ensure population persistence. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  19. Population genetics at three spatial scales of a rare sponge living in fragmented habitats

    Directory of Open Access Journals (Sweden)

    Uriz Maria J

    2010-01-01

    Full Text Available Abstract Background Rare species have seldom been studied in marine habitats, mainly because it is difficult to formally assess the status of rare species, especially in patchy benthic organisms, for which samplings are often assumed to be incomplete and, thus, inappropriate for establishing the real abundance of the species. However, many marine benthic invertebrates can be considered rare, due to the fragmentation and rarity of suitable habitats. Consequently, studies on the genetic connectivity of rare species in fragmented habitats are basic for assessing their risk of extinction, especially in the context of increased habitat fragmentation by human activities. Sponges are suitable models for studying the intra- and inter-population genetic variation of rare invertebrates, as they produce lecitotrophic larvae and are often found in fragmented habitats. Results We investigated the genetic structure of a Mediterranean sponge, Scopalina lophyropoda (Schmidt, using the allelic size variation of seven specific microsatellite loci. The species can be classified as "rare" because of its strict habitat requirements, the low number of individuals per population, and the relatively small size of its distribution range. It also presents a strong patchy distribution, philopatric larval dispersal, and both sexual and asexual reproduction. Classical genetic-variance-based methods (AMOVA and differentiation statistics revealed that the genetic diversity of S. lophyropoda was structured at the three spatial scales studied: within populations, between populations of a geographic region, and between isolated geographic regions, although some stochastic gene flow might occur among populations within a region. The genetic structure followed an isolation-by-distance pattern according to the Mantel test. However, despite philopatric larval dispersal and fission events in the species, no single population showed inbreeding, and the contribution of clonality to the

  20. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    International Nuclear Information System (INIS)

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai

    2007-01-01

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice

  1. Analysis of genetic diversity identified by amplified fragment length polymorphism marker in hybrid wheat.

    Science.gov (United States)

    Ejaz, M; Qidi, Z; Gaisheng, Z; Na, N; Huiyan, Z; Qunzhu, W

    2015-08-07

    Amplified fragment length polymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment length polymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies.

  2. Degree of landscape fragmentation influences genetic isolation among populations of a gliding mammal.

    Directory of Open Access Journals (Sweden)

    Andrea C Taylor

    Full Text Available Forests and woodlands are under continuing pressure from urban and agricultural development. Tree-dependent mammals that rarely venture to the ground are likely to be highly sensitive to forest fragmentation. The Australian squirrel glider (Petaurus norfolcensis provides an excellent case study to examine genetic (functional connectivity among populations. It has an extensive range that occurs in a wide band along the east coast. However, its forest and woodland habitat has become greatly reduced in area and is severely fragmented within the southern inland part of the species' range, where it is recognised as threatened. Within central and northern coastal regions, habitat is much more intact and we thus hypothesise that genetic connectivity will be greater in this region than in the south. To test this we employed microsatellite analysis in a molecular population biology approach. Most sampling locations in the highly modified south showed signatures of genetic isolation. In contrast, a high level of genetic connectivity was inferred among most sampled populations in the more intact habitat of the coastal region, with samples collected 1400 km apart having similar genetic cluster membership. Nonetheless, some coastal populations associated with urbanisation and agriculture are genetically isolated, suggesting the historic pattern observed in the south is emerging on the coast. Our study demonstrates that massive landscape changes following European settlement have had substantial impacts on levels of connectivity among squirrel glider populations, as predicted on the basis of the species' ecology. This suggests that landscape planning and management in the south should be focused on restoring habitat connectivity where feasible, while along the coast, existing habitat connectivity must be maintained and recent losses restored. Molecular population biology approaches provide a ready means for identifying fragmentation effects on a species at

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

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

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

  4. Immunolymphoscintigraphy and immunoscintigraphy of ovarian and fallopian tube cancer using F(ab')2 fragments of monoclonal antibody OC 125

    International Nuclear Information System (INIS)

    Lehtovirta, P.; Kairemo, K.J.; Liewendahl, K.; Seppaelae, M.

    1990-01-01

    We have used immunolymphoscintigraphy (ILS) alone or in combination with immunoscintigraphy with 131 I-labeled F(ab')2 fragments of monoclonal OC 125 antibodies to improve detection of retroperitoneal lymph node metastases of ovarian and fallopian tube cancer. ILS was carried out with bilateral dorsopedal s.c. injections on nine patients and with bilateral iliopelvic injections into the ischiorectal fossa on two other patients. Radioimaging was performed 2-4 times between 0 and 5 days. An additional dose of labeled antibody fragments was given i.v., and imaging was done 2-3 days later. Conventional immunoscintigraphy without preceding ILS was carried out on another nine patients. Dorsopedal ILS improved detection of pelvic and paraaortic lymph node metastases. Malignant lymph nodes were detectable as early as 3 h after s.c. injection of the tracer. Combined results of ILS and immunoscintigraphy in 16 surgically verified cases indicated a true positive finding in 9 patients, true negative finding in 5, false positive in one, and false negative in 1. Calculated from these figures the sensitivity, specificity, and accuracy of the method were 90, 83, and 88%, respectively. Involved lymph nodes were found more frequently in those patients whose serum CA 125 concentration was elevated demonstrating that an elevated serum CA 125 level does not preclude successful radioimmunodetection

  5. Selection of monoclonal anti-CEA antibody fragments for tumor detection by immunoscintigraphy

    International Nuclear Information System (INIS)

    Mach, J.P.; Buchegger, F.

    1986-01-01

    It is described how individual MAb directed against carcinoembryotic antigen (CEA) is selected which does not crossreact with granulocytes and gives the best tumor localization in the model of nude mice grafted with human colon carcinoma. Using this model, the superiority of F(ab')/sub 2/ and particularly Fab fragments from high affinity MAb for the localization of relatively small tumor nodules is demonstrated. These MAb fragments are also successfully used in an ongoing clinical trial for the detection of primary and metastatic colorectal carcinomas

  6. Population genetics after fragmentation: the case of the endangered Spanish imperial eagle ( Aquila adalberti)

    OpenAIRE

    Martínez-Cruz, Begoña; Godoy, José A.; Negro, Juan J.

    2004-01-01

    The highly endangered Spanish imperial eagle, Aquila adalberti , has suffered from both population decline and fragmentation during the last century. Here we describe the current genetic status of the population using an extensive sampling of its current distribution range and both mitochondrial control region sequences and nuclear microsatellite markers. Results were evaluated in comparison to those obtained for the Eastern imperial eagle, Aquila heliaca , its nearest...

  7. The geography of malaria genetics in the Democratic Republic of Congo: A complex and fragmented landscape

    Science.gov (United States)

    Carrel, Margaret; Patel, Jaymin; Taylor, Steve M.; Janko, Mark; Mwandagalirwa, Melchior Kashamuka; Tshefu, Antoinette K.; Escalante, Ananias A.; McCollum, Andrea; Alam, Md Tauqeer; Udhayakumar, Venkatachalam; Meshnick, Steven; Emch, Michael

    2014-01-01

    Understanding how malaria parasites move between populations is important, particularly given the potential for malaria to be reintroduced into areas where it was previously eliminated. We examine the distribution of malaria genetics across seven sites within the Democratic Republic of Congo (DRC) and two nearby countries, Ghana and Kenya, in order to understand how the relatedness of malaria parasites varies across space, and whether there are barriers to the flow of malaria parasites within the DRC or across borders. Parasite DNA was retrieved from dried blood spots from 7 Demographic and Health Survey sample clusters in the DRC. Malaria genetic characteristics of parasites from Ghana and Kenya were also obtained. For each of 9 geographic sites (7 DRC, 1 Ghana and 1 Kenya), a pair-wise RST statistic was calculated, indicating the genetic distance between malaria parasites found in those locations. Mapping genetics across the spatial extent of the study area indicates a complex genetic landscape, where relatedness between two proximal sites may be relatively high (RST > 0.64) or low (RST < 0.05), and where distal sites also exhibit both high and low genetic similarity. Mantel’s tests suggest that malaria genetics differ as geographic distances increase. Principal Coordinate Analysis suggests that genetically related samples are not co-located. Barrier analysis reveals no significant barriers to gene flow between locations. Malaria genetics in the DRC have a complex and fragmented landscape. Limited exchange of genes across space is reflected in greater genetic distance between malaria parasites isolated at greater geographic distances. There is, however, evidence for close genetic ties between distally located sample locations, indicating that movement of malaria parasites and flow of genes is being driven by factors other than distance decay. This research demonstrates the contributions that spatial disease ecology and landscape genetics can make to

  8. Genetic variability of the stable fly assessed on a global scale using amplified fragment length polymorphism.

    Science.gov (United States)

    Kneeland, Kathleen M; Skoda, Steven R; Foster, John E

    2016-10-01

    The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is a blood-feeding, economically important pest of animals and humans worldwide. Improved management strategies are essential and their development would benefit from studies on genetic diversity of stable flies. Especially if done on a global scale, such research could generate information necessary for the development and application of more efficient control methods. Herein we report on a genetic study of stable flies using amplified fragment length polymorphism, with samples of 10-40 individuals acquired from a total of 25 locations in the Nearctic, Neotropic, Palearctic, Afrotropic and Australasian biogeographical regions. We hypothesized that genetic differentiation would exist across geographical barriers. Although FST (0.33) was moderately high, the GST (0.05; representing genetic diversity between individuals) was very low; Nm values (representing gene flow) were high (9.36). The mismatch distribution and tests of neutrality suggested population expansion, with no genetic differentiation between locations. The analysis of molecular variance (AMOVA) results showed the majority of genetic diversity was within groups. The mantel test showed no correlation between geographic and genetic distance; this strongly supports the AMOVA results. These results suggest that stable flies did not show genetic differentiation but are panmictic, with no evidence of isolation by distance or across geographical barriers. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  9. The effect of varying the peptide linker length in a single chain variable fragment antibody against wogonin glucuronide.

    Science.gov (United States)

    Paudel, Madan Kumar; Sakamoto, Seiichi; Van Huy, Le; Tanaka, Hiroyuki; Miyamoto, Tomofumi; Morimoto, Satoshi

    2017-06-10

    Peptide linkers of three different lengths were constructed to join the variable regions of the heavy chain (VH) and the light chain (VL) in a single-chain variable fragment antibody (scFv) specific for wogonin glucuronide (Wgn) that has the structure VH-(GGGGS) n -VL (n=3, 5, or 7). The scFv antibodies, which were expressed in Escherichia coli, were derived from an anti-Wgn monoclonal antibody (315A). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to evaluate their reactivity and sensitivity, which is also used for quantitative analysis of Wgn. Our results, showed that the reactivity and specificity of the three different scFvs were, in fact, similar. Subsequently, the scFv having a VH-(GGGGS) 3 -VL linker which was slightly better that other two scFvs against Wgn, was applied to indirect competitive ELISA (icELISA) to analyze Scutellariae Radix (S. Radix). The utility of the icELISA was demonstrated for quality control and analysis of S. Radix in this report. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    Directory of Open Access Journals (Sweden)

    Edyta Petters

    2015-08-01

    Full Text Available Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

  11. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

    Science.gov (United States)

    Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

    2013-08-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Genetics of Euglossini bees (Hymenoptera in fragments of the Atlantic Forest in the region of Viçosa, MG

    Directory of Open Access Journals (Sweden)

    A. M. Waldschmidt

    Full Text Available With uncontrolled deforestation, forest fragments remain, which in most cases are in different stages of regeneration and present isolated populations. In the present study we analyzed the genetic patterns of Eulaema nigrita populations in seven Atlantic Forest fragments of different sizes and successional stages in the region of Viçosa, MG. This was done by RAPD molecular markers. We observed that the area of the fragments had no effect on the genetic variability of E. nigrita in the direction predicted by meta-population models. Medium-sized well-preserved woods presented the lowest variability, whereas large and small woods were statistically identical. The evidence supports the notion that rural areas present greater dispersal among fragments, implying greater similarity between the populations of fragments located in rural areas when compared to fragments in urban areas.

  13. Population Genetic Structure of Glycyrrhiza inflata B. (Fabaceae) Is Shaped by Habitat Fragmentation, Water Resources and Biological Characteristics.

    Science.gov (United States)

    Yang, Lulu; Chen, Jianjun; Hu, Weiming; Yang, Tianshun; Zhang, Yanjun; Yukiyoshi, Tamura; Zhou, Yanyang; Wang, Ying

    2016-01-01

    Habitat fragmentation, water resources and biological characteristics are important factors that shape the genetic structure and geographical distribution of desert plants. Analysis of the relationships between these factors and population genetic variation should help to determine the evolutionary potential and conservation strategies for genetic resources for desert plant populations. As a traditional Chinese herb, Glycyrrhiza inflata B. (Fabaceae) is restricted to the fragmented desert habitat in China and has undergone a dramatic decline due to long-term over-excavation. Determining the genetic structure of the G. inflata population and identifying a core collection could help with the development of strategies to conserve this species. We investigated the genetic variation of 25 G. inflata populations based on microsatellite markers. A high level of population genetic divergence (FST = 0.257), population bottlenecks, reduced gene flow and moderate genetic variation (HE = 0.383) were detected. The genetic distances between the populations significantly correlated with the geographical distances, and this suggests that habitat fragmentation has driven a special genetic structure of G. inflata in China through isolation by distance. STRUCTURE analysis showed that G. inflata populations were structured into three clusters and that the populations belonged to multiple water systems, which suggests that water resources were related to the genetic structure of G. inflata. In addition, the biological characteristics of the perennial species G. inflata, such as its long-lived seeds, asexual reproduction, and oasis ecology, may be related to its resistance to habitat fragmentation. A core collection of G. inflata, that included 57 accessions was further identified, which captured the main allelic diversity of G. inflata. Recent habitat fragmentation has accelerated genetic divergence. The population genetic structure of G. inflata has been shaped by habitat

  14. A Role for Small Antibody Fragments to Bind and Neutralize HIV | Center for Cancer Research

    Science.gov (United States)

    The surface of the Human Immunodeficiency Virus (HIV) is studded with numerous copies of the glycoprotein Env. Each Env spike is composed of three copies of the proteins gp41, which sits in the viral membrane, and gp120, which rests on top of each gp41 molecule. Env is essential for HIV-mediated infection because the binding of gp120 to the T cell surface receptor CD4 initiates a conformational change in Env exposing the fusion peptide, which inserts into the T cell membrane and helps fuse the T cell and virus together. This makes Env an attractive target for designing therapeutic inhibitory antibodies. However, the complexities of the HIV surface proteins and the tight association of the virus and T cell during infection have hampered the identification of full-length antibodies with effective HIV neutralizing activity.

  15. Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity.

    Science.gov (United States)

    Funayama, Jaqueline C; Pucca, Manuela B; Roncolato, Eduardo C; Bertolini, Thaís B; Campos, Lucas B; Barbosa, José E

    2012-03-01

    The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

  16. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

  17. A rapid, strong, and convergent genetic response to urban habitat fragmentation in four divergent and widespread vertebrates

    Science.gov (United States)

    Delaney, Kathleen Semple; Riley, Seth P.D.; Fisher, Robert N.

    2010-01-01

    Background: Urbanization is a major cause of habitat fragmentation worldwide. Ecological and conservation theory predicts many potential impacts of habitat fragmentation on natural populations, including genetic impacts. Habitat fragmentation by urbanization causes populations of animals and plants to be isolated in patches of suitable habitat that are surrounded by non-native vegetation or severely altered vegetation, asphalt, concrete, and human structures. This can lead to genetic divergence between patches and in turn to decreased genetic diversity within patches through genetic drift and inbreeding. Methodology/Principal Findings: We examined population genetic patterns using microsatellites in four common vertebrate species, three lizards and one bird, in highly fragmented urban southern California. Despite significant phylogenetic, ecological, and mobility differences between these species, all four showed similar and significant reductions in gene flow over relatively short geographic and temporal scales. For all four species, the greatest genetic divergence was found where development was oldest and most intensive. All four animals also showed significant reduction in gene flow associated with intervening roads and freeways, the degree of patch isolation, and the time since isolation. Conclusions/Significance: Despite wide acceptance of the idea in principle, evidence of significant population genetic changes associated with fragmentation at small spatial and temporal scales has been rare, even in smaller terrestrial vertebrates, and especially for birds. Given the striking pattern of similar and rapid effects across four common and widespread species, including a volant bird, intense urbanization may represent the most severe form of fragmentation, with minimal effective movement through the urban matrix.

  18. A rapid, strong, and convergent genetic response to urban habitat fragmentation in four divergent and widespread vertebrates.

    Directory of Open Access Journals (Sweden)

    Kathleen Semple Delaney

    2010-09-01

    Full Text Available Urbanization is a major cause of habitat fragmentation worldwide. Ecological and conservation theory predicts many potential impacts of habitat fragmentation on natural populations, including genetic impacts. Habitat fragmentation by urbanization causes populations of animals and plants to be isolated in patches of suitable habitat that are surrounded by non-native vegetation or severely altered vegetation, asphalt, concrete, and human structures. This can lead to genetic divergence between patches and in turn to decreased genetic diversity within patches through genetic drift and inbreeding.We examined population genetic patterns using microsatellites in four common vertebrate species, three lizards and one bird, in highly fragmented urban southern California. Despite significant phylogenetic, ecological, and mobility differences between these species, all four showed similar and significant reductions in gene flow over relatively short geographic and temporal scales. For all four species, the greatest genetic divergence was found where development was oldest and most intensive. All four animals also showed significant reduction in gene flow associated with intervening roads and freeways, the degree of patch isolation, and the time since isolation.Despite wide acceptance of the idea in principle, evidence of significant population genetic changes associated with fragmentation at small spatial and temporal scales has been rare, even in smaller terrestrial vertebrates, and especially for birds. Given the striking pattern of similar and rapid effects across four common and widespread species, including a volant bird, intense urbanization may represent the most severe form of fragmentation, with minimal effective movement through the urban matrix.

  19. A rapid, strong, and convergent genetic response to urban habitat fragmentation in four divergent and widespread vertebrates.

    Science.gov (United States)

    Delaney, Kathleen Semple; Riley, Seth P D; Fisher, Robert N

    2010-09-16

    Urbanization is a major cause of habitat fragmentation worldwide. Ecological and conservation theory predicts many potential impacts of habitat fragmentation on natural populations, including genetic impacts. Habitat fragmentation by urbanization causes populations of animals and plants to be isolated in patches of suitable habitat that are surrounded by non-native vegetation or severely altered vegetation, asphalt, concrete, and human structures. This can lead to genetic divergence between patches and in turn to decreased genetic diversity within patches through genetic drift and inbreeding. We examined population genetic patterns using microsatellites in four common vertebrate species, three lizards and one bird, in highly fragmented urban southern California. Despite significant phylogenetic, ecological, and mobility differences between these species, all four showed similar and significant reductions in gene flow over relatively short geographic and temporal scales. For all four species, the greatest genetic divergence was found where development was oldest and most intensive. All four animals also showed significant reduction in gene flow associated with intervening roads and freeways, the degree of patch isolation, and the time since isolation. Despite wide acceptance of the idea in principle, evidence of significant population genetic changes associated with fragmentation at small spatial and temporal scales has been rare, even in smaller terrestrial vertebrates, and especially for birds. Given the striking pattern of similar and rapid effects across four common and widespread species, including a volant bird, intense urbanization may represent the most severe form of fragmentation, with minimal effective movement through the urban matrix.

  20. Population structure and genetic diversity of black redhorse (Moxostoma duquesnei) in a highly fragmented watershed

    Science.gov (United States)

    Reid, S.M.; Wilson, C.C.; Mandrak, N.E.; Carl, L.M.

    2008-01-01

    Dams have the potential to affect population size and connectivity, reduce genetic diversity, and increase genetic differences among isolated riverine fish populations. Previous research has reported adverse effects on the distribution and demographics of black redhorse (Moxostoma duquesnei), a threatened fish species in Canada. However, effects on genetic diversity and population structure are unknown. We used microsatellite DNA markers to assess the number of genetic populations in the Grand River (Ontario) and to test whether dams have resulted in a loss of genetic diversity and increased genetic differentiation among populations. Three hundred and seventy-seven individuals from eight Grand River sites were genotyped at eight microsatellite loci. Measures of genetic diversity were moderately high and not significantly different among populations; strong evidence of recent population bottlenecks was not detected. Pairwise FST and exact tests identified weak (global FST = 0.011) but statistically significant population structure, although little population structuring was detected using either genetic distances or an individual-based clustering method. Neither geographic distance nor the number of intervening dams were correlated with pairwise differences among populations. Tests for regional equilibrium indicate that Grand River populations were either in equilibrium between gene flow and genetic drift or that gene flow is more influential than drift. While studies on other species have identified strong dam-related effects on genetic diversity and population structure, this study suggests that barrier permeability, river fragment length and the ecological characteristics of affected species can counterbalance dam-related effects. ?? 2007 Springer Science+Business Media B.V.

  1. [Use of antibodies or their fragments for the treatment of tumors].

    Science.gov (United States)

    Douillard, J Y; Chatal, J F

    1998-11-01

    During the last 15 years, various antibodies specific for antigens associated with determined types of cancer have been used therapeutically, including some in unlabeled forms as immune effectors. The results of clinical studies have been unpromising for patients with colorectal cancer at the advanced metastatic stage but much more favorable in terms of increased survival for the adjuvant situation of residual microscopic disease. Antibodies have also been used as carriers for cytotoxic substances, but with rather disappointing clinical results when they were labeled with toxins or antimitotic agents. The results have been variable for labeling with radionuclides (mainly iodine-131), some-times proving quite favorable for refractory forms of non-hodgkin's lymphomas or acute leukemias. In this last indication, radioimmunotherapy has been associated with chemotherapy to enhance action before a bone-marrow graft. However, the clinical results have been disappointing in the treatment of solid tumors, showing responses only in the case of small targets. In the future, treatment with antibodies will focus on microscopic tumors, in association with other therapeutic modalities especially, chemotherapy and biotherapy.

  2. Pollen dispersal and genetic structure of the tropical tree Dipteryx panamensis in a fragmented Costa Rican landscape.

    Science.gov (United States)

    Hanson, Thor R; Brunsfeld, Steven J; Finegan, Bryan; Waits, Lisette P

    2008-04-01

    In the face of widespread deforestation, the conservation of rainforest trees relies increasingly on their ability to maintain reproductive processes in fragmented landscapes. Here, we analysed nine microsatellite loci for 218 adults and 325 progeny of the tree Dipteryx panamensis in Costa Rica. Pollen dispersal distances, genetic diversity, genetic structure and spatial autocorrelation were determined for populations in four habitats: continuous forest, forest fragments, pastures adjacent to fragments and isolated pastures. We predicted longer but less frequent pollen movements among increasingly isolated trees. This pattern would lead to lower outcrossing rates for pasture trees, as well as lower genetic diversity and increased structure and spatial autocorrelation among their progeny. Results generally followed these expectations, with the shortest pollen dispersal among continuous forest trees (240 m), moderate distances for fragment (343 m) and adjacent pasture (317 m) populations, and distances of up to 2.3 km in isolated pastures (mean: 557 m). Variance around pollen dispersal estimates also increased with fragmentation, suggesting altered pollination conditions. Outcrossing rates were lower for pasture trees and we found greater spatial autocorrelation and genetic structure among their progeny, as well as a trend towards lower heterozygosity. Paternal reproductive dominance, the pollen contributions from individual fathers, did not vary among habitats, but we did document asymmetric pollen flow between pasture and adjacent fragment populations. We conclude that long-distance pollen dispersal helps maintain gene flow for D. panamensis in this fragmented landscape, but pasture and isolated pasture populations are still at risk of long-term genetic erosion.

  3. Population Genetic Structure of Arctomecon californica Torrey & Fremont (Papaveraceae) in Fragmented and Unfragmented Habitat(Population Biology)

    OpenAIRE

    LAURA L., HICKERSON; PAUL G., WOLF; Department of Biology, Utah State University; Department of Biology, Utah State University

    1998-01-01

    Arctomecon californica (the Las Vegas bearpoppy) is endemic to gypsum outcrops of the northern Mojave Desert. Native habitat of this plant in the Las Vegas Valley has been severely fragmented, while relatively undisturbed, unfragmented habitat still exists in the Lake Mead National Recreation Area. Allozyme data from seven loci for 16 populations indicate high levels of genetic variability. Nei's genetic identity and G_ values suggest that populations in fragmented habitat are more differenti...

  4. Uptake of 99mTc labelled (Fab')2 fragments of monoclonal antibody 225.28S by a benign ocular naevus

    International Nuclear Information System (INIS)

    Bomanji, J.; Granowska, M.; Britton, K.E.; Hungerford, J.L.

    1988-01-01

    Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, 99m Tc radiolabelled (Fab') 2 fragments of monoclonal antibody 225.28S raised against cutaneous melanomas have been used for imaging uveal melanomas. We report here a case where uptake of radiolabelled antibody was observed in a choroidal melanoma of the right eye and a benign choroidal naevus of the left. (orig.)

  5. Population genetics of the wood-rotting basidiomycete Armillaria cepistipes in a fragmented forest landscape.

    Science.gov (United States)

    Heinzelmann, Renate; Rigling, Daniel; Prospero, Simone

    2012-09-01

    Armillaria cepistipes is a common wood-rotting basidiomycete fungus found in most forests in Central Europe. In Switzerland, the habitat of A. cepistipes is fragmented because of the presence of major geographical barriers, in particular the Alps, and past deforestation. We analysed the impact of habitat fragmentation on the current spatial genetic structure of the Swiss A. cepistipes population. A total of 167 isolates were sampled across an area of 41 000 km(2) and genotyped at seven microsatellite and four single nucleotide polymorphism (SNP) loci. All isolates belonged to different genotypes which, according to the Bayesian clustering algorithm implemented in Tess, originated from a single gene pool. Our analyses indicate that the overall A. cepistipes population shows little, but significant (F(ST)=0.02), genetic differentiation. Such a situation suggests gene flow is strong, possibly due to long-distance dispersal of airborne basidiospores. This hypothesis is supported by the fact that we could not detect a pattern of isolation by distance. Gene flow is partially restricted by the high mountain ranges of the Alps, as indicated by a signal of spatial autocorrelation detected among genotypes separated by less than about 80-130 km. In contrast, past deforestation seems to have no significant effect on the current spatial population structure of A. cepistipes. This might indicate the existence of a time lag between the current spatial genetic structure and the processes that have induced this specific structure. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  6. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  7. Low genetic diversity and strong population structure shaped by anthropogenic habitat fragmentation in a critically endangered primate, Trachypithecus leucocephalus.

    Science.gov (United States)

    Wang, W; Qiao, Y; Li, S; Pan, W; Yao, M

    2017-06-01

    Habitat fragmentation may strongly impact population genetic structure and reduce the genetic diversity and viability of small and isolated populations. The white-headed langur (Trachypithecus leucocephalus) is a critically endangered primate species living in a highly fragmented and human-modified habitat in southern China. We examined the population genetic structure and genetic diversity of the species and investigated the environmental and anthropogenic factors that may have shaped its population structure. We used 214 unique multi-locus genotypes from 41 social groups across the main distribution area of T. leucocephalus, and found strong genetic structure and significant genetic differentiation among local populations. Our landscape genetic analyses using a causal modelling framework suggest that a large habitat gap and geographical distance represent the primary landscape elements shaping genetic structure, yet high levels of genetic differentiation also exist between patches separated by a small habitat gap or road. This is the first comprehensive study that has evaluated the population genetic structure and diversity of T. leucocephalus using nuclear markers. Our results indicate strong negative impacts of anthropogenic land modifications and habitat fragmentation on primate genetic connectivity between forest patches. Our analyses suggest that two management units of the species could be defined, and indicate that habitat continuity should be enforced and restored to reduce genetic isolation and enhance population viability.

  8. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  9. Renal brush border enzyme-cleavable linkages for low renal radioactivity levels of radiolabeled antibody fragments.

    Science.gov (United States)

    Akizawa, Hiromichi; Imajima, Mitsuo; Hanaoka, Hirofumi; Uehara, Tomoya; Satake, Satoshi; Arano, Yasushi

    2013-02-20

    We previously demonstrated that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(ε)-maleoyl-l-lysine ([(131)I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[(131)I]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-l-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: l-tyosine O-methyl, l-asparagine, l-glutamine, and N(ε)-Boc-l-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[(125)I]iodohippuryl O-methyl-l-tyrosine (2c) liberated the highest amount of m-[(125)I]iodohippuric acid among the four substrates and the change in the linkage structure altered enzyme species responsible for the hydrolysis reaction. To further assess the applicability of the linkage, a radioiodination reagent containing a glycyl-tyrosine linkage, 3'-[(125)I]iodohippuryl O-((2-maleimidoethyl)carbamoyl)methyl-l-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [(125)I]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [(125)I]HML-Fab and directly radioiodinated Fab, while the blood clearance rates of the three were similar. The analyses of urine for 24 h postinjection of [(125)I]HMT-Fab showed that m-[(125)I]iodohippuric acid was excreted as the major radiometabolite. The findings indicated that glycyl-tyrosine linkage is also available to reduce renal radioactivity levels of radioiodinated Fab fragments, due to liberation of m-iodohippuric acid by the action of enzymes present on renal brush border membrane. These findings suggest that an appropriate selection of peptide linkages would allow the liberation of a designed radiolabeled compound from covalently conjugated

  10. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1.

    Science.gov (United States)

    Ascione, Alessandro; Capecchi, Barbara; Campitelli, Laura; Imperiale, Valentina; Flego, Michela; Zamboni, Silvia; Gellini, Mara; Alberini, Isabella; Pittiglio, Eliana; Donatelli, Isabella; Temperton, Nigel J; Cianfriglia, Maurizio

    2009-09-01

    Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

  11. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

    International Nuclear Information System (INIS)

    Demartis, S.; Tarli, L.; Neri, D.; Borsi, L.; Zardi, L.

    2001-01-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211 At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. (orig.)

  12. Study of the viability of technetium-99m labeling of whole antimyosin antibody and its fragment: development of radiopharmaceutical for cardiac survey

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro

    2007-01-01

    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled

  13. Genetic analyses of historic and modern marbled murrelets suggest decoupling of migration and gene flow after habitat fragmentation

    Science.gov (United States)

    M. Zachariah Peery; Laurie A. Hall; Sellas. Anna; Steven R. Beissinger; Craig Moritz; Martine Berube; Martin G. Raphael; S. Kim Nelson; Richard T. Golightly; Laura McFarlane-Tranquilla; Scott H. Newman; Per J. Palsboll

    2009-01-01

    The dispersal of individuals among fragmented populations is generally thought to prevent genetic and demographic isolation, and ultimately reduce extinction risk. In this study, we show that a century of reduction in coastal old-growth forests, as well as a number of other environmental factors, has probably resulted in the genetic divergence of marbled murrelets (...

  14. Murine CR1/2 targeted antigenized single-chain antibody fragments induce transient low affinity antibodies and negatively influence an ongoing immune response.

    Science.gov (United States)

    Prechl, József; Molnár, Eszter; Szekeres, Zsuzsanna; Isaák, Andrea; Papp, Krisztián; Balogh, Péter; Erdei, Anna

    2007-01-01

    We have generated a single-chain antibody which recognizes murine CR1/2 and carries a genetically fused influenza hemagglutinin derived peptide. Theoretically such a construct is able to crosslink the B cell antigen receptor and CR1/2 on peptide specific B cells. The construct was able to reach its CR1/2 positive target cells, yet intraperitoneal delivery of the construct elicited an IgM response only slightly exceeding that induced by the free peptide. Providing T cell help by the injection of peptide specific lymphocytes did not alter the response in essence, that is anti-peptide IgG was not detectable even after booster immunizations. When used as a booster vaccine following injection of the peptide in adjuvant, the construct even inhibited the development of IgG1 and IgG3 anti-peptide antibodies. These data indicate that although targeting of antigen to CR1/2 on B cells can enhance transient proliferation or differentiation of antigen specific B cells it cannot induce strong, longlasting humoral immune responses. Furthermore, CR1/2 targeting constructs may negatively influence an ongoing immune reaction.

  15. Kinetics and tissue distribution of the radiolabeled chimeric monoclonal antibody MOv18 IgG and F(ab')2 fragments in ovarian carcinoma patients

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; den Hollander, W.; Vermorken, J. B.; Molthoff, C. J.; Burger, C. W.; Helmerhorst, T. J.; Baak, J. P.; Roos, J. C.

    1993-01-01

    Twenty-four patients suspected of having ovarian carcinoma received i.v. injection with a combination of radiolabeled intact IgG (1 mg) and F(ab')2 fragments (1 mg) of the chimeric monoclonal antibody MOv18, each form labeled with 1.85 MBq 131I or 125I. Laparotomy was performed either 2 or 6 days

  16. Pharmacokinetics and tumor targeting of 131I-labeled F(ab')2 fragments of the chimeric monoclonal antibody G250: preclinical and clinical pilot studies.

    NARCIS (Netherlands)

    Brouwers, A.H.; Mulders, P.F.A.; Oosterwijk, E.; Buijs, W.C.A.M.; Corstens, F.H.M.; Boerman, O.C.; Oyen, W.J.G.

    2004-01-01

    INTRODUCTION: Clinical and animal studies of chimeric monoclonal antibody G250 (moAb cG250) for the targeting of clear-cell renal cell carcinoma (RCC), to date, have been with the intact IgG form. To determine whether F(ab')2 fragments are more suited for radioimmunotherapy (RIT) than intact IgG,

  17. Biokinetic studies and scintigraphic imaging of human pancreatic carcinoma xenografts with iodine 131-labeled F(ab')2-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Moellenstaedt, S.; Kriegel, H.; Baum, R.P.; Maul, F.D.; Hoer, G.; Wenisch, H.J.C.

    1985-01-01

    Biodistribution and tumor uptake of I-131 labeled F(ab') 2 -fragments of monoclonal antibodies to CA 125, CA 19-9 and CEA were investigated in nuce mice with human pancreatic carcinoma xenografts. To assess the immunological specificity of these antibody fragments their tumor uptake was compared with that of unspecific IgG F(ab') 2 . Additionally serum levels of CA 125 and CA 19-9 were determined in tumor bearing mice by radioimmunoassay and the expression of CA 125 and CA 19-9 in pancreatic carcinoma was demonstrated by immunhistochemicals studies. The rapid blood clearance of the antibody fragments leads to increasing tumor-to-blood ratios with time after injection for all antibody fragments. The highest ratios were found for OC 125 at all time points p.i., with mean values of 9.6 at 48 h p.i. and 11.8 at 96 h p.i. On scintigraphic images tumors with a weight of 100 mg could be localized between 24 and 48 h after injection of iodinated OC 125 F(ab') 2 . The serum levels of CA 125 and CA 19-9 were elevated only in some animals and no correlation between the antigen serum levels and the antigen expression in the tumor shown by immunhistochemical staining could be determined. (orig.) [de

  18. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display.

    Science.gov (United States)

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.

  19. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    Science.gov (United States)

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  20. The genetic impact of translocations and habitat fragmentation in chamois (Rupicapra) spp.

    Science.gov (United States)

    Crestanello, Barbara; Pecchioli, Elena; Vernesi, Cristiano; Mona, Stefano; Martínková, Natalia; Janiga, Marian; Hauffe, Heidi C; Bertorelle, Giorgio

    2009-01-01

    The chamois is a useful species with which to investigate the combined genetic impact of habitat fragmentation, over hunting, and translocations. Genetic variation within and between chamois (genus Rupicapra) populations was analyzed in 259 individuals from 16 sampling sites located in Italy, Spain, Slovakia, and the Czech Republic. Two mitochondrial DNA markers (control region and cytochrome b) and 11 nuclear microsatellites were typed. The principal results of this study can be summarized as follows: 1) high and significant differentiation between almost all chamois populations is observed even on a microgeographical scale, probably caused by the patchy distribution of this species, sharp geographical barriers to gene flow, and drift effects related to recent bottlenecks; 2) historical translocation events have left a clear genetic signature, including interspecific hybridization in some Alpine localities; 3) the Apennine subspecies of chamois, Rupicapra pyrenaica ornata, shows a high and similar level of divergence (about 1.5 My) from the Pyrenean (Rupicapra pyrenaica pyrenaica) and the Alpine (Rupicapra rupicapra) chamois; therefore, the specific status of these taxa should be revised. These results confirm the potential of population genetic analyses to dissect and interpret complex patterns of diversity in order to define factors important to conservation and management.

  1. Movement disorders with neuronal antibodies: syndromic approach, genetic parallels and pathophysiology.

    Science.gov (United States)

    Balint, Bettina; Vincent, Angela; Meinck, Hans-Michael; Irani, Sarosh R; Bhatia, Kailash P

    2018-01-01

    Movement disorders are a prominent and common feature in many autoantibody-associated neurological diseases, a group of potentially treatable conditions that can mimic infectious, metabolic or neurodegenerative disease. Certain movement disorders are likely to associate with certain autoantibodies; for example, the characteristic dyskinesias, chorea and dystonia associated with NMDAR antibodies, stiff person spectrum disorders with GAD, glycine receptor, amphiphysin or DPPX antibodies, specific paroxysmal dystonias with LGI1 antibodies, and cerebellar ataxia with various anti-neuronal antibodies. There are also less-recognized movement disorder presentations of antibody-related disease, and a considerable overlap between the clinical phenotypes and the associated antibody spectra. In this review, we first describe the antibodies associated with each syndrome, highlight distinctive clinical or radiological 'red flags', and suggest a syndromic approach based on the predominant movement disorder presentation, age, and associated features. We then examine the underlying immunopathophysiology, which may guide treatment decisions in these neuroimmunological disorders, and highlight the exceptional interface between neuronal antibodies and neurodegeneration, such as the tauopathy associated with IgLON5 antibodies. Moreover, we elaborate the emerging pathophysiological parallels between genetic movement disorders and immunological conditions, with proteins being either affected by mutations or targeted by autoantibodies. Hereditary hyperekplexia, for example, is caused by mutations of the alpha subunit of the glycine receptor leading to an infantile-onset disorder with exaggerated startle and stiffness, whereas antibodies targeting glycine receptors can induce acquired hyperekplexia. The spectrum of such immunological and genetic analogies also includes cerebellar ataxias and some encephalopathies. Lastly, we discuss how these pathophysiological considerations could

  2. Tuning the Hydrolytic Stability of Next Generation Maleimide Cross-Linkers Enables Access to Albumin-Antibody Fragment Conjugates and tri-scFvs.

    Science.gov (United States)

    Forte, Nafsika; Livanos, Maria; Miranda, Enrique; Morais, Maurício; Yang, Xiaoping; Rajkumar, Vineeth S; Chester, Kerry A; Chudasama, Vijay; Baker, James R

    2018-02-21

    We describe investigations to expand the scope of next generation maleimide cross-linkers for the construction of homogeneous protein-protein conjugates. Diiodomaleimides are shown to offer the ideal properties of rapid bioconjugation with reduced hydrolysis, allowing the cross-linking of even sterically hindered systems. The optimized linkers are exploited to link human serum albumin to antibody fragments (Fab or scFv) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability. Finally, a triprotein conjugate is formed, by linking scFv antibody fragments targeting carcinoembryonic antigen. This tri-scFv is shown to infer a combination of greater antigen avidity and increased in vivo half-life, representing a promising platform for antibody therapeutic development.

  3. Radioimmunoimaging of human colon carcinoma grafted into nudemice using 131I-labeled monoclonal anticea antibody and its F(ab')2 fragments

    International Nuclear Information System (INIS)

    Liu Guangda

    1988-01-01

    131 I-labeled monoclonal anti-CEA antibody and its F(ab') 2 fragments were injected into nude mice bearing human colon carcinoma xenografts for tumor localization and radioimmunoimaging studies. Transplanted tumors were visualized in 12 hours after injection of the labeled anti-CEA or its F(ab') 2 by gamma camera. Biodistribution data indicated that F(ab') 2 fragments were cleared more rapidly from blood (T 1/2 = 13.3 h for F(ab') 2 , T 1/2 = 21.1 h for intact antibody) over 6-24 h and had higher tumor blood ratios. The intact antibody was concentrated in the tumor better than F(ab') 2 . In double-label experiments, a nonspecific localization of the control ( 125 I-labeled anti-HCG) in the tumor was also observed

  4. Consequences of severe habitat fragmentation on density, genetics, and spatial capture-recapture analysis of a small bear population.

    Directory of Open Access Journals (Sweden)

    Sean M Murphy

    Full Text Available Loss and fragmentation of natural habitats caused by human land uses have subdivided several formerly contiguous large carnivore populations into multiple small and often isolated subpopulations, which can reduce genetic variation and lead to precipitous population declines. Substantial habitat loss and fragmentation from urban development and agriculture expansion relegated the Highlands-Glades subpopulation (HGS of Florida, USA, black bears (Ursus americanus floridanus to prolonged isolation; increasing human land development is projected to cause ≥ 50% loss of remaining natural habitats occupied by the HGS in coming decades. We conducted a noninvasive genetic spatial capture-recapture study to quantitatively describe the degree of contemporary habitat fragmentation and investigate the consequences of habitat fragmentation on population density and genetics of the HGS. Remaining natural habitats sustaining the HGS were significantly more fragmented and patchier than those supporting Florida's largest black bear subpopulation. Genetic diversity was low (AR = 3.57; HE = 0.49 and effective population size was small (NE = 25 bears, both of which remained unchanged over a period spanning one bear generation despite evidence of some immigration. Subpopulation density (0.054 bear/km2 was among the lowest reported for black bears, was significantly female-biased, and corresponded to a subpopulation size of 98 bears in available habitat. Conserving remaining natural habitats in the area occupied by the small, genetically depauperate HGS, possibly through conservation easements and government land acquisition, is likely the most important immediate step to ensuring continued persistence of bears in this area. Our study also provides evidence that preferentially placing detectors (e.g., hair traps or cameras primarily in quality habitat across fragmented landscapes poses a challenge to estimating density-habitat covariate relationships using spatial

  5. Consequences of severe habitat fragmentation on density, genetics, and spatial capture-recapture analysis of a small bear population.

    Science.gov (United States)

    Murphy, Sean M; Augustine, Ben C; Ulrey, Wade A; Guthrie, Joseph M; Scheick, Brian K; McCown, J Walter; Cox, John J

    2017-01-01

    Loss and fragmentation of natural habitats caused by human land uses have subdivided several formerly contiguous large carnivore populations into multiple small and often isolated subpopulations, which can reduce genetic variation and lead to precipitous population declines. Substantial habitat loss and fragmentation from urban development and agriculture expansion relegated the Highlands-Glades subpopulation (HGS) of Florida, USA, black bears (Ursus americanus floridanus) to prolonged isolation; increasing human land development is projected to cause ≥ 50% loss of remaining natural habitats occupied by the HGS in coming decades. We conducted a noninvasive genetic spatial capture-recapture study to quantitatively describe the degree of contemporary habitat fragmentation and investigate the consequences of habitat fragmentation on population density and genetics of the HGS. Remaining natural habitats sustaining the HGS were significantly more fragmented and patchier than those supporting Florida's largest black bear subpopulation. Genetic diversity was low (AR = 3.57; HE = 0.49) and effective population size was small (NE = 25 bears), both of which remained unchanged over a period spanning one bear generation despite evidence of some immigration. Subpopulation density (0.054 bear/km2) was among the lowest reported for black bears, was significantly female-biased, and corresponded to a subpopulation size of 98 bears in available habitat. Conserving remaining natural habitats in the area occupied by the small, genetically depauperate HGS, possibly through conservation easements and government land acquisition, is likely the most important immediate step to ensuring continued persistence of bears in this area. Our study also provides evidence that preferentially placing detectors (e.g., hair traps or cameras) primarily in quality habitat across fragmented landscapes poses a challenge to estimating density-habitat covariate relationships using spatial capture

  6. Stuck in fragments: Population genetics of the Endangered collared brown lemur Eulemur collaris in the Malagasy littoral forest.

    Science.gov (United States)

    Bertoncini, Stefania; D'Ercole, Jacopo; Brisighelli, Francesca; Ramanamanjato, Jean-Baptiste; Capelli, Cristian; Tofanelli, Sergio; Donati, Giuseppe

    2017-07-01

    The Endangered collared brown lemur (Eulemur collaris) is the largest primate living in the littoral forest of southeastern Madagascar, a top priority habitat for biodiversity conservation on the island. Because this lemur is a key seed-disperser, an evaluation of the structure and connectivity of the populations surviving in the forest fragments is urgently needed to guide conservation plans. Genetic variability at autosomal microsatellites and mitochondrial DNA was investigated in a total of 49 collared brown lemurs sampled by non-invasive methods in three littoral forest fragments and in the nearby lowland humid forest. The overall genetic diversity of E. collaris in the southeastern coastal region of Madagascar was lower than in other populations, as well as in other lemur species. The population appears highly structured, with less variable and more inbred groups inhabiting the littoral forest fragments compared to the inland area. Major barriers to gene flow were identified isolating littoral forest fragments from each other and from the inland lowland humid forest. Medium to long-term drift and scarce gene flow is the scenario that best explains the current genetic distribution. Habitat discontinuities such as rivers and grassland between forest fragments played a major role in structuring the population. A common history of size contraction is pointed out by several genetic estimators, indicating a possible ecological crisis triggered around 1,300 years ago. The adoption of strategies aimed at facilitating gene flow and population growth appears crucial to delay further loss of genetic diversity. © 2017 Wiley Periodicals, Inc.

  7. The relative effects of habitat loss and fragmentation on population genetic variation in the red-cockaded woodpecker (Picoides borealis).

    Science.gov (United States)

    Bruggeman, Douglas J; Wiegand, Thorsten; Fernández, Néstor

    2010-09-01

    The relative influence of habitat loss, fragmentation and matrix heterogeneity on the viability of populations is a critical area of conservation research that remains unresolved. Using simulation modelling, we provide an analysis of the influence both patch size and patch isolation have on abundance, effective population size (N(e)) and F(ST). An individual-based, spatially explicit population model based on 15 years of field work on the red-cockaded woodpecker (Picoides borealis) was applied to different landscape configurations. The variation in landscape patterns was summarized using spatial statistics based on O-ring statistics. By regressing demographic and genetics attributes that emerged across the landscape treatments against proportion of total habitat and O-ring statistics, we show that O-ring statistics provide an explicit link between population processes, habitat area, and critical thresholds of fragmentation that affect those processes. Spatial distances among land cover classes that affect biological processes translated into critical scales at which the measures of landscape structure correlated best with genetic indices. Therefore our study infers pattern from process, which contrasts with past studies of landscape genetics. We found that population genetic structure was more strongly affected by fragmentation than population size, which suggests that examining only population size may limit recognition of fragmentation effects that erode genetic variation. If effective population size is used to set recovery goals for endangered species, then habitat fragmentation effects may be sufficiently strong to prevent evaluation of recovery based on the ratio of census:effective population size alone.

  8. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.

    Science.gov (United States)

    Rea, Jennifer C; Lou, Yun; Cuzzi, Joel; Hu, Yuhua; de Jong, Isabella; Wang, Yajun Jennifer; Farnan, Dell

    2012-12-28

    Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 μL HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Guus Koch

    2013-04-01

    Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

  10. Highly specific PET imaging of prostate tumors in mice with an iodine-124-labeled antibody fragment that targets phosphatidylserine.

    Directory of Open Access Journals (Sweden)

    Jason H Stafford

    Full Text Available Phosphatidylserine (PS is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab'2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab'2 was labeled with the positron-emitting isotope iodine-124 ((124I and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124I-PGN635 F(ab'2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124I-PGN635 F(ab'2 is a promising new imaging agent for predicting tumor response to therapy.

  11. Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms.

    Science.gov (United States)

    Datwyler, Shannon L; Weiblen, George D

    2006-03-01

    Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.

  12. Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

    Science.gov (United States)

    Bagheri, Salman; Yousefi, Mehdi; Safaie Qamsari, Elmira; Riazi-Rad, Farhad; Abolhassani, Mohsen; Younesi, Vahid; Dorostkar, Ruhollah; Movassaghpour, Ali Akbar; Sharifzadeh, Zahra

    2017-03-01

    The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

  13. Forest fragmentation in Vietnam : Effects on tree diversity, populations and genetics

    NARCIS (Netherlands)

    Ha, V.T.

    2015-01-01

    Millions of square kilometers of the Earth’s surface is covered by forest fragments, and a quarter of remaining tropical forest has been fragmented. In Southeast Asia, about 650,000 ha of natural forests are fragmented per year. Fragmentation of old growth forests is considered to be the greatest

  14. Potential Population Genetic Consequences of Habitat Fragmentation in Central European Forest Trees and Associated Understorey Species—An Introductory Survey

    Directory of Open Access Journals (Sweden)

    Christoph Dobeš

    2017-02-01

    Full Text Available Habitat fragmentation threatens the maintenance of genetic diversity of affected populations. Assessment of the risks associated with habitat fragmentation is a big challenge as the change in population genetic diversity is a dynamic process, often acting over long time periods and depending on various characteristics pertaining to both species (life history traits and their populations (extrinsic characteristics. With this survey, we provide an introductory overview for persons who have to make or are interested in making predictions about the fate of forest-dwelling plant populations which have recently become fragmented and isolated from their main occurrences. We provide a concise introduction to the field of population genetics focusing on terms, processes and phenomena relevant to the maintenance of genetic diversity and vitality of plant populations. In particular the antagonistic effects of gene flow and random genetic drift are covered. A special chapter is devoted to Central European tree species (including the Carpathians which we treat in detail with reference to an extensive literature survey on population genetic studies assembled from the whole of Europe. We further provide an overview of the population biology of associated understorey species. We conclude with recommended steps to be taken for the evaluation of potential perils of habitat fragmentation or population thinning for the genetics of tree populations. The complexity of effects exerted by life history traits and extrinsic characteristics of populations suggest population genetic development is strongly situation dependent. Therefore, we recommend following a case-by-case approach ideally supported by computer simulations to predict future population genetic development of both trees and associated understorey species.

  15. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

    Science.gov (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

    2014-01-01

    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

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

    Science.gov (United States)

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

    2015-05-27

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

  17. Significant genetic boundaries and spatial dynamics of giant pandas occupying fragmented habitat across southwest China.

    Science.gov (United States)

    Zhu, Lifeng; Zhang, Shanning; Gu, Xiaodong; Wei, Fuwen

    2011-03-01

    Understanding population history and genetic structure are key drivers of ecological research. Here, we studied two highly fragmented and isolated populations (Xiaoxiangling and Daxiangling) of giant pandas (Ailuropoda melanoleuca) at the extreme southwestern edge of their distribution. This area also contains the Dadu River, national road 108 and various human infrastructure and development, providing an ideal region in which we can identify the effects of different barriers on animal movements. We used partial mitochondrial control region (mtDNA) and nine microsatellite loci (nuclear DNA) data derived from 192 faecal and one blood sample collected from the wild. We found 136 genotypes corresponding to 53 unique multilocus genotypes and eight unique control region haplotypes (653 bp). Significant genetic boundaries correlated spatially with the Dadu River (K = 2). We estimate that a major divergence took place between these populations 26,000 years bp, at around the similar time the rock surface of valley bottom formed in Dadu River. The national road has resulted in further recent population differentiation (Pairwise F(S) on mtDNA and nuclear DNA) so that in effect, four smaller sub-populations now exist. Promisingly, we identified two possible first-generation migrants and their migration paths, and recommended the immediate construction of a number of corridors. Fortunately, the Chinese government has accepted our advice and is now planning corridor construction. © 2011 Blackwell Publishing Ltd.

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

    DEFF Research Database (Denmark)

    Goris, An; Pauwels, Ine; Gustavsen, Marte W

    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...... 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 as female gender, age at onset and severity. This is the largest study population so far investigated for the genetic influence on antibody levels...

  19. Genetic differences in natural antibody levels in common carp (Cyprinus carpio L.)

    NARCIS (Netherlands)

    Kachamakova, N.M.; Irnazarow, I.; Parmentier, H.K.; Savelkoul, H.F.J.; Pilarczyk, A.; Wiegertjes, G.F.

    2006-01-01

    In mammals, natural antibodies (Nabs) are mostly of the IgM isotype and can bind to a particular antigen or pathogen even if the host has never been exposed. Despite their early detection and abundance, the exact role and genetic control of Nabs remain unclear. We have used an indirect ELISA with

  20. Genetic parameters for natural antibody isotype titers in milk of Dutch Holstein-Friesians

    NARCIS (Netherlands)

    Wijga, S.; Bovenhuis, H.; Bastiaansen, J.W.M.; Arendonk, van J.A.M.; Ploegaert, T.C.W.; Tijhaar, E.; Poel, van der J.J.

    2013-01-01

    The objective of the present study was to estimate genetic parameters for natural antibody isotypes immunoglobulin (Ig) A, IgG1 and IgM titers binding the bacterial antigens lipopolysaccharide, peptidoglycan and the model antigen keyhole limpet hemocyanin in Dutch Holstein-Friesian cows (n = 1695).

  1. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.

    1991-01-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats

  2. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Habitat loss other than fragmentation per se decreased nuclear and chloroplast genetic diversity in a monoecious tree.

    Science.gov (United States)

    Zhang, Xin; Shi, Miao-Miao; Shen, Dong-Wei; Chen, Xiao-Yong

    2012-01-01

    Generally, effect of fragmentation per se on biodiversity has not been separated from the effect of habitat loss. In this paper, using nDNA and cpDNA SSRs, we studied genetic diversity of Castanopsis sclerophylla (Lindl. & Paxton) Schotty populations and decoupled the effects of habitat loss and fragmentation per se. We selected seven nuclear and six cpDNA microsatellite loci and genotyped 460 individuals from mainland and island populations, which were located in the impoundment created in 1959. Number of alleles per locus of populations in larger habitats was significantly higher than that in smaller habitats. There was a significant relationship between the number of alleles per locus and habitat size. Based on this relationship, the predicted genetic diversity of an imaginary population of size equaling the total area of the islands was lower than that of the global population on the islands. Re-sampling demonstrated that low genetic diversity of populations in small habitats was caused by unevenness in sample size. Fisher's α index was similar among habitat types. These results indicate that the decreased nuclear and chloroplast genetic diversity of populations in smaller habitats was mainly caused by habitat loss. For nuclear and chloroplast microsatellite loci, values of F(ST) were 0.066 and 0.893, respectively, and the calculated pollen/seed dispersal ratio was 162.2. When separated into pre-and post-fragmentation cohorts, pollen/seed ratios were 121.2 and 189.5, respectively. Our results suggest that habitat loss explains the early decrease in genetic diversity, while fragmentation per se may play a major role in inbreeding and differentiation among fragmented populations and later loss of genetic diversity.

  4. Population genetics after fragmentation: the case of the endangered Spanish imperial eagle (Aquila adalberti).

    Science.gov (United States)

    Martinez-Cruz, B; Godoy, J A; Negro, J J

    2004-08-01

    The highly endangered Spanish imperial eagle, Aquila adalberti, has suffered from both population decline and fragmentation during the last century. Here we describe the current genetic status of the population using an extensive sampling of its current distribution range and both mitochondrial control region sequences and nuclear microsatellite markers. Results were evaluated in comparison to those obtained for the Eastern imperial eagle, Aquila heliaca, its nearest extant relative. Mitochondrial haplotype diversity was lower in the Spanish than in the Eastern species whereas microsatellite allelic richness and expected heterozygosity did not differ. Both allelic richness and expected heterozygosity were lower in the small Parque Nacional de Doñana breeding nucleus compared to the remaining nuclei. A signal for a recent genetic bottleneck was not detected in the current Spanish imperial eagle population. We obtained low but significant pairwise FST values that were congruent with a model of isolation by distance. FST and exact tests showed differentiation among the peripheral and small Parque Nacional de Doñana population and the remaining breeding subgroups. The centrally located Montes de Toledo population did not differ from the surrounding Centro, Extremadura and Sierra Morena populations whereas the latter were significantly differentiated. On the other hand, a Bayesian approach identified two groups, Parque Nacional de Doñana and the rest of breeding nuclei. Recent migration rates into and from Parque Nacional de Doñana and the rest of breeding nuclei were detected by assignment methods and estimated as 2.4 and 5.7 individuals per generation, respectively, by a Bayesian approach. We discuss how management strategies should aim at the maintenance of current genetic variability levels and the avoidance of inbreeding depression through the connection of the different nuclei. Copyright 2004 Blackwell Publishing Ltd

  5. Radioimmunoimaging of human breast carcinoma xenografts in nude mouse model with 111In-labeled new monoclonal antibody EBA-1 and F(ab')2 fragments

    International Nuclear Information System (INIS)

    Yemul, Shrishailam; Leon, J.A.; Pozniakoff, Ted; Esser, P.D.; Estabrook, Alison; Met-Path Inc., Teterboro, NJ

    1993-01-01

    Radioimmunoimaging characteristics of a new monoclonal antibody EBA-1 and its F(ab') 2 fragments utilizing nu/nu mice bearing human breast carcinoma xenografts are described. 111 In-DPTA conjugates of EBA-1 localized with tumor/blood ratios of 0.99 ± 0.10 (P 2 radioconjugates at 48 h. These results suggest that EBA-1 and its F(ab') 2 might be useful reagents in radioimmunoimaging and radioimmunotherapy. (author)

  6. A comparative study on genetic effects of artificial and natural habitat fragmentation on Loropetalum chinense (Hamamelidaceae) in Southeast China.

    Science.gov (United States)

    Yuan, N; Comes, H P; Cao, Y N; Guo, R; Zhang, Y H; Qiu, Y X

    2015-06-01

    Elucidating the demographic and landscape features that determine the genetic effects of habitat fragmentation has become fundamental to research in conservation and evolutionary biology. Land-bridge islands provide ideal study areas for investigating the genetic effects of habitat fragmentation at different temporal and spatial scales. In this context, we compared patterns of nuclear microsatellite variation between insular populations of a shrub of evergreen broad-leaved forest, Loropetalum chinense, from the artificially created Thousand-Island Lake (TIL) and the Holocene-dated Zhoushan Archipelago of Southeast China. Populations from the TIL region harboured higher levels of genetic diversity than those from the Zhoushan Archipelago, but these differences were not significant. There was no correlation between genetic diversity and most island features, excepting a negative effect of mainland-island distance on allelic richness and expected heterozygosity in the Zhoushan Archipelago. In general, levels of gene flow among island populations were moderate to high, and tests of alternative models of population history strongly favoured a gene flow-drift model over a pure drift model in each region. In sum, our results showed no obvious genetic effects of habitat fragmentation due to recent (artificial) or past (natural) island formation. Rather, they highlight the importance of gene flow (most likely via seed) in maintaining genetic variation and preventing inter-population differentiation in the face of habitat 'insularization' at different temporal and spatial scales.

  7. scFv Antibody: Principles and Clinical Application

    OpenAIRE

    Ahmad, Zuhaida Asra; Yeap, Swee Keong; Ali, Abdul Manaf; Ho, Wan Yong; Alitheen, Noorjahan Banu Mohamed; Hamid, Muhajir

    2012-01-01

    To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional prot...

  8. How protein recognizes ladder-like polycyclic ethers. Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9.

    Science.gov (United States)

    Ui, Mihoko; Tanaka, Yoshikazu; Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Hirama, Masahiro; Tsumoto, Kouhei

    2008-07-11

    Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 angstroms, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 degrees C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.

  9. Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

    International Nuclear Information System (INIS)

    Ditzel, H.; Erb, K.; Rasmussen, J.W.; Jensenius, J.C.

    1992-01-01

    Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-1 at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients. (orig.)

  10. Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds.

    Science.gov (United States)

    Melchinger, A E; Lee, M; Lamkey, K R; Hallauer, A R; Woodman, W L

    1990-10-01

    Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment length polymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F1; F2, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster

  11. Landscape genetic analyses reveal fine-scale effects of forest fragmentation in an insular tropical bird.

    Science.gov (United States)

    Khimoun, Aurélie; Peterman, William; Eraud, Cyril; Faivre, Bruno; Navarro, Nicolas; Garnier, Stéphane

    2017-10-01

    Within the framework of landscape genetics, resistance surface modelling is particularly relevant to explicitly test competing hypotheses about landscape effects on gene flow. To investigate how fragmentation of tropical forest affects population connectivity in a forest specialist bird species, we optimized resistance surfaces without a priori specification, using least-cost (LCP) or resistance (IBR) distances. We implemented a two-step procedure in order (i) to objectively define the landscape thematic resolution (level of detail in classification scheme to describe landscape variables) and spatial extent (area within the landscape boundaries) and then (ii) to test the relative role of several landscape features (elevation, roads, land cover) in genetic differentiation in the Plumbeous Warbler (Setophaga plumbea). We detected a small-scale reduction of gene flow mainly driven by land cover, with a negative impact of the nonforest matrix on landscape functional connectivity. However, matrix components did not equally constrain gene flow, as their conductivity increased with increasing structural similarity with forest habitat: urban areas and meadows had the highest resistance values whereas agricultural areas had intermediate resistance values. Our results revealed a higher performance of IBR compared to LCP in explaining gene flow, reflecting suboptimal movements across this human-modified landscape, challenging the common use of LCP to design habitat corridors and advocating for a broader use of circuit theory modelling. Finally, our results emphasize the need for an objective definition of landscape scales (landscape extent and thematic resolution) and highlight potential pitfalls associated with parameterization of resistance surfaces. © 2017 John Wiley & Sons Ltd.

  12. Serum concentration of immunoglobulin G-type antibodies against the whole Epstein-Barr nuclear antigen 1 and its aa35-58 or aa398-404 fragments in the sera of patients with systemic lupus erythematosus and multiple sclerosis.

    Science.gov (United States)

    Csuka, D; Simon, D; Hóbor, R; Uray, K; Prohászka, Z; Bánlaki, Z; Jani, P K; Szilágyi, Á; Hudecz, F; Rajczy, K; Beke, G; Boros Major, A; Tordai, A; Illés, Z; Berki, T; Czirják, L; Füst, G

    2013-03-01

    Several studies suggest that infection by Epstein-Barr virus (EBV) might be one of the environmental factors which facilitates the development of autoimmune disorders in genetically susceptible individuals. Recent data indicate that high anti-Epstein-Barr nuclear antigen 1 (EBNA)-1 immunoglobulin (Ig)G titre is a strong risk factor for multiple sclerosis (MS) in patients both with and without the main genetic predisposing trait, human leucocyte antigen (HLA)-DRB1*15:01. Because no similar studies have been published in systemic lupus erythematosus (SLE) patients, we determined the HLA-DRB1*15:01 carrier state and the serum titres against the whole EBNA-1 and its small fragments aa35-58 and aa398-404 in 301 SLE patients, 135 MS patients and in 345 healthy controls. The carrier state of the HLA-DRB1*15:01 allele was deduced from genotyping of a tagSNP (rs3135388) by applying a Taqman-based assay. The serum concentrations of antibodies to EBNA-1 and its aa35-58 or aa398-404 fragments were determined using a commercial assay (ETI-EBNA-G) and home-made enzyme-linked immunosorbent assays, respectively. The serum concentration of anti-EBNA-1 antibodies was significantly (P HLA-DRB1*15:01 carriers and non-carriers. Furthermore, titres of antibodies against the aa35-58 EBNA-1 fragment were elevated both in MS and SLE patients. By contrast, the levels of aa398-404 EBNA-1 antibodies were elevated significantly only in the SLE patients. These findings indicate that high anti-EBNA-1 IgG titres are HLA-DRB1*15:01-independent risk factors not only for MS, but also for SLE, while high antibody titres against the aa398-404 fragment are characteristic for SLE. © 2012 British Society for Immunology.

  13. Phylogeography and genetic effects of habitat fragmentation on endemic Urophysa (Ranunculaceae) in Yungui Plateau and adjacent regions.

    Science.gov (United States)

    Xie, Deng-Feng; Li, Min-Jie; Tan, Jin-Bo; Price, Megan; Xiao, Qun-Ying; Zhou, Song-Dong; Yu, Yan; He, Xing-Jin

    2017-01-01

    Urophysa is a Chinese endemic genus with only two species (U. rockii and U. henryi) distributed in Yungui Plateau (Guizhou Province) and adjacent regions (i.e., Provinces of Hunan, Hubei, Chongqing and Sichuan). The aim of this study was to determine the genetic diversity and population differentiation within Urophysa and investigate the effect of the Yungui Plateau uplift and climate oscillations on evolution of Urophysa. In this study, micro-morphological characteristics, nine microsatellite loci (SSR), two nuclear loci (ITS and ETS) and two chloroplast fragments (psbA-trnH and trnL-trnF) were used to analyze the phylogenetic relationships and assess genetic and phylogeographical structure of Urophysa. Isolation by distance (IBD) was performed to research the effects of geographical isolation. We detected high genetic diversity at the species level but low genetic diversity within populations. Striking genetic differentiation (AMOVA) among populations and a significant phylogeographical structure (NST > GST, p habitat fragmentation played an important role in the genetic diversity and population differentiation of U. rockii and U. henryi. Heterogenous geomorphological configuration and complicated environment resulted from rapid uplift of the Yungui Plateau were inferred as important incentives for the modern phylogeograhpical pattern and species divergence of Urophysa. The geographical isolation, limited gene flow, specialized morphologies and the Pleistocene climatic oscillation greatly contributed to the allopatric divergence of U. rockii. Significant genetic drift and inbreeding were detected in these two species, in situ measures should be implemented to protect them.

  14. Landscape genetics indicate recently increased habitat fragmentation in African forest-associated chafers.

    Science.gov (United States)

    Eberle, Jonas; Rödder, Dennis; Beckett, Marc; Ahrens, Dirk

    2017-05-01

    Today, indigenous forests cover less than 0.6% of South Africa's land surface and are highly fragmented. Most forest relicts are very small and typically occur in fire-protected gorges along the eastern Great Escarpment. Yet, they hold a unique and valuable fauna with high endemism and ancient phylogenetic lineages, fostered by long-term climatic stability and complex microclimates. Despite numerous studies on southern African vegetation cover, the current state of knowledge about the natural extension of indigenous forests is rather fragmentary. We use an integrated approach of population-level phylogeography and climatic niche modeling of forest-associated chafer species to assess connectivity and extent of forest habitats since the last glacial maximum. Current and past species distribution models ascertained potential fluctuations of forest distribution and supported a much wider potential current extension of forests based on climatic data. Considerable genetic admixture of mitochondrial and nuclear DNA among many populations and an increase in mean population mutation rate in Extended Bayesian Skyline Plots of all species indicated more extended or better connected forests in the recent past (habitat succession scenarios suggests considerable loss of habitat connectivity. As major anthropogenic influence is likely, conservational actions need to be considered. © 2017 John Wiley & Sons Ltd.

  15. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

    Science.gov (United States)

    Heo, Mi-Ae; Kim, Su-Hyun; Kim, So-Yeon; Kim, Yu-Jin; Chung, Junho; Oh, Min-Kyu; Lee, Sun-Gu

    2006-05-01

    c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction

  16. Immune System and Genetics: A Different Approach to the Diversity of Antibodies

    International Nuclear Information System (INIS)

    Matta Camacho, Nubia Estela

    2011-01-01

    It is common to find in immunology or genetic books a chapter entitled immune system and genetics; this association focuses on how the generation of antibodies broke the paradigm one gene, one protein, since in this case one gene generates millions of proteins. However, the immune system has many more links to genetics and heredity. For example, any substance or compound that an organism produces is a potential antigen, when it is recognized as foreign by the immune system of another organism from the same or different species. The proteins that are potentially antigenic are encoded by the individual's genotype. The ability of the immune system to respond to antigenic proteins, as well as the type and intensity of that response, are also correlated with the organism's genotype. In addition, deficiencies in the immune response may be associated with mutations or genetic polymorphisms, which result in susceptibility to infection diseases.

  17. SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells.

    Science.gov (United States)

    Hussain, Ahmad Fawzi; Kampmeier, Florian; von Felbert, Verena; Merk, Hans-F; Tur, Mehmet Kemal; Barth, Stefan

    2011-12-21

    Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.

  18. Application of a single-chain fragment variable (scFv antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

    Directory of Open Access Journals (Sweden)

    Xiaobo Xu

    2017-09-01

    Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P < 0.05. The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

  19. Comparative Population Genetic Structure of the Endangered Southern Brown Bandicoot, Isoodon obesulus, in Fragmented Landscapes of Southern Australia.

    Directory of Open Access Journals (Sweden)

    You Li

    Full Text Available Genetic connectivity is a key factor for maintaining the persistence of populations in fragmented landscapes. In highly modified landscapes such us peri-urban areas, organisms' dispersal among fragmented habitat patches can be reduced due to the surrounding matrix, leading to subsequent decreased gene flow and increased potential extinction risk in isolated sub-populations. However, few studies have compared within species how dispersal/gene flow varies between regions and among different forms of matrix that might be encountered. In the current study, we investigated gene flow and dispersal in an endangered marsupial, the southern brown bandicoot (Isoodon obesulus in a heavily modified peri-urban landscape in South Australia, Australia. We used 14 microsatellite markers to genotype 254 individuals which were sampled from 15 sites. Analyses revealed significant genetic structure. Our analyses also indicated that dispersal was mostly limited to neighbouring sites. Comparisons of these results with analyses of a different population of the same species revealed that gene flow/dispersal was more limited in this peri-urban landscape than in a pine plantation landscape approximately 400 km to the south-east. These findings increase our understanding of how the nature of fragmentation can lead to profound differences in levels of genetic connectivity among populations of the same species.

  20. Selection criteria for scoring amplified fragment length polymorphisms (AFLPs) positively affect the reliability of population genetic parameter estimates.

    Science.gov (United States)

    Herrmann, Doris; Poncet, Bénédicte N; Manel, Stéphanie; Rioux, Delphine; Gielly, Ludovic; Taberlet, Pierre; Gugerli, Felix

    2010-04-01

    A reliable data set is a fundamental prerequisite for consistent results and conclusions in population genetic studies. However, marker scoring of genetic fingerprints such as amplified fragment length polymorphisms (AFLPs) is a highly subjective procedure, inducing inconsistencies owing to personal or laboratory-specific criteria. We applied two alternative marker selection algorithms, the newly developed script scanAFLP and the recently published AFLPScore, to a large AFLP genome scan to test how population genetic parameters and error rates were affected. These results were confronted with replicated random selections of marker subsets. We show that the newly developed marker selection criteria reduced the mismatch error rate and had a notable influence on estimates of genetic diversity and differentiation. Both effects are likely to influence biological inference. For example, genetic diversity (HS) was 29% lower while genetic differentiation (FST) was 8% higher when applying scanAFLP compared with AFLPScore. Likewise, random selections of markers resulted in substantial deviations of population genetic parameters compared with the data sets including specific selection criteria. These randomly selected marker sets showed surprisingly low variance among replicates. We conclude that stringent marker selection and phenotype calling reduces noise in the data set while retaining patterns of population genetic structure.

  1. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

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    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  2. Genetic control of the radiosensitivity of lymphoid cells for antibody formation ability in mice

    International Nuclear Information System (INIS)

    Okumoto, Masaaki; Mori, Nobuko; Esaki, Kozaburo; Imai, Shunsuke; Haga, Satomi; Hilgers, Jo; Takamori, Yasuhiko.

    1994-01-01

    To analyze the genetic basis of the relationship between the radiosensitivity of the immune response and radiation lymphomagenesis, we examined the radiosensitivity of lymphoid cells for antibody formation in BALB/cHeA, STS/A, F 1 hybrids, and their recombinant inbred mouse strains. The decrease in the number of plaque-forming spleen cells in BALB/cHeA mice exposed to 3 Gy X-irradiation was more than tenfold that in STS/A mice. The phenotype of radioresistance was dominant over sensitivity. The coincidence between the strain distribution patterns of the genetic markers and radiosensitivities of antibody formation in the various recombinant inbred strains was in the region with the lgh locus on chromosome 12. There was obvious difference between the patterns in the region containing the lfa locus on chromosome 4 which has been shown to be related to the incidence of radiation-induced lymphomas. These results indicate that the region on chromosome 12 may contain major gene(s) related to radiosensitivity for antibody formation. (author)

  3. Sampling scheme on genetic structure of tree species in fragmented tropical dry forest: an evaluation from landscape genetic simulations

    Science.gov (United States)

    Yessica Rico; Marie-Stephanie. Samain

    2017-01-01

    Investigating how genetic variation is distributed across the landscape is fundamental to inform forest conservation and restoration. Detecting spatial genetic discontinuities has value for defining management units, germplasm collection, and target sites for reforestation; however, inappropriate sampling schemes can misidentify patterns of genetic structure....

  4. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

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    Urszula Jarocka

    2014-08-01

    Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

  5. Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice.

    Science.gov (United States)

    Bugli, Francesca; Graffeo, Rosalia; Paroni Sterbini, Francesco; Torelli, Riccardo; Masucci, Luca; Sali, Michela; Grasso, Alfonso; Rufini, Stefano; Ricci, Enzo; Fadda, Giovanni; Pescatori, Mario

    2008-03-15

    Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

  6. Life history and past demography maintain genetic structure, outcrossing rate, contemporary pollen gene flow of an understory herb in a highly fragmented rainforest

    Directory of Open Access Journals (Sweden)

    Pilar Suárez-Montes

    2016-12-01

    Full Text Available Introduction Theory predicts that habitat fragmentation, by reducing population size and increasing isolation among remnant populations, can alter their genetic diversity and structure. A cascade of effects is expected: genetic drift and inbreeding after a population bottleneck, changes in biotic interactions that may affect, as in the case of plants, pollen dynamics, mating system, reproductive success. The detection of the effects of contemporary habitat fragmentation on the genetic structure of populations are conditioned by the magnitude of change, given the few number of generations since the onset of fragmentation, especially for long-lived organisms. However, the present-day genetic structure of populations may bear the signature of past demography events. Here, we examine the effects of rainforest fragmentation on the genetic diversity, population structure, mating system (outcrossing rate, indirect gene flow and contemporary pollen dynamics in the understory herb Aphelandra aurantiaca. Also, we assessed its present-day genetic structure under different past demographic scenarios. Methods Twelve populations of A. aurantiaca were sampled in large (4, medium (3, and small (5 forest fragments in the lowland tropical rainforest at Los Tuxtlas region. Variation at 11 microsatellite loci was assessed in 28–30 reproductive plants per population. In two medium- and two large-size fragments we estimated the density of reproductive plants, and the mating system by analyzing the progeny of different mother plants per population. Results Despite prevailing habitat fragmentation, populations of A. aurantiaca possess high genetic variation (He = 0.61, weak genetic structure (Rst = 0.037, and slight inbreeding in small fragments. Effective population sizes (Ne were large, but slightly lower in small fragments. Migrants derive mostly from large and medium size fragments. Gene dispersal is highly restricted but long distance gene dispersal events

  7. Life history and past demography maintain genetic structure, outcrossing rate, contemporary pollen gene flow of an understory herb in a highly fragmented rainforest.

    Science.gov (United States)

    Suárez-Montes, Pilar; Chávez-Pesqueira, Mariana; Núñez-Farfán, Juan

    2016-01-01

    Theory predicts that habitat fragmentation, by reducing population size and increasing isolation among remnant populations, can alter their genetic diversity and structure. A cascade of effects is expected: genetic drift and inbreeding after a population bottleneck, changes in biotic interactions that may affect, as in the case of plants, pollen dynamics, mating system, reproductive success. The detection of the effects of contemporary habitat fragmentation on the genetic structure of populations are conditioned by the magnitude of change, given the few number of generations since the onset of fragmentation, especially for long-lived organisms. However, the present-day genetic structure of populations may bear the signature of past demography events. Here, we examine the effects of rainforest fragmentation on the genetic diversity, population structure, mating system (outcrossing rate), indirect gene flow and contemporary pollen dynamics in the understory herb Aphelandra aurantiaca . Also, we assessed its present-day genetic structure under different past demographic scenarios. Twelve populations of A. aurantiaca were sampled in large (4), medium (3), and small (5) forest fragments in the lowland tropical rainforest at Los Tuxtlas region. Variation at 11 microsatellite loci was assessed in 28-30 reproductive plants per population. In two medium- and two large-size fragments we estimated the density of reproductive plants, and the mating system by analyzing the progeny of different mother plants per population. Despite prevailing habitat fragmentation, populations of A. aurantiaca possess high genetic variation ( H e  = 0.61), weak genetic structure ( R st  = 0.037), and slight inbreeding in small fragments. Effective population sizes ( N e ) were large, but slightly lower in small fragments. Migrants derive mostly from large and medium size fragments. Gene dispersal is highly restricted but long distance gene dispersal events were detected. Aphelandra

  8. Isolation and characterisation of a human-like antibody fragment (scFv that inactivates VEEV in vitro and in vivo.

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    Torsten Rülker

    Full Text Available Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv, ToR67-3B4, from a non-human primate (NHP antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

  9. Phylogeography and genetic effects of habitat fragmentation on endangered Taxus yunnanensis in southwest China as revealed by microsatellite data.

    Science.gov (United States)

    Miao, Y C; Lang, X D; Zhang, Z Z; Su, J R

    2014-03-01

    It is not known how the profoundly complex topography and habitat heterogeneity generated by the uplift of the Qinghai-Tibetan Plateau (QTP) during the late Tertiary affected population genetic structure of endangered Taxus yunnanensis. In addition, the effects of habitat fragmentation due to anthropogenic disturbance on genetic diversity and population differentiation of this species have not been studied. T. yunnanensis is an ancient tree/shrub mainly distributed in southwest China. Recently, the species has suffered a sharp decline due to excessive logging for its famous anticancer metabolite taxol, resulting in smaller and more isolated populations. To understand the phylogeography and genetic consequences of habitat fragmentation of this endangered species, using 11 polymorphic microsatellites, we genotyped 288 individuals from 14 populations from a range-wide sampling in China. Our results suggest that two different population groups that were once isolated have persisted in situ during glacial periods in both areas, and have not merged since. Habitat fragmentation has led to significant genetic bottlenecks, high inbreeding and population divergence in this species. The two different population groups of T. yunnanensis could be attributed to restricted gene flow caused through isolation by geographical barriers and by habitat heterogeneity during uplift of the QTP, or the existence of two separate glacial refugia during the Pleistocene. In situ and ex situ conservation of the two Evolutionarily Significant Units (ESUs), artificial gene flow between populations and a comprehensive understanding of the pollination system in this endangered species are suggested from this study. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  10. Fragmentation-related patterns of genetic differentiation in pedunculate oak (Quercus robur) at two hierarchical scales

    OpenAIRE

    Pohjanmies, Tähti; Elshibli, Sakina; Pulkkinen, Pertti; Rusanen, Mari; Vakkari, Pekka; Korpelainen, Helena; Roslin, Tomas

    2016-01-01

    Populations at species' range margins are expected to show lower genetic diversity than populations at the core of the range. Yet, long-lived, widespread tree species are expected to be resistant to genetic impoverishment, thus showing comparatively high genetic diversity within populations and low differentiation among populations. Here, we study the distribution of genetic variation in the pedunculate oak (Quercus robur L.) at its range margin in Finland at two hierarchical scales using 15 ...

  11. A high-density genetic map of cucumber derived from Specific Length Amplified Fragment sequencing (SLAF-seq).

    Science.gov (United States)

    Xu, Xuewen; Xu, Ruixue; Zhu, Biyun; Yu, Ting; Qu, Wenqin; Lu, Lu; Xu, Qiang; Qi, Xiaohua; Chen, Xuehao

    2014-01-01

    High-density genetic map provides an essential framework for accurate and efficient genome assembly and QTL fine mapping. Construction of high-density genetic maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. In this research, a high-density genetic map of cucumber (Cucumis sativus L.) was successfully constructed across an F2 population by a recently developed Specific Length Amplified Fragment sequencing (SLAF-seq) method. In total, 18.69 GB of data containing 93,460,000 paired-end reads were obtained after preprocessing. The average sequencing depth was 44.92 in the D8 (female parent), 42.16 in the Jin5-508 (male parent), and 5.01 in each progeny. 79,092 high-quality SLAFs were detected, of which 6784 SLAFs were polymorphic, and 1892 of the polymorphic markers met the requirements for constructing genetic map. The genetic map spanned 845.87 cm with an average genetic distance of 0.45 cm. It is a reliable linkage map for fine mapping and molecular breeding of cucumber for its high marker density and well-ordered markers.

  12. Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

    Science.gov (United States)

    Demezas, David H.; Reardon, Terry B.; Watson, John M.; Gibson, Alan H.

    1991-01-01

    Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships. PMID:16348600

  13. Population genetics data help to guide the conservation of palm species with small population sizes and fragmented habitats in Madagascar

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    Lauren M. Gardiner

    2017-05-01

    Full Text Available Background The need to incorporate genetic data into conservation management decisions is increasingly recognised. However, many published studies represent a ‘gold standard’ of sampling, techniques, and analyses. Such rigour is often not possible with limited funding and resourcing available for developing plans for the increasing number of threatened species requiring conservation management. Two endemic palm species of the Itremo Massif in central Madagascar, Dypsis ambositrae and D. decipiens, are known to be threatened with extinction and conservation management for these species is a priority for the newly created protected area in the region. Methods The genetic diversity of these two species was studied using the relatively low-cost and rapid AFLP technique. DNA fragments generated using three primer combinations were analysed for 20 and 50 individuals of the two species, respectively, from across their ranges. Results Genetic diversity was relatively low for both species. The two sites where the highly restricted D. ambositrae grows were found to be genetically distinct (although overall heterozygosity was low. Despite having a much wider distribution and relatively large population, D. decipiens did not show clear geographical nor genetic groupings and had similarly low genetic heterozygosity to D. ambositrae. Discussion and Recommendations With so few individuals remaining in the wild and two genetically distinct subpopulations, it is recommended that both sites of D. ambositrae are conserved and that seed are collected from both for ex situ conservation and potential future reintroduction. It may be less important to focus resources on conserving or collecting ex situ material from all sites where D. decipiens is found, as the genetic diversity represented by each subpopulation is limited and increasing sampling may not protect significantly higher levels of genetic diversity. This study provides data that inform and support

  14. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  15. EXPRESSION OF RECOMBINANT ANTIBODY FRAGMENT, ANTI BNP-SCFV ON THE PERIPLASM OF Escherichia coli FOR THE DETECTION OF HEART FAILURE

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    Shabarni Gaffar

    2017-05-01

    Full Text Available Basic natriuretic peptide (BNP is a polypeptide hormone consist of 32 amino acids that secreted by the heart ventricle to respond the excessive stretching of heart muscle cells. BNP can be used as prognostic marker for patients with heart failure. The presence of BNP in blood can be detected by BNP antibody, which is anti BNP-single chain variable fragment (Anti BNP-SCFV. The antibody is a combination of polypeptides between varying region on the heavy chain (VH and the light chain (VL of immunoglobulin. Anti BNP-SCFV will bind to BNP through the antigen-antibody interaction. Concentration of BNP in a patient’s blood can be detected through the interaction of BNP with Anti BNP-SCFV using immunosensor method. Production of recombinant Anti BNP-SCFV in Escherichia coli as host is reported in the present study. Anti BNP-SCFV was expressed in fusion form with OmpC signal peptide that direct the protein to a periplasmic space. Expression was performed under RhaBad promoter as control using L-rhamnose as inducer. SDS-PAGE characterization showed consistent band at 28 kDa, which was assumed as Anti BNP-SCFV. The optimum expression was found at four hours after induction with 4 mM inducer. Anti BNP-SCFV was secreted from the cell as characterized by the presence of the protein on periplasmic membrane and extracellular fraction.

  16. Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

    Science.gov (United States)

    McLean, Michael D; Chen, Rongji; Yu, Deqiang; Mah, Kor-Zheng; Teat, John; Wang, Haifeng; Zaplachinski, Steve; Boothe, Joseph; Hall, J Christopher

    2012-12-01

    Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

  17. Genetic structure and diversity of Copaifera langsdorffii Desf. in Cerrado fragments of the São Paulo State, Brazil

    Directory of Open Access Journals (Sweden)

    Lia Maris Orth Ritter Antiqueira

    2014-08-01

    Full Text Available The loss of large areas of Cerrado (Brazilian savanna in Brazil can lead to reduced biodiversity and to the extinction of species. Therefore, the present study aimed to investigate the genetic fragility of populations of Copaifera langsdorffii Desf exposed to different anthropic conditions in fragments of Cerrado in the state of São Paulo. The study was carried out in two Experimental Stations operated by the Forest Institute (Assis and Itirapina, in one fully protected conservation unit (Pedregulho and in one private property (Brotas. Analyses were conducted using leaf samples from 353 adult specimens and eight pairs of microsatellite loci. The number of alleles per locus ranged from 13 to 15 in all populations, but the mean number of effective alleles was approximately half this value (7.2 to 9-1. Observed heterozygosity was significant and lower than the expected in all populations. Consequently, all populations deviated from Hardy-Weinberg expected frequencies. Fixation indexes were significant for all populations, with the Pedregulho population having the lowest value (0.189 and Itirapina having the highest (0.283. The analysis of spatial genetic structure detected family structures at distance classes of 20 to 65 m in the populations studied. No clones were detected in the populations. Estimates of effective population size were low, but the area occupied by each population studied was large enough for conservation, medium and long term. Recent reductions or bottlenecks were detected in all four populations. Mean Gst’ (genetic divergence indicated that most of the variation was within populations. Cluster structure analysis based on the genotypes detected K= 4 clusters with distinct allele frequencies patterns. The genetic differentiation observed among populations is consistent with the hypothesis of genetic and geographic isolation. Therefore, it is essential to adopt conservation strategies that raise the gene flow between fragments.

  18. Patterns of genetic diversity and migration in increasingly fragmented and declining orang-utan (Pongo pygmaeus) populations from Sabah, Malaysia.

    Science.gov (United States)

    Goossens, B; Chikhi, L; Jalil, M F; Ancrenaz, M; Lackman-Ancrenaz, I; Mohamed, M; Andau, P; Bruford, M W

    2005-02-01

    We investigated the genetic structure within and among Bornean orang-utans (Pongo pygmaeus) in forest fragments of the Lower Kinabatangan flood plain in Sabah, Malaysia. DNA was extracted from hair and faecal samples for 200 wild individuals collected during boat surveys on the Kinabatangan River. Fourteen microsatellite loci were used to characterize patterns of genetic diversity. We found that genetic diversity was high in the set of samples (mean H(E) = 0.74) and that genetic differentiation was significant between the samples (average F(ST) = 0.04, P < 0.001) with F(ST) values ranging from low (0.01) to moderately large (0.12) values. Pairwise F(ST) values were significantly higher across the Kinabatangan River than between samples from the same river side, thereby confirming the role of the river as a natural barrier to gene flow. The correlation between genetic and geographical distance was tested by means of a series of Mantel tests based on different measures of geographical distance. We used a Bayesian method to estimate immigration rates. The results indicate that migration is unlikely across the river but cannot be completely ruled out because of the limited F(ST) values. Assignment tests confirm the overall picture that gene flow is limited across the river. We found that migration between samples from the same side of the river had a high probability indicating that orang-utans used to move relatively freely between neighbouring areas. This strongly suggests that there is a need to maintain migration between isolated forest fragments. This could be done by restoring forest corridors alongside the river banks and between patches.

  19. Predicting Landscape-Genetic Consequences of Habitat Loss, Fragmentation and Mobility for Multiple Species of Woodland Birds

    Science.gov (United States)

    Amos, J. Nevil; Bennett, Andrew F.; Mac Nally, Ralph; Newell, Graeme; Pavlova, Alexandra; Radford, James Q.; Thomson, James R.; White, Matt; Sunnucks, Paul

    2012-01-01

    Inference concerning the impact of habitat fragmentation on dispersal and gene flow is a key theme in landscape genetics. Recently, the ability of established approaches to identify reliably the differential effects of landscape structure (e.g. land-cover composition, remnant vegetation configuration and extent) on the mobility of organisms has been questioned. More explicit methods of predicting and testing for such effects must move beyond post hoc explanations for single landscapes and species. Here, we document a process for making a priori predictions, using existing spatial and ecological data and expert opinion, of the effects of landscape structure on genetic structure of multiple species across replicated landscape blocks. We compare the results of two common methods for estimating the influence of landscape structure on effective distance: least-cost path analysis and isolation-by-resistance. We present a series of alternative models of genetic connectivity in the study area, represented by different landscape resistance surfaces for calculating effective distance, and identify appropriate null models. The process is applied to ten species of sympatric woodland-dependant birds. For each species, we rank a priori the expectation of fit of genetic response to the models according to the expected response of birds to loss of structural connectivity and landscape-scale tree-cover. These rankings (our hypotheses) are presented for testing with empirical genetic data in a subsequent contribution. We propose that this replicated landscape, multi-species approach offers a robust method for identifying the likely effects of landscape fragmentation on dispersal. PMID:22363508

  20. Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Samah, S; Valadez-Moctezuma, E; Peláez-Luna, K S; Morales-Manzano, S; Meza-Carrera, P; Cid-Contreras, R C

    2016-06-03

    Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

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

    Science.gov (United States)

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

    2013-11-01

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

  2. The genetic defect of fragmented coronoid process in Labrador retrievers and other skeletal diseases in dogs

    NARCIS (Netherlands)

    Temwichitr, J.

    2009-01-01

    Fragmented medial coronoid process (FCP) is the main component of elbow dysplasia (ED) in dogs, which also includes osteochondrosis of the humeral condyle (OCD), elbow incongruity (INC), and ununited anconeal process (UAP). FCP is recognized as a hereditary disease in many breeds and is a major

  3. Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

    Science.gov (United States)

    Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C

    2015-12-02

    Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

  4. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.

    Science.gov (United States)

    Kaiser, Philipp D; Maier, Julia; Traenkle, Bjoern; Emele, Felix; Rothbauer, Ulrich

    2014-11-01

    In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

    Science.gov (United States)

    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.

  6. Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

    Directory of Open Access Journals (Sweden)

    Els Beirnaert

    2017-07-01

    Full Text Available The activity of tumor necrosis factor (TNF, a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™ were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3 were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  9. Separating the effects of habitat area, fragmentation and matrix resistance on genetic differentiation in complex landscapes

    Science.gov (United States)

    Samuel A. Cushman; Andrew J. Shirk; Erin L. Landguth

    2012-01-01

    Little is known about how variation in landscape mosaics affects genetic differentiation. The goal of this paper is to quantify the relative importance of habitat area and configuration, as well as the contrast in resistance between habitat and non-habitat, on genetic differentiation. We hypothesized that habitat configuration would be more influential than habitat...

  10. Genetic Diversity and Sectional Relationships from an Amplified Fragment Length Polymorphism Analysis of Taiwan Bananas

    OpenAIRE

    Chang, Shu-Fen; Chang, Yueh-Long; Yen, Yung-Fu; Miyajima, Ikuo; Huang, Kuang-Liang

    2017-01-01

    Phylogenetic relationships among 19 Musa species or cultivars were examined through DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis. The AFLP analysis was performed on the Musa species or cultivars with 21 primer combinations, yielding a total of 6,348 DNA bands, among which 6,113 (96.3%) were polymorphic. M. itinerans var. formosana demonstrated 133 monomorphic bands, which is the most among all samples. Unweighted pair–group method with arithmetic averages was...

  11. Genetic structure of fragmented populations of red squirrel (Sciurus vulgaris) in the UK.

    Science.gov (United States)

    Barratt, E M; Gurnell, J; Malarky, G; Deaville, R; Bruford, M W

    1999-12-01

    The relationships among 207 squirrels from 12 locations in the UK and three in mainland Europe were examined using mitochondrial DNA (mtDNA) control region sequence. Twenty-six haplotypes were detected, many of which were population specific. Eighty per cent of the populations analysed contained two or more haplotypes. Hierarchical analysis of molecular variance showed the majority of genetic variation to be partitioned among populations. Genetic diversity varied considerably within the UK, and conformed to no obvious geographical trend. The populations in Argyll and Spadeadam Forest showed the highest levels of variation in the UK. However, the greatest genetic diversity was seen in Bavaria, southern Germany where six unique alleles were detected in a sample of 10 individuals. Phylogenetic analysis revealed no evolutionary divergence between UK and mainland European haplotypes. We conclude that, within the UK, the genetic patterns observed are most likely to be explained by the effects of genetic drift which has occurred since the isolation of populations during the past few hundred years, hence we cannot detect any underlying phylogeographic pattern. Therefore, the use of larger, geographically distinct populations within the UK for augmentation of small isolated populations is unlikely to pose problems of genetic incompatibility. Further, the role that demographic factors may have in complicating the application of current genetically based management unit criteria is likely to need further attention.

  12. Genetic variation and heritability of the antibody response to Mycobacterium avium subspecies paratuberculosis in Danish Hostein cows

    DEFF Research Database (Denmark)

    Mortensen, Hanne; Nielsen, Søren Saxmose; Berg, Peer

    2004-01-01

    The purpose of this study was to estimate the genetic variation and the heritability of the ability to establish an immune response by producing antibodies to Mycobacterium avium subsp. paratuberculosis. Antibody levels were determined using an ELISA and measuring optical density (OD) values from...... milk samples of 11,535 cows from 99 herds. The pedigree of the 11,535 cows and information about days in milk, parity, milk yield, and others were obtained from the Danish Cattle database. The statistical analyses were made using a bivariate mixed animal model. The bivariate model with daily milk yield...... and OD as dependent variables showed a significant heritability of the ability to produce Mycobacterium avium subsp. paratuberculosis antibodies of 0.102 (genetic variance = 0.054) and a nonsignificant genetic correlation of −0.037 between daily milk yield and OD. When a sire model was used...

  13. Genetic Diversity in Relict and Fragmented Populations of Ulmus glabra Hudson in the Central System of the Iberian Peninsula

    Directory of Open Access Journals (Sweden)

    María Martín del Puerto

    2017-04-01

    Full Text Available Ulmus glabra Hudson, or Wych elm, occurs as fragmented and relict natural populations in the Central System, which acts as a refugium in the Iberian Peninsula. Considering the importance of the Central System populations of U. glabra, the main objective was to assess their genetic diversity using nuclear microsatellite markers. A total of 360 different genotypes were detected in the 427 U. glabra individuals analyzed. Wych elm populations showed a highly significant genetic differentiation (24%; p = 0.0001. Of the 22 populations studied, population of Rozas de Puerto Real (ROZ showed the highest values of effective number of alleles (2.803, mean Shannon’s diversity (1.047 and expected heterozygosity (0.590. Populations of ROZ and Mombeltrán (MOM showed the highest values of observed heterozygosity (0.838 and 0.709, respectively, and highly negative values for inbreeding coefficient (−0.412 and −0.575, respectively. Also, most of putative hybrids (50 of 55 were observed in these two populations. Demographic analysis revealed signals for recent (four populations and ancestral (fifteen populations bottlenecks. Fragmented populations with diminishing number of individuals, along with anthropogenic intervention and Dutch elm disease (DED, are the main threats to U. glabra populations. From a future perspective, the information generated can be considered in the formulation of conservation strategies for U. glabra populations in the Central System.

  14. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    Science.gov (United States)

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  15. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    Science.gov (United States)

    Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  16. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    Science.gov (United States)

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Amplified fragment length polymorphism used to investigate genetic variability of the stable fly (Diptera: Muscidae) across North America.

    Science.gov (United States)

    Kneeland, K M; Skoda, S R; Foster, J E

    2013-09-01

    The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan pest of livestock and humans. The pestiferous nature and painful bite cause stress to cattle and other animals. The stress and resulting avoidance behaviors manifest as reductions in weight gain or milk production in cattle; estimated annual economic loss in the United States exceeds US$2 billion. Understanding the population genetics of stable flies could provide information on their population dynamics, origins of outbreaks, and geographical patterns of insecticide resistance, resulting in a tactical advantage for developing management strategies. Previous studies, mostly on a local scale, reported a high level of gene flow between locations. Here, we report results wherein amplified fragment length polymorphism was used to determine genetic diversity of stable fly samples consisting of 11-40 individuals from 12 locations representing the United States, Canada, and Panama. The Analysis of Molecular Variance showed that the majority of genetic diversity was within groups; very little was among groups. The F(ST) and G(ST) values were low ( 1.0). The tests of neutrality suggested population expansion, and no genetic differentiation was found between locations. These results show that stable flies have a high level of gene flow on a continental scale, with limited isolation owing to distance or geographical barriers.

  18. Detecting Instability in Animal Social Networks: Genetic Fragmentation Is Associated with Social Instability in Rhesus Macaques

    OpenAIRE

    Beisner, Brianne A.; Jackson, Megan E.; Cameron, Ashley N.; McCowan, Brenda

    2011-01-01

    The persistence of biological systems requires evolved mechanisms which promote stability. Cohesive primate social groups are one example of stable biological systems, which persist in spite of regular conflict. We suggest that genetic relatedness and its associated kinship structure are a potential source of stability in primate social groups as kinship structure is an important organizing principle in many animal societies. We investigated the effect of average genetic relatedness per matri...

  19. Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

    Science.gov (United States)

    Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

    2018-04-01

    Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

  20. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    Science.gov (United States)

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-05-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  1. Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia.

    Science.gov (United States)

    Pechar, Michal; Pola, Robert; Janoušková, Olga; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Tomalová, Barbora; Kovář, Marek

    2018-01-01

    A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    International Nuclear Information System (INIS)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-01-01

    Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  3. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    Science.gov (United States)

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-09

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

  4. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); Li, Lin [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Shibo, E-mail: sjiang@nybloodcenter.org [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China)

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  5. Data on crystal organization in the structure of the Fab fragment from the NIST reference antibody, RM 8671.

    Science.gov (United States)

    Gallagher, D T; Karageorgos, I; Hudgens, J W; Galvin, C V

    2018-02-01

    The reported data describe the crystallization, crystal packing, structure determination and twinning of the unliganded Fab (antigen-binding fragment) from the NISTmAb (standard reference material 8671). The raw atomic coordinates are available as Protein Data Bank structure 5K8A and biological aspects are described in the article, (Karageorgos et al., 2017) [1]. Crystal data show that the packing is unique, and show the basis for the crystal's twinned growth. Twinning is a common and often serious problem in protein structure determination by x-ray crystallography [2]. In the present case the twinning is due to a small deviation (about 0.3 nm) from 4-fold symmetry in the primary intermolecular interface. The deviation produces pseudosymmetry, generating slightly different conformations of the protein, and alternating strong and weak forms of key packing interfaces throughout the lattice.

  6. Occurrence of anti-Leptospira spp. antibodies in Rhipidomys spp. from a forest fragment of the Brazilian Cerrado.

    Science.gov (United States)

    Gomes, D O; Ramos, G B; Alves, V B A; Ciuffa, A Z; Cuccato, L P; Dos Reis, T F M; Lima, A M C; Gonçalves, M C; Tolesano, G V; Rodrigues, V S; Szabó, M P J

    2017-03-01

    Leptospirosis is a zoonotic disease of world importance, and its transmission depends on the interaction between humans and animals. Given the necessity to investigate potential hosts of Leptospira spp., this study verified the prevalence of different serovars in the species of Rhipidomys spp., a widespread sigmodont rodent in Brazil. The studied population originates from a semi-evergreen forest located in the county of Uberlândia, in the state of Minas Gerais. The microscopic agglutination test (MAT) was performed with 14 serovars. Thirteen out of the 43 wild rodents captured showed a positive agglutination reaction, with a greater prevalence of the serovars Pyrogenes, Copenhageni, and Canicola. This study found a prevalence of 30.3% anti-Leptospira spp. antibodies; all positive animals were reactive to more than one serovar.

  7. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

    Science.gov (United States)

    Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

    2008-07-22

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

  8. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    Directory of Open Access Journals (Sweden)

    Soraya S Pereira

    Full Text Available In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅ of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB, surface plasmon resonance (SPR device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with

  9. Recent habitat fragmentation caused by major roads leads to reduction of gene flow and loss of genetic variability in ground beetles.

    Science.gov (United States)

    Keller, Irene; Largiadèr, Carlo R

    2003-02-22

    Although habitat fragmentation is suspected to jeopardize the long-term survival of many species, few data are available on its impact on the genetic variability of invertebrates. We assess the genetic population structure of the flightless ground beetle Carabus violaceus L., 1758 in a Swiss forest, which is divided into several fragments by a highway and two main roads. Eight samples were collected from different forest fragments and analysed at six microsatellite loci. The largest genetic differentiation was observed between samples separated by roads and in particular by the highway. The number of roads between sites explained 44% of the variance in pairwise F(ST) estimates, whereas the age of the road and the geographical distance between locations were not significant factors. Furthermore, a comparison of allelic richness showed that the genetic variability in a small forest fragment isolated by the highway was significantly lower than in the rest of the study area. These findings strongly support the hypothesis that large roads are absolute barriers to gene flow in C. violaceus, which may lead to a loss of genetic variability in fragmented populations.

  10. Fibroblasts of skin fragments as a tool for the investigation of genetic diseases: technical recommendations

    Directory of Open Access Journals (Sweden)

    Coelho Janice Carneiro

    2000-01-01

    Full Text Available Skin biopsies are frequently indicated for investigation and/or confirmation of genetic disorders. Although relatively simple and noninvasive, these procedures require care in order to increase probability of success and to avoid patient discomfort and unnecessary repeated analyses and associated laboratory fees. The present report highlights the importance of skin biopsies in genetic disorder diagnosis and presents general rules for collecting, storing, transporting and processing samples. We recommend its reading to professionals intending to use this important and sometimes fundamental diagnostic tool.

  11. Detecting instability in animal social networks: genetic fragmentation is associated with social instability in rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Brianne A Beisner

    2011-01-01

    Full Text Available The persistence of biological systems requires evolved mechanisms which promote stability. Cohesive primate social groups are one example of stable biological systems, which persist in spite of regular conflict. We suggest that genetic relatedness and its associated kinship structure are a potential source of stability in primate social groups as kinship structure is an important organizing principle in many animal societies. We investigated the effect of average genetic relatedness per matrilineal family on the stability of matrilineal grooming and agonistic interactions in 48 matrilines from seven captive groups of rhesus macaques. Matrilines with low average genetic relatedness show increased family-level instability such as: more sub-grouping in their matrilineal groom network, more frequent fighting with kin, and higher rates of wounding. Family-level instability in multiple matrilines within a group is further associated with group-level instability such as increased wounding. Stability appears to arise from the presence of clear matrilineal structure in the rhesus macaque group hierarchy, which is derived from cohesion among kin in their affiliative and agonistic interactions with each other. We conclude that genetic relatedness and kinship structure are an important source of group stability in animal societies, particularly when dominance and/or affilative interactions are typically governed by kinship.

  12. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  13. Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages.

    Science.gov (United States)

    Romero-Martínez, Rafael; Curiel-Quesada, Everardo; Becerril-Luján, Baltazar; Flores-Carreón, Arturo; Pérez-Torres, Armando; Taylor, Maria Lucia

    2007-06-01

    A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

    2016-01-01

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  16. Genetic manipulation of B cells for the isolation of rare therapeutic antibodies from the human repertoire

    NARCIS (Netherlands)

    Kwakkenbos, Mark J.; Bakker, Arjen Q.; van Helden, Pauline M.; Wagner, Koen; Yasuda, Etsuko; Spits, Hergen; Beaumont, Tim

    2014-01-01

    Antibody based therapies are increasingly applied to prevent and treat human disease. While the majority of antibodies currently on the market are chimeric or humanized antibodies from rodents, the focus has now shifted to the isolation and development of fully human antibodies. By retroviral

  17. Genetic Characterization Of Syrian Erwinia Amylovora Strains By Amplified Fragment Length Polymorphism Technique

    International Nuclear Information System (INIS)

    Ammouneh, H.; Arabi, M.; Shoaib, A.

    2011-01-01

    Thirty Erwinia amylovora strains, collected from the main rosaceous crop-growing regions in Syria, were chosen as representatives of all major pathogenicity groups and were genetically studied by AFLP. Eight primer combinations were utilized and approximately 300 scorable bands in total were generated. Based on similarity coefficient, E. amylovora strains were placed into a main cluster containing two sub clusters, indicating very low genetic variations among the studied pathogen. The existence of two plasmids, pEA29 (present in nearly all E. amylovora isolates) and pEL60 (present mainly in Lebanese strains), was confirmed using multiplex PCR in all tested Syrian E. amylovora strains, indicating that Lebanese and Syrian isolates may share a common origin.(author)

  18. The genetic impact of translocations and habitat fragmentation in chamois (Rupicapra) spp

    Czech Academy of Sciences Publication Activity Database

    Crestanello, B.; Pecchioli, E.; Vernesi, C.; Mona, S.; Martínková, Natália; Janiga, M.; Hauffe, H. C.; Bertorelle, G.

    2009-01-01

    Roč. 100, č. 6 (2009), s. 691-708 ISSN 0022-1503 R&D Projects: GA ČR GA206/01/0562; GA AV ČR IAA600930609 Institutional research plan: CEZ:AV0Z60930519 Keywords : conservation * hybridization * management * microsatellites * mtDNA * taxonomy Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.052, year: 2009

  19. Genetic relationship and diversity in a sesame (Sesamum indicum L. germplasm collection using amplified fragment length polymorphism (AFLP

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2006-02-01

    Full Text Available Abstract Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia, and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP. Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA. This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres

  20. Genetic relationship and diversity in a sesame (Sesamum indicum L.) germplasm collection using amplified fragment length polymorphism (AFLP)

    Science.gov (United States)

    Laurentin, Hernán E; Karlovsky, Petr

    2006-01-01

    Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia), and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP). Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA). This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres. Future germplasm

  1. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    Science.gov (United States)

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  2. Genetic evidence of tiger population structure and migration within an isolated and fragmented landscape in Northwest India.

    Directory of Open Access Journals (Sweden)

    Patlolla Anuradha Reddy

    Full Text Available Majority of the tiger habitat in Indian subcontinent lies within high human density landscapes and is highly sensitive to surrounding pressures. These forests are unable to sustain healthy tiger populations within a tiger-hostile matrix, despite considerable conservation efforts. Ranthambore Tiger Reserve (RTR in Northwest India is one such isolated forest which is rapidly losing its links with other tiger territories in the Central Indian landscape. Non-invasive genetic sampling for individual identification is a potent technique to understand the relationships between threatened tiger populations in degraded habitats. This study is an attempt to establish tiger movement across a fragmented landscape between RTR and its neighboring forests, Kuno-Palpur Wildlife Sanctuary (KPWLS and Madhav National Park (MNP based on non-invasively obtained genetic data.Data from twelve microsatellite loci was used to define population structure and also to identify first generation migrants and admixed individuals in the above forests.Population structure was consistent with the Central Indian landscape and we could determine significant gene flow between RTR and MNP. We could identify individuals of admixed ancestry in both these forests, as well as first generation migrants from RTR to KPWLS and MNP.Our results indicate reproductive mixing between animals of RTR and MNP in the recent past and migration of animals even today, despite fragmentation and poaching risk, from RTR towards MNP. Substantial conservation efforts should be made to maintain connectivity between these two subpopulations and also higher protection status should be conferred on Madhav National Park.

  3. Genetic Evidence of Tiger Population Structure and Migration within an Isolated and Fragmented Landscape in Northwest India

    Science.gov (United States)

    Bhavanishankar, Maradani; Jaggi, Kanika; Hussain, Shaik Mohammed; Harika, Katakam; Shivaji, Sisinthy

    2012-01-01

    Background Majority of the tiger habitat in Indian subcontinent lies within high human density landscapes and is highly sensitive to surrounding pressures. These forests are unable to sustain healthy tiger populations within a tiger-hostile matrix, despite considerable conservation efforts. Ranthambore Tiger Reserve (RTR) in Northwest India is one such isolated forest which is rapidly losing its links with other tiger territories in the Central Indian landscape. Non-invasive genetic sampling for individual identification is a potent technique to understand the relationships between threatened tiger populations in degraded habitats. This study is an attempt to establish tiger movement across a fragmented landscape between RTR and its neighboring forests, Kuno-Palpur Wildlife Sanctuary (KPWLS) and Madhav National Park (MNP) based on non-invasively obtained genetic data. Methods Data from twelve microsatellite loci was used to define population structure and also to identify first generation migrants and admixed individuals in the above forests. Results Population structure was consistent with the Central Indian landscape and we could determine significant gene flow between RTR and MNP. We could identify individuals of admixed ancestry in both these forests, as well as first generation migrants from RTR to KPWLS and MNP. Conclusions Our results indicate reproductive mixing between animals of RTR and MNP in the recent past and migration of animals even today, despite fragmentation and poaching risk, from RTR towards MNP. Substantial conservation efforts should be made to maintain connectivity between these two subpopulations and also higher protection status should be conferred on Madhav National Park. PMID:22253791

  4. Genetic variation of fish parasite populations in historically connected habitats: undetected habitat fragmentation effect on populations of the nematode Procamallanus fulvidraconis in the catfish Pelteobagrus fulvidraco.

    Science.gov (United States)

    Li, Wen X; Wang, Gui T; Nie, P

    2008-06-01

    Habitat fragmentation may have some significant effects on population genetic structure because geographic distance and physical barriers may impede gene flow between populations. In this study, we investigated whether recent habitat fragmentation affected genetic structure and diversity of populations of the nematode Procamallanus fulvidraconis in the yellowhead catfish, Pelteobagrus fulvidraco. The nematode was collected from 12 localities in 7 floodplain lakes of the Yangtze River. Using 11 intersimple sequence repeat markers, analysis of molecular variance showed that genetic diversity occurred mainly within populations (70.26%). Expected heterozygosity (He) of P. fulvidraconis was barely different between connected (0.2105) and unconnected lakes (0.2083). Population subdivision (Fst) between connected lakes (0.2177) was higher than in unconnected lakes (0.1676). However, the connected and unconnected lakes did not cluster into 2 clades. A Mantel test revealed significant positive correlation between genetic and geographic distances (R = 0.5335, P < 0.01). These results suggest that habitat fragmentation did not cause genetic differentiation among populations or a reduction of diversity in isolated populations of P. fulvidraconis. At least 2 factors may increase the dispersal range of the nematode, i.e., flash flooding in summer and other species of fish that may serve as the definitive hosts. Moreover, lake fragmentation is probably a recent process; population size of the nematode in these lakes is large enough to maintain population structure.

  5. High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli.

    Science.gov (United States)

    Napathorn, Suchada Chanprateep; Kuroki, Motomu; Kuroki, Masahide

    2014-08-01

    The aim of this study was to establish a strategy for high-level production of single-chain variable fragment (scFv) antibodies fused with interleukin-2 (IL-2) in Escherichia coli. We constructed two fusion sequences consisting of a scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (MK-1) and human Interleukin-2(IL-2) gene, ligated the fusions into pET15b and transformed into three different E. coli strains. The effects of temperature, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and duration of IPTG induction were investigated. Employing E. coli strain Rosetta-gami B, which has an oxidizing cytoplasm that facilitates cytoplasmic disulfide bond formation, improved the level of soluble protein expression. Under optimal conditions, the highest levels of fusion protein expression and high percentages of the proteins were found in their soluble form. Specifically, 89.29% (0.28 g/l) of one fusion protein was soluble after a 10-h induction and 84.61% (0.26 g/l) of the other fusion protein was soluble after a separate 10-h induction. When analyzed by enzyme-linked immunosorbent assay, the partially-purified fusion proteins retained a specific binding activity to the cell lysate of Chinese hamster ovary (CHO) cells expressing MK-1. Taken together, the methods described herein permit the production of substantial amounts of the fusion proteins for conducting functional studies on the biological role of these fusion proteins. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  6. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    Science.gov (United States)

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  7. A Comparative Analysis of Genetic Diversity and Structure in Jaguars (Panthera onca, Pumas (Puma concolor, and Ocelots (Leopardus pardalis in Fragmented Landscapes of a Critical Mesoamerican Linkage Zone.

    Directory of Open Access Journals (Sweden)

    Claudia Wultsch

    Full Text Available With increasing anthropogenic impact and landscape change, terrestrial carnivore populations are becoming more fragmented. Thus, it is crucial to genetically monitor wild carnivores and quantify changes in genetic diversity and gene flow in response to these threats. This study combined the use of scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of multiple Neotropical felids coexisting across a fragmented landscape in Belize, Central America. We analyzed data from 14 polymorphic microsatellite loci in 1053 scat samples collected from wild jaguars (Panthera onca, pumas (Puma concolor, and ocelots (Leopardus pardalis. We assessed levels of genetic diversity, defined potential genetic clusters, and examined gene flow for the three target species on a countrywide scale using a combination of individual- and population-based analyses. Wild felids in Belize showed moderate levels of genetic variation, with jaguars having the lowest diversity estimates (HE = 0.57 ± 0.02; AR = 3.36 ± 0.09, followed by pumas (HE = 0.57 ± 0.08; AR = 4.20 ± 0.16, and ocelots (HE = 0.63 ± 0.03; AR = 4.16 ± 0.08. We observed low to moderate levels of genetic differentiation for all three target species, with jaguars showing the lowest degree of genetic subdivision across the country, followed by ocelots and pumas. Although levels of genetic diversity and gene flow were still fairly high, we detected evidence of fine-scale genetic subdivision, indicating that levels of genetic connectivity for wild felids in Belize are likely to decrease if habitat loss and fragmentation continue at the current rate. Our study demonstrates the value of understanding fine-scale patterns of gene flow in multiple co-occurring felid species of conservation concern, which is vital for wildlife movement corridor planning and prioritizing future conservation and management efforts within human-impacted landscapes.

  8. A Comparative Analysis of Genetic Diversity and Structure in Jaguars (Panthera onca), Pumas (Puma concolor), and Ocelots (Leopardus pardalis) in Fragmented Landscapes of a Critical Mesoamerican Linkage Zone.

    Science.gov (United States)

    Wultsch, Claudia; Waits, Lisette P; Kelly, Marcella J

    2016-01-01

    With increasing anthropogenic impact and landscape change, terrestrial carnivore populations are becoming more fragmented. Thus, it is crucial to genetically monitor wild carnivores and quantify changes in genetic diversity and gene flow in response to these threats. This study combined the use of scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of multiple Neotropical felids coexisting across a fragmented landscape in Belize, Central America. We analyzed data from 14 polymorphic microsatellite loci in 1053 scat samples collected from wild jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis). We assessed levels of genetic diversity, defined potential genetic clusters, and examined gene flow for the three target species on a countrywide scale using a combination of individual- and population-based analyses. Wild felids in Belize showed moderate levels of genetic variation, with jaguars having the lowest diversity estimates (HE = 0.57 ± 0.02; AR = 3.36 ± 0.09), followed by pumas (HE = 0.57 ± 0.08; AR = 4.20 ± 0.16), and ocelots (HE = 0.63 ± 0.03; AR = 4.16 ± 0.08). We observed low to moderate levels of genetic differentiation for all three target species, with jaguars showing the lowest degree of genetic subdivision across the country, followed by ocelots and pumas. Although levels of genetic diversity and gene flow were still fairly high, we detected evidence of fine-scale genetic subdivision, indicating that levels of genetic connectivity for wild felids in Belize are likely to decrease if habitat loss and fragmentation continue at the current rate. Our study demonstrates the value of understanding fine-scale patterns of gene flow in multiple co-occurring felid species of conservation concern, which is vital for wildlife movement corridor planning and prioritizing future conservation and management efforts within human-impacted landscapes.

  9. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Science.gov (United States)

    Kuroki, Motomu; Kuroki, Masahide

    2017-01-01

    Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. PMID:28860806

  10. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  11. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts

    DEFF Research Database (Denmark)

    Noël, D; Pelegrin, M; Brockly, F

    2000-01-01

    In vivo production and systemic delivery of therapeutic antibodies by engineered cells might advantageously replace injection of purified antibodies for treating a variety of life-threatening diseases, including cancer, acquired immunodeficiency syndrome, and autoimmune diseases. We report here...... that skin fibroblasts retrovirally transduced to express immunoglobulin genes can be used for sustained long-term systemic delivery of cloned antibodies in immunocompetent mice. Importantly, no anti- idiotypic response against the ectopically expressed model antibody used in this study was observed...

  12. Introduction of five potentially metabolizable linking groups between [sup 111]In-cyclohexyl EDTA derivatives and F(ab')[sub 2] fragments of anti-carcinoembryonic antigen antibody: Pt. 1; A new reproducible synthetic method

    Energy Technology Data Exchange (ETDEWEB)

    Gestin, J.F.; Faivre-Chauvet, A.; Sai-Maurel, C.; Thedrez, P.; Slinkin, M.; Chatal, J.F. (INSERM, 44 - Nantes (France)); Mease, R.C.; Meinken, G.E.; Srivastava, S.C. (Brookhaven National Lab., Upton, NY (United States))

    1993-08-01

    The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of [sup 111]In-labeled antibodies. A general simple synthetic method of preparing cyclohexyl EDTA (CDTA) derivatives containing a linker/spacer group is described. Linkers prepared included a diester, a six carbon aliphatic chain, two thioethers and a disulfide group. The CDTA-linker compounds were coupled to F(Ab')[sub 2] fragments of anti-carcinoembryonic antigen monoclonal antibody and labeled with [sup 111]In with good retention of immunoreactivity. (author).

  13. Introduction of five potentially metabolizable linking groups between 111In-cyclohexyl EDTA derivatives and F(ab')2 fragments of anti-carcinoembryonic antigen antibody--I. A new reproducible synthetic method.

    Science.gov (United States)

    Gestin, J F; Faivre-Chauvet, A; Mease, R C; Sai-Maurel, C; Thédrez, P; Slinkin, M; Meinken, G E; Srivastava, S C; Chatal, J F

    1993-08-01

    The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of 111In-labeled antibodies. A general simple synthetic method of preparing cyclohexyl EDTA (CDTA) derivatives containing a linker/spacer group is described. Linkers prepared included a diester, a six carbon aliphatic chain, two thioethers and a disulfide group. The CDTA-linker compounds were coupled to F(Ab')2 fragments of anti-carcinoembryonic antigen monoclonal antibody and labeled with 111In with good retention of immunoreactivity.

  14. Effects of an Aβ-antibody fragment on Aβ aggregation and astrocytic uptake are modulated by apolipoprotein E and J mimetic peptides.

    Directory of Open Access Journals (Sweden)

    Laia Montoliu-Gaya

    Full Text Available Aβ-Immunotherapy has long been studied in the treatment of Alzheimer's disease (AD, but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aβ-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE and J (apoJ concentrations to non-pathological values, and Aβ internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aβ aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs. Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aβ were studied by CD. The conformational change induced by the interaction of Aβ with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aβ complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aβ uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aβ uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides.

  15. Genetic effects of recent habitat fragmentation in the Thousand-Island Lake region of southeast China on the distylous herb Hedyotis chrysotricha (Rubiaceae).

    Science.gov (United States)

    Yuan, Na; Comes, Hans Peter; Mao, Yun-Rui; Qi, Xin-Shuai; Qiu, Ying-Xiong

    2012-10-01

    Known-age artificial-lake islands provide ideal model systems to elucidate the genetic and evolutionary consequences of anthropogenic habitat fragmentation on very recent time scales. Here, we studied a distylous herb, Hedyotis chrysotricha (Rubiaceae), in the artificially created Thousand-Island Lake (TIL) region of southeast China to explore the genetic consequences of islanding for this species. • Seven microsatellite loci were used to genotype 384 individuals of H. chrysotricha from 18 populations to estimate genetic diversity, population structure, and demographic parameters. • Island populations had significantly lower mean genetic diversity than those from the western/eastern mainland (e.g., H(E) = 0.381 vs. 0.461) and also displayed higher mean subdivision (F(ST) = 0.12 vs. 0.042/0.051). BayesAss analyses indicated moderate levels of migration rates among most populations, whereas Bottleneck did not provide strong evidence for such effects. In consequence, 2MOD strongly favored a gene flow-drift model over a pure drift model in the study area, but concomitantly revealed a relatively greater influence of drift in the island populations as evidenced by their significantly higher probabilities of allelic coancestry (F = 0.184 vs. 0.085). • The observed genetic patterns in H. chrysotricha indicate that recent anthropogenic habitat fragmentation in the TIL region can lead to significant loss of genetic diversity in isolated fragments (islands) due to ongoing drift. By contrast, patterns of random mating, gene flow, and population connectivity have not greatly been modified yet, possibly owing to the species' fruit (seed) dispersal capabilities providing resilience in the face of habitat fragmentation.

  16. Genetic Population Structure of the Ground Beetle, Pterostichus oblongopunctatus, Inhabiting a Fragmented and Polluted Landscape: Evidence for Sex-Biased Dispersal

    Science.gov (United States)

    Lagisz, Malgorzata; Wolff, Kirsten; Sanderson, Roy A; Laskowski, Ryszard

    2010-01-01

    Ground beetles are an integral and functionally important part of many terrestrial ecosystems. Habitat change often influences population genetic structure of carabid beetles. In this study, genetic variation, population differentiation, and sex-specific dispersal patterns were studied in the forest ground beetle, Pterostichus oblongopunctatus F. (Coleoptera: Carabidae), in a fragmented and metal-polluted landscape to assess the consequences of human-induced changes on the population genetic structure. Genotypic variation at five microsatellite loci was screened in 309 beetles from 21 sample locations around zinc-and-lead smelter in southern Poland. Low levels of genetic differentiation among sampling sites were observed, suggesting high gene flow among populations. A negative correlation was found between levels of genetic differentiation and habitat patch size. No significant effects of metal pollution, in terms of genetic bottlenecks and genetic differentiation, were observed. Analyses revealed weak genetic clustering that is loosely tied to the geographic position of the sampled populations. Several tests of sex-biased dispersal were conducted. Most of them indicated male-biased dispersal. Differing levels of dispersal between females and males resulted in sex-specific spatial genetic patterns. Genetic differentiation was significantly correlated with geographical distance for males, but not for females, who were more diverged locally. Also, the effect of habitat patch size was sex-dependent, supporting the finding of different dispersal patterns between the sexes. This study demonstrated the application of microsatellite markers to answer questions regarding complex interactions between population structure and physical properties of the landscape. In the study system, migration appears to be sufficient to override potential effects of environmental pollution as well as habitat fragmentation. This investigation of population genetic structure indicated, for

  17. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    OpenAIRE

    Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-01-01

    International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...

  18. Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen

    2017-08-01

    Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

  19. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

    Directory of Open Access Journals (Sweden)

    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

  20. Effects of digoxin-specific antibody Fab fragment (Digibind) on blood pressure and renal water-sodium metabolism in 5/6 reduced renal mass hypertensive rats.

    Science.gov (United States)

    Kaide, J; Ura, N; Torii, T; Nakagawa, M; Takada, T; Shimamoto, K

    1999-06-01

    The importance of increased endogenous digitalis-like factor (EDLF) in volume-expanded hypertension has been generally agreed. To further clarify the role of EDLF on the development of hypertension and renal water-sodium handling in 5/6 reduced renal mass hypertensive rats (RRM), we studied the effects of acute administration of digoxin-specific antibody Fab fragment (Digibind) in the early phase and the chronic phase of hypertension in RRM. RRM and sham-operated rats were given 1% saline for 1 or 4 weeks. RRM were injected Digibind (60 mg/kg) or vehicle (0.9% saline) intravenously in the first or fourth week under thiobutabarbital anesthesia. All sham-operated rats were administered Digibind under the same condition. Digibind altered neither blood pressure, heart rate, urine volume, nor urinary sodium excretion in sham-operated rats. However, Digibind produced a gradual but significant decline in mean arterial pressure to the level slightly above that in sham-operated rats from 153 +/- 5 to 131 +/- 5 mm Hg in the first week and from 181 +/- 6 to 129 +/- 4 mm Hg in the fourth week without any significant change in heart rate. The decrease in mean arterial pressure at 160 min after Digibind administration in the fourth week (-48 +/- 5 mm Hg) was greater than that in the first week (-22 +/- 4 mm Hg). No differences were observed in urine volume, urinary sodium excretion, or plasma norepinephrine concentration between Digibind and vehicle-treated RRM in either week. These data suggest that EDLF would contribute to both the early and chronic phase in the development of hypertension in RRM.

  1. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  2. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  3. scFv Antibody: Principles and Clinical Application

    Directory of Open Access Journals (Sweden)

    Zuhaida Asra Ahmad

    2012-01-01

    Full Text Available To date, generation of single-chain fragment variable (scFv has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

  4. scFv antibody: principles and clinical application.

    Science.gov (United States)

    Ahmad, Zuhaida Asra; Yeap, Swee Keong; Ali, Abdul Manaf; Ho, Wan Yong; Alitheen, Noorjahan Banu Mohamed; Hamid, Muhajir

    2012-01-01

    To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

  5. Identification of novel genetic Loci associated with thyroid peroxidase antibodies and clinical thyroid disease.

    Directory of Open Access Journals (Sweden)

    Marco Medici

    2014-02-01

    Full Text Available Autoimmune thyroid diseases (AITD are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis, as well as autoimmune hyperthyroidism (Graves' disease. As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10(-8 were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores of these variants on (subclinical hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68-2.81, P = 8.1×10(-8, a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26-1.82, P = 2.9×10(-6, as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66-0.89, P = 6.5×10(-4. The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves' disease (OR: 1.37, 95% CI 1.22-1.54, P = 1.2×10(-7 and OR: 1.25, 95% CI 1.12-1.39, P = 6.2×10(-5. The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18-2.10, P = 1.9×10(-3. This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why

  6. Genetic consequences of fragmentation in "arbor vitae," eastern white cedar (Thuja occidentalis L.), toward the northern limit of its distribution range.

    Science.gov (United States)

    Xu, Huaitong; Tremblay, Francine; Bergeron, Yves; Paul, Véronique; Chen, Cungen

    2012-10-01

    We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive F(is) values and heterozygote deficiency were observed in populations from the marginal (F(is) = 0.244; P(HW) = 0.0042) and discontinuous zones (F(is) = 0.166; P(HW) = 0.0042). However, populations from the continuous zone were in HW equilibrium (F(is) = -0.007; P(HW) = 0.3625). There were no significant latitudinal effects on gene diversity (H(s)), allelic richness (AR), or population differentiation (F(st)). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.

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

    Science.gov (United States)

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

    2017-01-01

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

  8. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    Science.gov (United States)

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection. © 2015 Wiley Periodicals, Inc.

  9. Critical Effects of Urbanization on a Charismatic Carnivore: Genetic Change, Disease and Toxicant Exposure, and Disease Susceptibility in Bobcat Populations in an Urban, Fragmented Landscape

    OpenAIRE

    Serieys, Laurel EK

    2014-01-01

    Urbanization has profound ecological impacts that reach beyond city boundaries. Obvious ecological consequences of urbanization include habitat loss and fragmentation. Anthropogenic barriers reduce habitat connectivity, impede gene flow between populations and accelerate the loss of genetic diversity in populations due to drift. Urbanization may have also cryptic consequences such as the effects of human-introduced toxicants on wildlife populations. Toxicants are a leading cause of population...

  10. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab')2

    International Nuclear Information System (INIS)

    Anderson, C.J.; Schwarz, S.W.; Connett, J.M.

    1995-01-01

    Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab') 2 was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of 64 Cu-BAT-2IT-1A3-F(ab') 2 was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N double-prime,N'double-prime-tetraacetic acid (BAT) was conjugated to 1A3-F(ab') 2 using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab'. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab') 2 from TETA-1A3-Fab' after conjugation prior to labeling with 64 Cu. The biodistribution of 64 Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab') 2 , the immunoreactivity of 64 Cu-labeled 1A3-F(ab') 2 was significantly improved over that of non-FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 , and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 .The improved dosimetry of 64 Cu-labeled FPLC purified BAT-2IT-1A3-F(ab') 2 should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs

  11. Radiolabeled Antibody Fragment for Preparation of (177Lu-DOTAm-PAMAM G3.0-F(ab’2 trastuzumab as a Radiopharmaceutical for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    R.D. Haryuni

    2017-06-01

    Full Text Available Several radiolabeled monoclonal antibodies (mAbs have been used as radioimmunotherapy (RIT agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α particles, beta (β particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of (177Lu-DOTAm-PAMAM G3-F(ab'2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2. Due to its reduced molecular weight, the use of F(ab'2-trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH2 functional groups that are able to bind many DOTAs (£ 31 which in turn can bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab'2-trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab'2-trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC to produce SMCC-F(ab'2-trastuzumab. The next reaction was to conjugate SMCC-F(ab'2-trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab. Finally, the (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab was radiolabelled with 177Lu to produce (177Lu-DOTAm-PAMAM G3.0-F(ab'2-trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column.Received: 31 October 2015; Revised: 30 June 2016; Accepted: 25 September 2016

  12. Genetic relations between natural antibodies binding keyhole limpet hemocyanin and production traits in a purebred layer chicken line.

    Science.gov (United States)

    van der Klein, S A S; Berghof, T V L; Arts, J A J; Parmentier, H K; van der Poel, J J; Bovenhuis, H

    2015-05-01

    Natural antibodies (NAb) are an important component of the first line of immune defense. Selective breeding for enhanced NAb levels in chickens may improve general disease resistance. It is unknown what the consequences of selection for NAb will be on the productive performance of laying hens. In this paper we describe the genetic relations between NAb titers binding keyhole limpet hemocyanin at 19 wk age and production traits in a white purebred leghorn chicken line observed in several time periods. A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. Negative genetic correlations were found between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive genetic correlations were found between the feed conversion ratio (consumed feed/egg mass produced) and NAb titers, and egg production and NAb titers. Negative phenotypic correlations were found between body weight and NAb titers, between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive phenotypic correlations were found between egg production and NAb titers, and feed conversion ratio and NAb titers. In general, phenotypic correlations were more often significant, but less pronounced than genetic correlations. Other production traits were not found to be significant related to NAb titers. These findings suggest that there is a genetic tradeoff between levels of immunity and some production traits, although the underlying mechanism(s) remain(s) unclear. The results suggest possible consequences for production efficiency as a result of selective breeding for improved general disease resistance by natural antibodies. © 2015 Poultry Science Association Inc.

  13. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage...

  14. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

  15. Fate of transgenic deoxyribonucleic acid fragments in digesta and tissues of rabbits fed genetically modified soybean meal.

    Science.gov (United States)

    Morera, P; Basiricò, L; Ronchi, B; Bernabucci, U

    2016-03-01

    Numerous animal feeding studies have investigated the presence of DNA from transgenic plants in tissues from different animal species, but the data reported are sometimes controversial. The aim of this study was to investigate the presence of transgenic DNA (tDNA) in the digesta and tissues of a meat rabbit breed fed genetically modified (GM) soybean meal. Fifteen male New Zealand White rabbits were used for the experimental trial. Ten rabbits (treated group [TG]) were fed a mixed feed containing 10% GM soybean meal and 5 rabbits (control group [CG]) received a mixed feed containing conventional soybean meal, both from weaning (28 d of age) to slaughter (80 ± 3 d). Samples of blood, liver, kidney, heart, stomach, intestine (jejunum), lateral quadricep muscle, longissimus muscle, and perirenal adipose tissue were collected to assess the possible DNA transfer from GM feed to animal tissues. Samples of stomach contents and feces were also taken to study the degradability of ingested tDNA from feed in the digestive tract of rabbit. Moreover, samples of hair were collected to determine the possible environmental contamination from feed powders present on the farm. The DNA extraction was performed using specific genomic DNA kits. All samples were monitored, by using real-time PCR, for oligonucleotide primers and probes specific for the transgenic Roundup Ready soybean 40-3-2 and for the endogenous () gene. As an internal control of rabbit tissues, the presence of the () gene was used. In this study, no fragments of tDNA were detectable in tissue DNA samples of rabbits except in the extracted DNA from stomach digesta, feces, and hair of rabbits fed with GM soybean. Similar results were found for the reference gene, whereas the presence of the gene was detected in all rabbit tissues. The lack of tDNA of soybean in rabbit tissues represents an important result, which demonstrates that meat from rabbits fed a diet containing GM feed is as that derived from rabbits fed

  16. Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis ( Vitis vinifera L. × Vitis amurensis Rupr.

    Directory of Open Access Journals (Sweden)

    yinshan eGuo

    2015-06-01

    Full Text Available In this study, 149 F1 plants from the interspecific cross between ‘Red Globe’ (Vitis vinifera L. and ‘Shuangyou’ (Vitis amurensis Rupr. and the parent were used to construct a molecular genetic linkage map by using the specific length amplified fragment sequencing technique. DNA sequencing generated 41.282 Gb data consisting of 206,411,693 paired-end reads. The average sequencing depths were 68.35 for ‘Red Globe,’ 63.65 for ‘Shuangyou,’ and 8.01 for each progeny. In all, 115,629 high-quality specific length amplified fragments were detected, of which 42,279 were polymorphic. The genetic map was constructed using 7,199 of these polymorphic markers. These polymorphic markers were assigned to 19 linkage groups; the total length of the map was 1929.13 cM, with an average distance of 0.28 cM between each maker. To our knowledge, the genetic maps constructed in this study contain the largest number of molecular markers. These high-density genetic maps might form the basis for the fine quantitative trait loci mapping and molecular-assisted breeding of grape.

  17. Diversification in continental island archipelagos: new evidence on the roles of fragmentation, colonization and gene flow on the genetic divergence of Aegean Nigella (Ranunculaceae).

    Science.gov (United States)

    Jaros, Ursula; Tribsch, Andreas; Comes, Hans Peter

    2018-02-12

    Disentangling the relative roles of past fragmentation (vicariance), colonization (dispersal) and post-divergence gene flow in the genetic divergence of continental island organisms remains a formidable challenge. Amplified fragment length polymorphisms (AFLPs) were used to (1) gain further insights into the biogeographical processes underlying the Pleistocene diversification of the Aegean Nigella arvensis complex; (2) evaluate the role of potential key factors driving patterns of population genetic variability (mating system, geographical isolation and historical contingencies); and (3) test the robustness of conclusions previously drawn from chloroplast (cp) DNA. Genetic diversity was analysed for 235 AFLP markers from 48 populations (497 individuals) representing 11 taxa of the complex using population genetic methods and Bayesian assignment tests. Most designated taxa are identifiable as genetically distinct units. Both fragmentation and dispersal-driven diversification processes occurred at different geological time scales, from Early to Late Pleistocene, specifically (1) sea barrier-induced vicariant speciation in the Cyclades, the Western Cretan Strait and Ikaria; and (2) bi-regional colonizations of the 'Southern Aegean Island Arc' from the Western vs. Eastern Aegean mainland, followed by allopatric divergences in Crete vs. Rhodos and Karpathos/Kasos. Outcrossing island taxa experienced drift-related demographic processes that are magnified in the two insular selfing species. Population genetic differentiation on the mainland seems largely driven by dispersal limitation, while in the Central Aegean it may still be influenced by historical events (island fragmentation and sporadic long-distance colonization). The biogeographical history of Aegean Nigella is more complex than expected for a strictly allopatric vicariant model of divergence. Nonetheless, the major phylogeographical boundaries of this radiation are largely congruent with the geography and

  18. Genetic fusion protein 3×STa-ovalbumin is an effective coating antigen in ELISA to titrate anti-STa antibodies.

    Science.gov (United States)

    Duan, Qiangde; Zhang, Weiping

    2017-07-01

    Heat-stable toxin type I (STa)-ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti-STa antibodies for ETEC vaccine candidates. STa-ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti-STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa-ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti-STa antibody titration ELISA. Data showed fusion protein 3×STa-ovalbumin was effectively expressed and extracted, and anti-STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa-ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti-STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa-ovalbumin is an effective ELISA coating antigen for anti-STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  19. Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development

    Directory of Open Access Journals (Sweden)

    Victor Greiff

    2017-05-01

    Full Text Available Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1 B cell development (pre-B cell, naive B cell, plasma cell, (2 antigen exposure (three structurally different proteins, and (3 four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size extracted from antibody repertoire sequencing data (400 million reads. Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells and antigen-driven (e.g., 40% for clonal diversity in plasma cells predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.

  20. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...... the development of lupus nephritis at an early stage....

  1. [Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25-35)].

    Science.gov (United States)

    Kolobov, V V; Davydova, T V; Fomina, V G

    2014-01-01

    Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp 1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ25-35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp 1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.

  2. Genetic parameters of IgM and IgG antibodies binding autoantigens in healthy chickens

    NARCIS (Netherlands)

    Bao, Mandy; Bovenhuis, H.; Nieuwland, M.G.B.; Parmentier, H.K.; Poel, van der J.J.

    2016-01-01

    Levels of natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH) in layers were shown to be heritable and to be potential indicative parameters for survival. A proportion of NAb are directed to self-molecules, or slightly changed self-molecules (neo-epitopes), labeled as natural

  3. Encapsulation dehydration colligative cryoprotective strategies and amplified fragment length polymorphism markers to verify the identity and genetic stability of euglenoids following cryopreservation.

    Science.gov (United States)

    Harding, Keith; Miller, Julia; Timmermann, Hella; Lorenz, Maike; Day, John G; Friedl, Thomas

    2010-01-01

    An encapsulation/dehydration procedure was developed for Euglena gracilis Klebs as a 'model alga' to examine various cryoprotective regimes combined with controlled rate cooling to cryopreserve other Euglenoid taxa. Cryoprotective variables were optimised to enable reproducible growth following a combination of alginate encapsulation, sucrose osmotic dehydration, air desiccation, methanol treatment, cooling to -40 degrees C and plunging into liquid nitrogen (LN). Amplified Fragment Length Polymorphism (AFLP) analysis was adapted to: (i) verify algal identity by discriminating between different Euglenoids and (ii) examine the genetic stability of algal cultures prior to various stages of cryoprotective treatments and following exposure to LN. AFLPs were highly reproducible (> 99%) as reliable diagnostic markers, where a single DNA fragment change accounted for -0.4% of the detectable variation in an AFLP pattern. AFLP changes were detected in cryoprotective treatments following LN exposure. Successive stages of the dehydration and desiccation treatments did not accumulate AFLP changes indicating these are random events.

  4. Cell-free DNA fragmentation patterns in amniotic fluid identify genetic abnormalities and changes due to storage.

    Science.gov (United States)

    Peter, Inga; Tighiouart, Hocine; Lapaire, Olav; Johnson, Kirby L; Bianchi, Diana W; Terrin, Norma

    2008-09-01

    Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N=36) and aneuploid (N=29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (-80 degrees C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification.

  5. Within-population spatial genetic structure in four naturally fragmented species of a neotropical inselberg radiation, Alcantarea imperialis, A. geniculata, A. glaziouana and A. regina (Bromeliaceae).

    Science.gov (United States)

    Barbará, T; Lexer, C; Martinelli, G; Mayo, S; Fay, M F; Heuertz, M

    2008-09-01

    Studies of organisms on 'terrestrial islands' can improve our understanding of two unresolved issues in evolutionary genetics: the likely long-term effects of habitat fragmentation and the genetic underpinnings of continental species radiations in island-like terrestrial habitats. We have addressed both issues for four closely related plant species of the adaptive radiation Bromeliaceae, Alcantarea imperialis, A. geniculata, A. regina and A. glaziouana. All four are adapted to ancient, isolated inselberg rock outcrops in the Brazilian Atlantic rainforest and are thus long-term fragmented by nature. We used eight nuclear microsatellites to study within-population spatial genetic structure (SGS) and historical gene dispersal in nine populations of these species. Within-population SGS reflected known between-species differences in mating systems. The strongest SGS observed in A. glaziouana (Sp=0.947) was stronger than literature estimates available for plants. Analysis of short- and long-distance components of SGS identified biparental inbreeding, selfing and restricted seed dispersal as main determinants of SGS, with restricted pollen dispersal by bats contributing in some localities. The ability of Alcantarea spp. to colonize isolated inselbergs probably stems from their flexible mating systems and an ability to tolerate inbreeding. Short-ranging gene dispersal (average sigma=7-27 m) is consistent with a loss of dispersal power in terrestrial island habitats. Population subdivision associated with sympatric colour morphs in A. imperialis is accompanied by between-morph differences in pollen and seed dispersal. Our results indicate a high potential for divergence with gene flow in inselberg bromeliads and they provide base-line data about the long-term effects of fragmentation in plants.

  6. Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles

    DEFF Research Database (Denmark)

    Siemer, B.L.; Harrington, C.S.; Nielsen, E.M.

    2004-01-01

    health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters......Aims: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. Methods and Results: Field strains (n = 207) from human...... diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical...

  7. Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

    Science.gov (United States)

    Poussier, Stephane; Vandewalle, Peggy; Luisetti, Jacques

    1999-01-01

    The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas. PMID:10224018

  8. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    S Gnanakaran

    Full Text Available A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an

  9. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; van der Graaf, W.T.; Oyen, W.J.G.; Boerman, O.C.

    2012-01-01

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  10. Comparison of IgG and F(ab')2 fragments of bispecific anti-RCCxanti-DTIn-1 antibody for pretargeting purposes.

    NARCIS (Netherlands)

    Schaijk, F.G. van; Boerman, O.C.; Soede, A.C.; McBride, W.J.; Goldenberg, D.M.; Corstens, F.H.M.; Oosterwijk, E.

    2005-01-01

    PURPOSE: An effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCCxanti-DTPA(In) (bsMAb: G250xDTIn-1). Tumour uptake of a (111)In-labelled bivalent peptide after pretargeting with bsMAb G250xDTIn-1 was

  11. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol...

  12. Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

    Science.gov (United States)

    Nunoura, Takuro; Hirayama, Hisako; Takami, Hideto; Oida, Hanako; Nishi, Shinro; Shimamura, Shigeru; Suzuki, Yohey; Inagaki, Fumio; Takai, Ken; Nealson, Kenneth H; Horikoshi, Koki

    2005-12-01

    Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

  13. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    Energy Technology Data Exchange (ETDEWEB)

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E. [Wales Institute, Clwyd (United Kingdom)] [and others

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  14. Genetic and genomic basis of antibody response to porcine reproductive and respiratory syndrome (PRRS) in gilts and sows.

    Science.gov (United States)

    Serão, Nick V L; Kemp, Robert A; Mote, Benny E; Willson, Philip; Harding, John C S; Bishop, Stephen C; Plastow, Graham S; Dekkers, Jack C M

    2016-07-14

    Our recent research showed that antibody response to porcine reproductive and respiratory syndrome (PRRS), measured as sample-to-positive (S/P) ratio, is highly heritable and has a high genetic correlation with reproductive performance during a PRRS outbreak. Two major quantitative trait loci (QTL) on Sus scrofa chromosome 7 (SSC7; QTLMHC and QTL130) accounted for ~40 % of the genetic variance for S/P. Objectives of this study were to estimate genetic parameters for PRRS S/P in gilts during acclimation, identify regions associated with S/P, and evaluate the accuracy of genomic prediction of S/P across populations with different prevalences of PRRS and using different single nucleotide polymorphism (SNP) sets. Phenotypes and high-density SNP genotypes of female pigs from two datasets were used. The outbreak dataset included 607 animals from one multiplier herd, whereas the gilt acclimation (GA) dataset included data on 2364 replacement gilts from seven breeding companies placed on health-challenged farms. Genomic prediction was evaluated using GA for training and validation, and using GA for training and outbreak for validation. Predictions were based on SNPs across the genome (SNPAll), SNPs in one (SNPMHC and SNP130) or both (SNPSSC7) QTL, or SNPs outside the QTL (SNPRest). Heritability of S/P in the GA dataset increased with the proportion of PRRS-positive animals in the herd (from 0.28 to 0.47). Genomic prediction accuracies ranged from low to moderate. Average accuracies were highest when using only the 269 SNPs in both QTL regions (SNPSSC7, with accuracies of 0.39 and 0.31 for outbreak and GA validation datasets, respectively. Average accuracies for SNPALL, SNPMHC, SNP130, and SNPRest were, respectively, 0.26, 0.39, 0.21, and 0.05 for the outbreak, and 0.28, 0.25, 0.22, and 0.12, for the GA validation datasets. Moderate genomic prediction accuracies can be obtained for PRRS antibody response using SNPs located within two major QTL on SSC7, while the rest of

  15. [HLA genetic markers and auto-antibody profile in a Mapuche family with a case affected of type 1 diabetes].

    Science.gov (United States)

    Asenjo, Sylvia; Gleisner, Andrea; Pérez, Francisco

    2004-01-01

    Type 1 diabetes (DM1) is caused by an autoimmune process that destroys beta cells of pancreas. Not all carriers of susceptible HLA genes and positive for autoantibodies develop the disease. Environmental factors play a role in triggering the autoimmune process. To analyze an exceptional case of DM1 in a Mapuche family in the context of genetic, immunological and environmental factors. A study of a family with an affected female child was carried out in a Mapuche community in Southern Chile (VIII region). This is an unique and sporadic DM1 case with Mapuche heritage. Nutritional and viral infections data were collected by interview and clinical records. A genetic analysis by PCR was done to detect class I and II HLA genes by reverse dot blot. The proband, her mother and sister had positive islet cell antibodies (ICA). Her father and brother were negative. All thefamily was positive for anti glutamic decarboxylase antibodies (GAD65). All subjects had HLA-DRB1 0407/0407 and HLA-DQB1 0302/0302 alleles. The index case and her father were homozygotes for the HLA-A1:A*68012/A*68012 allele. Mean breastfeeding lapse was 18 months in all children. No evidences for viral infections such as rubella, mumps or measles were found in this family. There was an altered profile of autoantibodies in the family of the index case. All genotypes were comparable with the European population where the diabetogenic combination DR4/DQB1*0302 is the most prevalent. No environmental factors could be incriminated as triggers of the disease.

  16. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

    Science.gov (United States)

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold

    2002-04-01

    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  17. Genetic differentiation of Liparus glabrirostris (Curculionidae: Molytinae) populations from the fragmented habitats of the Alps and Carpathian Mounatains

    Czech Academy of Sciences Publication Activity Database

    Mitrović, M.; Tomanović, Ž.; Jakovljević, M.; Radović, D.; Havelka, Jan; Starý, Petr

    2016-01-01

    Roč. 106, č. 5 (2016), s. 651-662 ISSN 0007-4853 Institutional support: RVO:60077344 Keywords : Liparus glabrirostris * mitochondrial DNA * allopatric speciation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.758, year: 2016

  18. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  19. Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease

    DEFF Research Database (Denmark)

    Medici, Marco; Porcu, Eleonora; Pistis, Giorgio

    2014-01-01

    Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease.......12-1.39, P = 6.2×10(-5)). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18-2.10, P = 1.9×10(-3)). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease....... With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb...

  20. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  1. Gegenees: fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups.

    Directory of Open Access Journals (Sweden)

    Joakim Agren

    Full Text Available The rapid development of Next Generation Sequencing technologies leads to the accumulation of huge amounts of sequencing data. The scientific community faces an enormous challenge in how to deal with this explosion. Here we present a software tool, 'Gegenees', that uses a fragmented alignment approach to facilitate the comparative analysis of hundreds of microbial genomes. The genomes are fragmented and compared, all against all, by a multithreaded BLAST control engine. Ready-made alignments can be complemented with new genomes without recalculating the existing data points. Gegenees gives a phylogenomic overview of the genomes and the alignment can then be mined for genomic regions with conservation patterns matching a defined target group and absent from a background group. The genomic regions are given biomarker scores forming a uniqueness signature that can be viewed and explored, graphically and in tabular form. A primer/probe alignment tool is also included for specificity verification of currently used or new primers. We exemplify the use of Gegenees on the Bacillus cereus group, on Foot and Mouth Disease Viruses, and on strains from the 2011 Escherichia coli O104:H4 outbreak. Gegenees contributes towards an increased capacity of fast and efficient data mining as more and more genomes become sequenced.

  2. Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake

    International Nuclear Information System (INIS)

    Rotman, Maarten; Welling, Mick M.; Boogaard, Marlinde L. van den; Moursel, Laure Grand; Graaf, Linda M. van der; Buchem, Mark A. van; Maarel, Silvère M. van der; Weerd, Louise van der

    2015-01-01

    Introduction: Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab′)2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. Methods: To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ( 111 In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 μg VHH-pa2H-Fc-DTPA- 111 In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. Results: We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50 mg/L growth medium. The fusion protein showed homodimerization – necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA- 111 In and the VHH-Fc-DTPA- 111 In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA- 111 In

  3. Genetic Characterization of Campylobacter Jejuni and C. coli Isolated From Broilers Using flaA PCR-Restriction Fragment Length Polymorphism Method in Shiraz, Southern Iran.

    Science.gov (United States)

    Khoshbakht, Rahem; Tabatabaei, Mohammad; Hosseinzadeh, Saeid; Shirzad Aski, Hesamaddin; Seifi, Saeed

    2015-05-01

    Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders. The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran. Ninety Campylobacter isolates were recovered from broiler feces using enrichment process followed by cultivation method. The isolates were species typing on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR for determining two thermophilic species. To evaluate strain diversity of thermophilic Campylobacter isolates, flaA PCR-Restriction Fragment Length Polymorphism (RFLP) was performed using DdeI restriction enzyme. All 90 Campylobacter isolates confirmed by m-PCR were successfully typed using flaA-PCR-RFLP. Eleven different types were defined according to flaA-typing method and the RFLP patterns were located at three separate clusters in RFLP image analysis dendrogram. Campylobacter jejuni isolates significantly showed more variety than C. coli isolates. A relatively low genetic diversity existed among C. jejuni and C. coli isolated from broilers in Shiraz, southern Iran. In our knowledge, this was the first report of genetic diversity among broiler originated human pathogen thermophilic campylobacters in Shiraz, southern Iran.

  4. Restarting and recentering genetic algorithm variations for DNA fragment assembly: The necessity of a multi-strategy approach.

    Science.gov (United States)

    Hughes, James Alexander; Houghten, Sheridan; Ashlock, Daniel

    2016-12-01

    DNA Fragment assembly - an NP-Hard problem - is one of the major steps in of DNA sequencing. Multiple strategies have been used for this problem, including greedy graph-based algorithms, deBruijn graphs, and the overlap-layout-consensus approach. This study focuses on the overlap-layout-consensus approach. Heuristics and computational intelligence methods are combined to exploit their respective benefits. These algorithm combinations were able to produce high quality results surpassing the best results obtained by a number of competitive algorithms specially designed and tuned for this problem on thirteen of sixteen popular benchmarks. This work also reinforces the necessity of using multiple search strategies as it is clearly observed that algorithm performance is dependent on problem instance; without a deeper look into many searches, top solutions could be missed entirely. Copyright © 2016. Published by Elsevier Ireland Ltd.

  5. The preservation of the Agoudal impact crater, Morocco, under a landslide: Indication of a genetic link between shatter cones and meteorite fragments

    Science.gov (United States)

    Nachit, Hassane; Abia, El Hassan; Bonadiman, Costanza; Di Martino, Mario; Vaccaro, Carmela

    2017-10-01

    Geological studies and tomographic profiles of a locality nearby the Agoudal village (Morocco) showed the presence of a single impact crater, 500-600 m diameter, largely hidden by a limestone block, 220 m long and 40 m deep. The site was interpreted as a landslide that followed the fall of a cosmic body. The Agoudal impact crater was not affected by intense erosion. The lack of an evident impact structure, as well as the sporadic distribution of impactites and their limited occurrence, can be explained by a complex geological framework and by recent tectonics. The latter is the result of the sliding of limestone block, which hides almost two-thirds of the crater's depression, and the oblique fall of the meteoroid on sloping ground. In addition, some impact breccia dikes sharply cut the host rock in the Agoudal impact structure. They do not show any genetic relationship with tectonics or hydrothermal activity, nor are they related to any karst or calcrete formations. Altogether, the overlapping of the meteorite strewn field (11 km long and 3 km wide) with the area of occurrence of shatter cones and impact breccias, together with the presence of meteorite fragments (shrapnel) ejected from the crater, the presence of shatter cones contaminated by products of iron meteorites and the presence of impact breccias that contain meteorite fragments of the same chemical composition of the Agoudal meteorite indicate that the fall of this meteorite can be responsible for the formation of the impact structure.

  6. High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes▿

    Science.gov (United States)

    Kütahya, Oylum Erkus; Starrenburg, Marjo J. C.; Rademaker, Jan L. W.; Klaassen, Corné H. W.; van Hylckama Vlieg, Johan E. T.; Smid, Eddy J.; Kleerebezem, Michiel

    2011-01-01

    A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers. PMID:21666014

  7. Genetic Typing of Bovine Viral Diarrhoea Virus (BVDV by Restriction Fragment Length Polymorphism (RFLP and Identification of a New Subtype in Poland

    Directory of Open Access Journals (Sweden)

    Kuta Aleksandra

    2015-04-01

    Full Text Available Restriction fragment length polymorphism (RFLP analysis was developed for genetic typing of Polish strains of bovine viral diarrhoea virus (BVDV. The method was applied using 60 BVDV isolates, which included BVDV genotype 1, subtypes a, b, d, e, f, and g, and genotype 2a. RT-PCR products of the 5’untranslated region (5’UTR were digested using three enzymes. Restriction patterns classified the strains into seven groups, each with a specific and different pattern from other subtypes. These findings were confirmed by nucleotide sequencing and phylogenetic analysis. The results suggest that RFLP analysis is a simple, reliable, and fast genotyping method for BVDV strains in comparison with sequencing. This method can distinguish six subtypes of BVDV-1 including a new subtype 1e, identified exclusively by this method, and it allows differentiation of BVDV-1 from BVDV-2 genotype.

  8. Genetic analysis of autoimmune gld mice. I. Identification of a restriction fragment length polymorphism closely linked to the gld mutation within a conserved linkage group

    Science.gov (United States)

    1988-01-01

    A linkage map of distal mouse chromosome 1 was generated using restriction fragment length polymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease. PMID:2894402

  9. Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

    Directory of Open Access Journals (Sweden)

    Patrícia Carvalho de Sequeira

    2005-11-01

    Full Text Available Simple double repetitive element polymerase chain reaction (MaDRE-PCR and Pvu II-IS1245 restriction fragment length polymorphism (RFLP typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

  10. Inferring the evolutionary history of Indian Plasmodium vivax from population genetic analyses of multilocus nuclear DNA fragments.

    Science.gov (United States)

    Gupta, Bhavna; Srivastava, Nalini; DAS, Aparup

    2012-04-01

    The human malaria parasite Plasmodium vivax is globally widespread, causing high malaria morbidity. As P. vivax is highly endemic to India, and previous reports indicate genetic homogeneity in population samples, we tested the hypothesis of no genetic structuring in Indian P. vivax. Further, based on the reports of increasing incidence of Plasmodium falciparum infection in comparison with P. vivax in recent years in India, it was important to understand whether reduction in population size has resulted in decrease in P. vivax infection rate in India. For this, we utilized recently developed putatively neutral markers from chromosome 13 of P. vivax to score single nucleotide polymorphisms in 126 P. vivax isolates collected from 10 different places in India. The overall results indicated that Indian P. vivax bears high nucleotide diversity within population samples but moderate amount of genetic differentiation between population samples. STRUCTURE analysis grouped 10 population samples into three clusters based on the proportion of the genetic ancestries in each population. However, the pattern of clustering does not correlate with sampling locations in India. Furthermore, analyses of past demographic events indicated reduction in population size in majority of population samples, but when isolates from all the 10 samples were considered as a single population, the data fit to the demographic equilibrium model. All these observations clearly indicate that Indian P. vivax presents complex evolutionary history but possesses several features of being a part of ancestral distribution range of this species. © 2012 Blackwell Publishing Ltd.

  11. Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice

    International Nuclear Information System (INIS)

    Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P.

    1990-01-01

    During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-( 131 I) labeled intact antibodies or 131 I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2

  12. Analysis of genetic variability in endemic medicinal plants of genus Chlorophytum from the Indian subcontinent using amplified fragment length polymorphism marker.

    Science.gov (United States)

    Patil, Swapnil Mahadeo; Chandanshive, Vishal Vinayak; Tamboli, Asif Shabodin; Adsul, Avinash Asraji; Yadav, Shrirang Ramchandra; Govindwar, Sanjay Prabhu

    2015-12-01

    The genus Chlorophytum consists of medicinally important species like Chlorophytum borivilianum, C. tuberosum and C. attenuatum. Uncontrolled harvest of this plant from wild habitat due to its high commercial value made the species of this genus be listed in the Red Data Book of Indian plants as an endangered species. In India, approximately nineteen species of Chlorophytum are found; out of these, only C. borivilianum is cultivated commercially. The objective of this study was to measure genetic diversity, population structure and phylogenetic relationship among the species using Amplified Fragment Length Polymorphisms (AFLP). Fifteen pairs of primer (out of 64 primer pairs screened) were used to analyse the genetic diversity in eighteen species of genus Chlorophytum. Cluster analysis, estimation of the gene flow among the species and of the phylogeographic distribution of this genus were carried out using an AFLP data matrix. A high level of genetic diversity was observed on the basis of the percentage of polymorphic bands (99.91%), Shannon's information index (0.3592) and Nei's gene diversity (0.2085) at species level. Cluster analysis of UPGMA dendrogram, principal component analysis and Bayesian method analysis resolved these species in three different clusters, which was supported by morphological information. The Mantel test (r=0.4432) revealed a significant positive correlation between genetic and geographic distances. The collected data have an important implication in the identification, authentication, and conservation of the species of the genus Chlorophytum. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  13. Crystal structure of snake venom acetylcholinesterase in complex with inhibitory antibody fragment Fab410 bound at the peripheral site: evidence for open and closed states of a back door channel.

    Science.gov (United States)

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-16

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  15. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    International Nuclear Information System (INIS)

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.

    1988-01-01

    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging

  16. The future of monoclonal antibody technology

    OpenAIRE

    Zider, Alexander; Drakeman, Donald L

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  17. Population Genetic Patterns of Threatened European Mudminnow (Umbra krameri Walbaum, 1792 in a Fragmented Landscape: Implications for Conservation Management.

    Directory of Open Access Journals (Sweden)

    Péter Takács

    Full Text Available The European mudminnow (Umbra krameri is a Middle Danubian endemic fish species, which is characterised by isolated populations living mainly in artificial habitats in the centre of its range, in the Carpathian Basin. For their long term preservation, reliable information is needed about the structure of stocks and the level of isolation. The recent distribution pattern, and the population genetic structure within and among regions were investigated to designate the Evolutionary Significant, Conservation and Management Units (ESUs, CUs, MUs and to explore the conservation biological value of the shrinking populations. In total, eight microsatellite loci were studied in 404 specimens originating from eight regions. The results revealed a pronounced population structure, where strictly limited gene flow was detected among regions, as well as various strengths of connections within regions. Following the results of hierarchical structure analyses, two ESUs were supposed in the Carpathian Basin, corresponding to the Danube and Tisza catchments. Our results recommend designating the borders of CUs in an 80-90km range and 16 clusters should be set up as MUs for the 33 investigated populations. How these genetic findings can be used to better allocate conservation resources for the long term maintenance of the metapopulation structure of this threathened endemic fish is discussed.

  18. Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

    Science.gov (United States)

    Hu, Jiemiao; Vien, Long T; Xia, Xueqing; Bover, Laura; Li, Shulin

    2014-02-04

    Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

  19. Construction of a high-density genetic map based on large-scale marker development in mango using specific-locus amplified fragment sequencing (SLAF-seq

    Directory of Open Access Journals (Sweden)

    Chun Luo

    2016-08-01

    Full Text Available Genetic maps are particularly important and valuable tools for quantitative trait locus (QTL mapping and marker assisted selection (MAS of plant with desirable traits. In this study, 173 F1 plants from a cross between Mangifera indica L. ‘Jin-Hwang’ and M. indica L. ‘Irwin’ and their parent plants were subjected to high-throughput sequencing and specific-locus amplified fragment (SLAF library construction. After preprocessing, 66.02 Gb of raw data containing 330.64 M reads were obtained. A total of 318,414 SLAFs were detected, of which 156,368 were polymorphic. Finally, 6,594 SLAFs were organized into a linkage map consisting of 20 linkage groups (LGs. The total length of the map was 3148.28 cM and the average distance between adjacent markers was 0.48 cM. This map could be considered, to our knowledge, the first high-density genetic map of mango, and might form the basis for fine QTL mapping and MAS of mango.

  20. Nuclear fragmentation

    International Nuclear Information System (INIS)

    Chung, K.C.

    1989-01-01

    An introduction to nuclear fragmentation, with emphasis in percolation ideas, is presented. The main theoretical models are discussed and as an application, the uniform expansion approximation is presented and the statistical multifragmentation model is used to calculate the fragment energy spectra. (L.C.)

  1. Sortilin Fragments Deposit at Senile Plaques in Human Cerebrum

    Directory of Open Access Journals (Sweden)

    Xia Hu

    2017-06-01

    Full Text Available Genetic variations in the vacuolar protein sorting 10 protein (Vps10p family have been linked to Alzheimer’s disease (AD. Here we demonstrate deposition of fragments from the Vps10p member sortilin at senile plaques (SPs in aged and AD human cerebrum. Sortilin changes were characterized in postmortem brains with antibodies against the extracellular and intracellular C-terminal domains. The two antibodies exhibited identical labeling in normal human cerebrum, occurring in the somata and dendrites of cortical and hippocampal neurons. The C-terminal antibody also marked extracellular lesions in some aged and all AD cases, appearing as isolated fibrils, mini-plaques, dense-packing or circular mature-looking plaques. Sortilin and β-amyloid (Aβ deposition were correlated overtly in a region/lamina- and case-dependent manner as analyzed in the temporal lobe structures, with co-localized immunofluorescence seen at individual SPs. However, sortilin deposition rarely occurred around the pia, at vascular wall or in areas with typical diffuse Aβ deposition, with the labeling not enhanced by section pretreatment with heating or formic acid. Levels of a major sortilin fragment ~15 kDa, predicted to derive from the C-terminal region, were dramatically elevated in AD relative to control cortical lysates. Thus, sortilin fragments are a prominent constituent of the extracellularly deposited protein products at SPs in human cerebrum.

  2. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    Science.gov (United States)

    Welfringer, Frédéric; d'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-12-20

    Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

  3. Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

    Energy Technology Data Exchange (ETDEWEB)

    Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Weisgraber, Karl [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Department of Pathology, University of California, San Francisco, CA 94143 (United States); Finkbeiner, Steven, E-mail: sfinkbeiner@gladstone.ucsf.edu [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Departments of Neurology, Physiology and Neuroscience and Biomedical Sciences Program, University of California, San Francisco, CA 94143 (United States)

    2005-12-01

    Optimization of crystallization conditions and cryoprotectants decreased the anisotropy of the diffraction obtained from 3B5H10 Fab crystals. Dehydration improved the resolution of cryoprotected 3B5H10 crystals from 2.6 to 1.9 Å, but changed the space group of the crystals from P2{sub 1}2{sub 1}2 to P2{sub 1}. Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 Å resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 Å and space group P2{sub 1}2{sub 1}2. Dehydrated crystals diffracted isotropically to 1.9 Å with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 Å, β = 90.3° and space group P2{sub 1}.

  4. In Vivo HER2-Targeted Magnetic Resonance Tumor Imaging Using Iron Oxide Nanoparticles Conjugated with Anti-HER2 Fragment Antibody.

    Science.gov (United States)

    Ding, Ning; Sano, Kohei; Kanazaki, Kengo; Ohashi, Manami; Deguchi, Jun; Kanada, Yuko; Ono, Masahiro; Saji, Hideo

    2016-12-01

    The feasibility of iron oxide nanoparticles (IONPs) conjugated with anti-epidermal growth factor receptor 2 (HER2) single-chain antibody (scFv-IONPs) as novel HER2-targeted magnetic resonance (MR) contrast agents was investigated. The scFv-IONPs were prepared and identified. For in vitro MRI, NCI-N87 (HER2 high expression) and SUIT2 (low expression) cells were incubated with scFv-IONPs. For in vivo MRI, NCI-N87 and SUIT2 tumor-bearing mice were intravenously injected with scFv-IONPs and imaged before and 24 h post-injection. The scFv-IONPs demonstrated high transverse relaxivity (296.3 s -1  mM -1 ) and affinity toward HER2 (K D  = 11.7 nM). In the in vitro MRI, NCI-N87 cells treated with scFv-IONPs exhibited significant MR signal reduction (44.6 %) than SUIT2 cells (6.8 %). In the in vivo MRI, decrease of MR signals in NCI-N87 tumors (19.3 %) was more notable than that in SUIT2 tumors (6.2 %). The scFv-IONPs enabled HER2-specific tumor MR imaging, suggesting the potential of scFv-IONPs as a robust HER2-targeted MR contrast agent.

  5. The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing.

    Science.gov (United States)

    Cai, Changfu; Cheng, Fang-Yun; Wu, Jing; Zhong, Yuan; Liu, Gaixiu

    2015-01-01

    Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti 'FenDanBai' × P. × suffruticosa 'HongQiao', to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 'SNP-only' markers, 18 'InDel-only' markers, and 56 'SNP&InDel' markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony.

  6. Nucleotide sequence and genetic organization of a 7.3 kb region (map unit 47 to 52.5) of Autographa californica nuclear polyhedrosis virus fragment EcoRI-C

    NARCIS (Netherlands)

    Kool, M.; Broer, R.; Zuidema, D.; Goldbach, R. W.; Vlak, J. M.

    1994-01-01

    The nucleotide sequence and genetic organization of a 7297 bp region within the EcoRI-C fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) are presented. Eight putative open reading frames were found and their respective amino acid sequences compared with a

  7. Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Pfister, C.; Fournier, K.; Prevel, F.; Schreyer, M.; Carrel, S.; Mach, J.P.

    1989-01-01

    Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131 I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131 I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131 I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation

  8. Genetic control of the radiosensitivity of lymphoid cells for antibody-forming ability in CXS series of recombinant inbred mouse strains

    International Nuclear Information System (INIS)

    Okumoto, M.; Mori, N.; Nishikawa, R.; Imai, S.; Hilgers, J.; Takamori, Y.; Yagasaki, O.

    1992-01-01

    Incidence of radiation-induced lymphomas differs remarkably among various mouse strains. BALB/cHeA (C) mice are highly susceptible to radiation induction of lymphomas, while STS/A (S) mice are resistant. Thus, the induction of the disease is controlled by some genetic factors. To examine an involvement of radiosensitivity of lymphoid cells in lymphomagenesis, we have compared genetic control of the radiosensitivity for antibody-forming ability with that of lymphoma development in BALB/cHeA, STS/A, (CXS)F 1 hybrids and CXS series of recombinant inbred strains. Decrease of number of splenic plaque-forming cell (PFC) in Jerne's method by 3 Gy of X-irradiation for BALB/cHeA mice was larger than that for STS/A mice by more than one order of magnitude. (CXS)F 1 hybrid mice showed small number of decrease of PFC similar to STS/A mice suggesting that phenotype of radioresistance was dominant over sensitivity. The best concordance between genetic markers and radiosensitivities of antibody-forming ability in recombinant inbred strains was observed in a region containing Igh locus on chromosome 12. The results show that one locus controlling the radioresistance of lymphoid cells for antibody-forming ability might exist in the region containing Igh locus, and that this region clearly differ from a region with Ifa locus on chromosome 4 which regulate the susceptibility to radiation-induced lymphomagenesis. (author)

  9. Recombinant monovalent llama-derived antibody fragments (VHH to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Directory of Open Access Journals (Sweden)

    Celina G Vega

    Full Text Available Group A Rotavirus (RVA is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH to protect against human rotavirus in gnotobiotic (Gn piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256 for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  10. Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Science.gov (United States)

    Vega, Celina G; Bok, Marina; Vlasova, Anastasia N; Chattha, Kuldeep S; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M; Garaicoechea, Lorena L; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  11. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

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

  12. Impact of genetic profiles on the efficacy of anti-EGFR antibodies in metastatic colorectal cancer with KRAS mutation.

    Science.gov (United States)

    Kishiki, Tomokazu; Ohnishi, Hiroaki; Masaki, Tadahiko; Ohtsuka, Kouki; Ohkura, Yasuo; Furuse, Jyunji; Sugiyama, Masanori; Watanabe, Takashi

    2014-07-01

    Reports indicate that, even in KRAS-mutated colon cancer, there are subsets of patients who benefit from anti-EGFR monoclonal antibody (MoAb) treatment. The aim of the present study was to identify genetic profiles that contribute to the responsiveness of metastatic colorectal cancer (mCRC) to anti-EGFR MoAb. We retrospectively evaluated the efficacy of anti-EGFR MoAb in mCRC patients with KRAS mutations according to KRAS mutational subtypes, BRAF and PIK3CA mutational status and PTEN and MET expression. Among 21 patients with KRAS-mutant tumors, 8 (38%) harbored p.G13D, 7 (33%) harbored p.G12V, 5 (24%) harbored p.G12D, and 1 (5%) harbored p.G12C mutation. Patients with the p.G13D mutation exhibited a significantly higher disease control rate than patients with other KRAS mutations (P=0.042), and tended to show a longer progression-free survival (PFS) than patients with other KRAS mutations with marginal significance (P=0.074). Patients with loss of PTEN had significantly shorter PFS than those with normal PTEN expression in patients with KRAS mutations (P=0.044). MET overexpression was significantly associated with shorter PFS compared to normal MET expression in patients with KRAS mutations (P=0.016). Our data demonstrated the potential utility of alterations in PTEN and MET expression as predictive markers for response to anti-EGFR MoAbs in mCRC patients with KRAS mutations. In addition, we confirmed the predictive value of the KRAS p.G13D mutation for better response to anti-EGFR therapies in comparison with other KRAS mutations.

  13. Evidence of a genetic basis for the different geographic occurrences of liver/kidney microsomal antibody type 1 in hepatitis C.

    Science.gov (United States)

    Muratori, Paolo; Czaja, Albert J; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Ferri, Silvia; Volta, Umberto; Mantovani, Wilma; Pappas, Georgios; Cassani, Fabio; Lenzi, Marco; Bianchi, Francesco B

    2007-01-01

    Antibodies to liver/kidney microsome type 1 occur in Italian patients with hepatitis C, but rarely develop in North American patients. Our goals were to compare the frequencies of the HLA markers associated with autoimmune expression in Italian and North American patients with chronic hepatitis C and to determine genetic bases for regional differences in antibody production. HLA B8, DR3, DR4, DR7, DR11, DR13, DQ2, and the B8-DR3-DQ2 haplotype were determined by microlymphocytotoxicity and polymerase chain reaction in 105 Italian patients (50 with microsomal antibodies), 100 North American patients (none with microsomal antibodies), and Italian and North American healthy control subjects. Italian patients with microsomal antibodies differed from North American patients without these antibodies by having a higher frequency of HLA DR7 (54% vs. 27%, P=0.002). HLA DR7 occurred more frequently in seropositive Italian patients than in seronegative counterparts (54% vs. 11% P < 0.0001), Italian healthy control subjects (54% vs. 29%, P=0.0009), and North American healthy control subjects (54% vs. 19%, P < 0.0001). The frequency of HLA DR7 was similar in North American patients and controls (27% vs. 19%, P=0.2), but it was lower than in Italian controls (19% vs. 29%, P=0.059). Seropositive Italian patients had a lower frequency of HLA DR11 than seronegative Italian patients and Italian controls (18% vs. 34%, P=0.07, and 18% vs. 35%, P=0.02, respectively). In contrast to seropositive Italian patients, North American patients had HLA DR4 (30% vs. 12%, P=0.02), HLA DR13 (29% vs. 10%, P=0.01), and the B8-DR3-DQ2 haplotype (23% vs. 6%, P=0.01) more often. Similarly, HLA DR4 and the B8-DR3-DQ2 phenotype were more frequent in North American patients than in Italian controls (30% vs. 16%, P=0.005, and 23% vs. 7%, P=0.00002, respectively). HLA DR7 is associated with the development of microsomal antibodies in Italian patients with chronic hepatitis C. The lower frequency of HLA DR7

  14. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

    Science.gov (United States)

    Alanen, Heli I; Walker, Kelly L; Lourdes Velez Suberbie, M; Matos, Cristina F R O; Bönisch, Sarah; Freedman, Robert B; Keshavarz-Moore, Eli; Ruddock, Lloyd W; Robinson, Colin

    2015-03-01

    Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Chameleon fragmentation

    International Nuclear Information System (INIS)

    Brax, Philippe; Upadhye, Amol

    2014-01-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ 4 and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments

  16. Chameleon fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brax, Philippe [Institut de Physique Théorique, CEA, IPhT, CNRS, URA 2306, F-91191Gif/Yvette Cedex (France); Upadhye, Amol, E-mail: philippe.brax@cea.fr, E-mail: aupadhye@anl.gov [Institute for the Early Universe, Ewha University, International Education, Building #601, 11-1, Daehyun-Dong Seodaemun-Gu, Seoul 120-750 (Korea, Republic of)

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  17. Genetic aspects of auto-immune profiles of healthy chickens.

    Science.gov (United States)

    Parmentier, Henk K; van der Vaart, Priscilla S; Nieuwland, Mike G B; Savelkoul, Huub F J

    2017-09-01

    Auto-antibody profiles binding liver antigens differed between chicken lines divergently selected for specific antibody responses to SRBC, and were affected by ageing suggesting both genetic and environmental effects. Presence and levels of IgM and IgG antibodies binding chicken liver cell lysate (CLL) fragments in plasma at 5 weeks of age from 10 individual full sibs and their parents from 5 H srbc and 5 L srbc line families was studied to reveal genetic relations. Non-genetic maternal effects were studied by comparing auto-antibody profiles of 36 weeks old hens from 2 other unrelated lines with the profiles from their chicks at hatch. IgM and IgG antibodies from parents and progeny from both H srbc and L srbc lines bound CLL fragments. Significant line and generation differences and their interactions were found for both isotypes. Higher staining of CLL fragments was usually found for H srbc line birds. Lines were clustered by auto-antibody profiles, but staining by birds of both lines in both generations was very individual for IgG and IgM. The current data with full sibs therefore not supported a genetic basis for auto-antibody profiles. IgG but not IgM auto-antibody profiles of chicks correlated with maternal auto-antibody profiles. The results suggest that the auto-antibody repertoire of healthy chickens is largely stochastically initiated and may be affected by environmental challenges during ageing, but genetic mechanisms may underlie staining intensity of individual bound CLL fragments. The present results suggest that identification of fragments or profiles to be used at early age for genetic selection for health traits is not feasible yet. Secondly, the IgM profile of neonatal chickens seems non-organised independent of the maternal profile, but the neonatal IgG profile is much more related with the maternal profile. Consequences of these findings for disease susceptibility or breeding for optimal health are discussed. Copyright © 2017 Elsevier Ltd. All

  18. Indication of viruses and virus-specific antibodies by ELISA using conjugates based on β-lactamase obtained by genetic engineering

    International Nuclear Information System (INIS)

    Kharitonenkov, I.G.; Kordym, V.A.; Khristova, M.L.; Leonov, S.V.; Kirillova, V.S.; Chernykh, S.I.

    1987-01-01

    The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses

  19. Intermediate Fragment

    DEFF Research Database (Denmark)

    Kruse Aagaard, Anders

    2015-01-01

    ‘Engaging Through Architecture’ in 2015 by Aarhus School of Architecture as a part of the Ventura Lambrate Milan Design Week, where it was exhibited under the name Concrete. The fundamental pool of techniques and knowledge that set the agenda for the fragment was established before the intentions...

  20. Framing Fragmentation

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte

    2009-01-01

    to create architectural meaning and give character to an architecture of fragmentation. Layers are both seen as conceptual as well as material frames which define certain strong properties or meanings in the architectural work. Defining layers is a way of separating and organizing; it both defines...

  1. Rock fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brown, W.S.; Green, S.J.; Hakala, W.W.; Hustrulid, W.A.; Maurer, W.C. (eds.)

    1976-01-01

    Experts in rock mechanics, mining, excavation, drilling, tunneling and use of underground space met to discuss the relative merits of a wide variety of rock fragmentation schemes. Information is presented on novel rock fracturing techniques; tunneling using electron beams, thermocorer, electric spark drills, water jets, and diamond drills; and rock fracturing research needs for mining and underground construction. (LCL)

  2. Role of antibodies in protection elicited by active vaccination with genetically inactivated alpha hemolysin in a mouse model of staphylococcus aureus skin and soft tissue infections.

    Science.gov (United States)

    Mocca, Christopher P; Brady, Rebecca A; Burns, Drusilla L

    2014-05-01

    Due to the emergence of highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, S. aureus has become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused by S. aureus USA300 strains that are known to produce high levels of alpha hemolysin (Hla). Therefore, vaccines that contain inactivated forms of this toxin are currently being developed. In this study, we sought to determine the immune mechanisms of protection for this antigen using a vaccine composed of a genetically inactivated form of Hla (HlaH35L). Using a murine model of skin and soft tissue infections (SSTI), we found that BALB/c mice were protected by vaccination with HlaH35L; however, Jh mice, which are deficient in mature B lymphocytes and lack IgM and IgG in their serum, were not protected. Passive immunization with anti-HlaH35L antibodies conferred protection against bacterial colonization. Moreover, we found a positive correlation between the total antibody concentration induced by active vaccination and reduced bacterial levels. Animals that developed detectable neutralizing antibody titers after active vaccination were significantly protected from infection. These data demonstrate that antibodies to Hla represent the major mechanism of protection afforded by active vaccination with inactivated Hla in this murine model of SSTI, and in this disease model, antibody levels correlate with protection. These results provide important information for the future development and evaluation of S. aureus vaccines.

  3. Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

    Science.gov (United States)

    Lomakin, Ya A; Shmidt, A A; Bobik, T V; Chernov, A S; Pyrkov, A Yu; Aleksandrova, N M; Okunola, D O; Vaskina, M I; Ponomarenko, N A; Telegin, G B; Dubina, M V; Belogurov, A A

    2017-10-01

    Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

  4. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    or from a position as business leader to a position in the state apparatus or in the Party and vice versa. To conceptualize the coexistence of the contradicting forces for further enterprise autonomy and continued central control that characterizes the evolving relationship between business groups...... and the Party-state, I suggest the notion of integrated fragmentation....

  5. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220.

    Science.gov (United States)

    Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo

    2011-03-01

    A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.

    Science.gov (United States)

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Kowalewsky, Frank; Vogel, Rudolf; Heinrich, Julia

    2013-01-01

    Generic immunoassay formats in animal serum have been described for pharmacokinetic (PK) analysis of human full-length antibodies, but not of human antigen binding fragment (Fab) proteins. Here we characterize two murine monoclonal antibodies (mAb) raised against human immunoglobulin G (IgG) which bind to unique epitopes in the Fab region of human IgG. mAb M-1.7.10 is directed against the constant domain of the kappa light chain and mAb M-1.19.31 binds to the constant domain 1 (CH1) of the heavy chain. Surface plasmon resonance analysis showed that mAb M-1.7.10 does not cross-react with sera from mouse, rat, rabbit, dog, marmoset, rhesus macaque, baboon and cynomolgus monkey, but binds to human and chimpanzee serum (dissociation constant K(D) of 6.8 × 10(-12) and 3.1 × 10(-11)M, respectively). mAb M-1.19.31 shows a higher K(D) for human and chimpanzee IgG (2.0 × 10(-9)M and 5.8 × 10(-10)M, respectively), but also does not bind to serum of the other species. Therefore, mAb M-1.7.10 was used as capture and mAb M-1.19.31 as detection reagent in a generic enzyme linked immunosorbent assay (ELISA) to quantify the human anti-IGF-1R Fab in mouse serum. The generic human Fab assay showed a limit of detection of 31.5 ng/mL anti-IGF-1R Fab. Intra- and inter-assay precision was less than 12% and the accuracy range for all controls was within ±20% of the target concentration. The generic human Fab ELISA was applied to determine serum levels of human anti-IGF-1R Fab after intravenous (iv) administration of 10mg/kg to mice. The resulting concentration-time profile was nearly identical to that obtained by analysis with a validated specific ELISA for anti-IGF-1R Fab. The mean relative concentration of anti-IGF-1R Fab analyzed by the generic assay was 82-118% of that of the specific assay. This equivalence was confirmed in a cynomolgus monkey study with the full length human mAb anti-TROP-2 IgG. Both specific ELISAs used mAb M-1.7.10 as detection reagent and their targets for

  7. Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

  8. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide......-antibody interface and the antibody intraface.the microenvironment and ecology of Acaryochloris and Prochloron, and in this thesis we attempted to further describe the distribution, growth characteristics and adaptive/regulatory mechanisms of these two cyanobacteria, both in their natural habitat and under defined...

  9. Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Juan C. Almagro

    2018-01-01

    Full Text Available The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life, which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

  10. Genetic Testing

    Science.gov (United States)

    ... risk factor for the development of celiac disease, genetic predisposition. Without this factor, it is impossible that the ... with antibody testing in the future. When the genetic predisposition for celiac disease was detected (on Chromosome 6) ...

  11. Fragmentation based

    Directory of Open Access Journals (Sweden)

    Shashank Srivastava

    2014-01-01

    Gaining the understanding of mobile agent architecture and the security concerns, in this paper, we proposed a security protocol which addresses security with mitigated computational cost. The protocol is a combination of self decryption, co-operation and obfuscation technique. To circumvent the risk of malicious code execution in attacking environment, we have proposed fragmentation based encryption technique. Our encryption technique suits the general mobile agent size and provides hard and thorny obfuscation increasing attacker’s challenge on the same plane providing better performance with respect to computational cost as compared to existing AES encryption.

  12. Architectural fragments

    DEFF Research Database (Denmark)

    Bang, Jacob Sebastian

    2018-01-01

    the photographs as a starting point for a series of paintings. This way of creating representations of something that already exists is for me to see a way forward in the "decoding" of my own models into other depictions. The models are analyzed through a series of representations in different types of drawings....... I try to invent the ways of drawing the models - that decode and unfold them into architectural fragments- into future buildings or constructions in the landscape. [1] Luigi Moretti: Italian architect, 1907 - 1973 [2] Man Ray: American artist, 1890 - 1976. in 2015, I saw the wonderful exhibition...

  13. Population genetics of chamois in the contact zone between the Alps and the Dinaric Mountains: uncovering the role of habitat fragmentation and past management

    Czech Academy of Sciences Publication Activity Database

    Buzan, E. V.; Bryja, Josef; Zemanová, Barbora; Kryštufek, B.

    2013-01-01

    Roč. 14, č. 2 (2013), s. 401-412 ISSN 1566-0621 Institutional support: RVO:68081766 Keywords : Rupicapra rupicapra * Microsatellites * Population structure * Fragmentation * Conservation management Subject RIV: EH - Ecology, Behaviour Impact factor: 1.846, year: 2013

  14. Targeting Malignant Brain Tumors with Antibodies

    Directory of Open Access Journals (Sweden)

    Rok Razpotnik

    2017-09-01

    Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

  15. Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad A

    2013-01-01

    Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking...... expressing tumor cells, the combination of CD3ζ, OX40, CD28 as well as the CH3-CH2-hinge-hinge domains most efficiently triggered T cell activation. Importantly, CAR mediated functions were not blocked by the soluble TAG-72 antigen at a supraphysiological concentration. Our approach may have the potential...... capacity of soluble antigen still remain. Here, we address these issues using a novel CAR binding moiety based on the oligoclonal camelid single domain antibodies. A unique set of 13 single domain antibodies were selected from an immunized camel phage library based on their target specificity and binding...

  16. Human antibody recognition of xenogeneic antigens (NeuGc and Gal) on porcine heart valves: could genetically modified pig heart valves reduce structural valve deterioration?

    Science.gov (United States)

    Lee, Whayoung; Long, Cassandra; Ramsoondar, Jagdeece; Ayares, David; Cooper, David K C; Manji, Rizwan A; Hara, Hidetaka

    2016-09-01

    Glutaraldehyde-fixed bioprosthetic heart valves (GBHVs) derived from wild-type (WT, genetically unmodified) pigs are widely used clinically for heart valve replacement. There is evidence that their failure is related to an immune response. The use of valves from genetically engineered pigs that do not express specific pig antigens may prolong GBHV survival. Our aims were to determine (i) expression of Gal and NeuGc on heart (aortic and pulmonary) valves and pericardium of WT, α1,3-galactosyltransferase gene knockout (GTKO) and GTKO/N-glycolylneuraminic acid gene-knockout (GTKO/NeuGcKO) pigs in comparison with three different commercially available GBHVs and (ii) to determine human antibody binding to these tissues. Wild-type, GTKO/CD46, and GTKO/CD46/NeuGcKO pig valves and pericardium were tested (i) fresh and (ii) after fixation with glutaraldehyde (0.02%, 0.2%, 2%). Sections of GBHVs, fresh and fixed valves, and pericardium were stained for Gal and NeuGc expression, and for human IgM and IgG antibody binding. Gal and NeuGc expression was high on all GBHVs and WT pig valves/pericardium, but was absent after antigen-specific-knockout. There was no difference in antigen expression or antibody binding among WT aortic, pulmonary valves, and pericardium as well as GBHVs. Glutaraldehyde fixation did not alter expression of Gal or NeuGc. After incubation with human serum, human IgM and IgG bound to all GBHVs and WT pig valves/pericardium. Valves from GTKO/CD46 pigs and, particularly, GTKO/CD46/NeuGcKO pigs (with/without glutaraldehyde fixation) showed less IgM and IgG binding. Compared to WT pigs, GTKO/CD46/NeuGcKO pigs would be preferable sources of GBHVs, because the absence of Gal/NeuGc expression reduces human antibody binding. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Disease ecology in the Galápagos Hawk (Buteo galapagoensis): host genetic diversity, parasite load and natural antibodies

    NARCIS (Netherlands)

    Whiteman, N.K.; Matson, K.D.; Bollmer, J.L.; Parker, P.G.

    2006-01-01

    An increased susceptibility to disease is one hypothesis explaining how inbreeding hastens extinction in island endemics and threatened species. Experimental studies show that disease resistance declines as inbreeding increases, but data from in situ wildlife systems are scarce. Genetic diversity

  18. Genetic investigations in immigration cases and frequencies of DNA fragments of the VNTR systems D2S44, D5S43, D7S21, D7S22, and D12S11 in Turks

    DEFF Research Database (Denmark)

    Hansen, Hanna Elsebeth; Morling, N

    1993-01-01

    We have included investigations of the DNA polymorphism of variable numbers of tandem repeat (VNTR) regions with restriction fragment length polymorphism (RFLP) in the genetic evaluations in immigrant cases. HinfI-digested DNA was separated by electrophoresis in agarose gels and hybridized...... with radiolabelled probes detecting the VNTR systems D2S44 (YNH24), D5S43 (MS8), D7S21 (MS31), D7S22 (g3), and D12S11 (MS43a). We used the matching criterion for paternity testing for the parent/child comparisons, i.e. non-match if the intra gel difference exceeded 1.25 mm. A total of 43 immigration cases involving...... putative father/child pairs. In a putative father/child combination with non-exclusion in 18 genetic systems, a single genetic inconsistency ('exclusion') in D7S21 (MS31) was observed. The frequency distributions of HinfI digested DNA fragments of the five VNTR systems in 105 Turks are presented....

  19. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    proved their influence by obstructing the creation of new ministries and regulatory commissions that would limit their powers. The heads of these business groups often outrank their counterparts in state administrative organs and bureaus that are supposed to regulate their activities. The increased role...... of these business leaders prompts the question of whether we are seeing the development of distinct interest groups that could challenge Party and state authority and create a fragmented polity. However, through the nomenklatura system the Party has an important instrument of control to wield over business groups....... Through this system the Party controls the appointment and promotion of the heads of the most important state-owned enterprises. The nomenklatura system also enables the Party to rotate leaders in big business from a position as CEO in one company to a similar position in another state-owned company...

  20. Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

    Science.gov (United States)

    Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

    2017-11-22

    Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

  1. Spatially-Explicit Assessments of Genetic Biodiversity and Dispersal in Gopher Tortoises for Evaluation of Habitat Fragmentation at DoD Sites

    Science.gov (United States)

    2008-10-01

    acute lethality), genetic drift caused by bottlenecks or founder events , extinction-recolonization, or dramatic alterations in the patterns of gene flow...there is variation from site to site but not much difference among sites. However, genetic diversity estimates are greatly affected by the number of...significantly different (p< 0.05, t-test with Bonferroni correction). Figure 6. Phylogenetic trees representing genetic relationships among colonies

  2. Anti-sulfotyrosine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  3. Bifunctional antibodies for radioimmunotherapy.

    Science.gov (United States)

    Chatal, J F; Faivre-Chauvet, A; Bardies, M; Peltier, P; Gautherot, E; Barbet, J

    1995-04-01

    In two-step targeting technique using bifunctional antibodies, a nonradiolabeled immunoconjugate with slow uptake kinetics (several days) is initially injected, followed by a small radiolabeled hapten with fast kinetics (several hours) that binds to the bispecific immunoconjugate already taken up by the tumor target. In patients with colorectal or medullary thyroid cancer, clinical studies performed with an anti-CEA/anti-DTPA-indium bifunctional antibody and an indium-111-labeled di-DTPA-TL bivalent hapten showed that tumor uptake was not modified compared to results for F(ab')2 fragments of the same anti-CEA antibody directly labeled with indium-111, whereas the radioactivity of normal tissues was significantly reduced (3- to 6-fold). The fast tumor uptake kinetics (several hours) and high or very high tumor-to-normal tissue ratios obtained with the bifunctional antibody technique are favorable parameters for efficient radioimmunotherapy.

  4. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas

    2017-11-01

    Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

  5. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

    Science.gov (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

    2017-01-01

    Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

  6. Effective multiple oral administration of reverse genetics engineered infectious bursal disease virus in mice in the presence of neutralizing antibodies.

    Science.gov (United States)

    Hornyák, Ákos; Lipinski, Kai S; Bakonyi, Tamás; Forgách, Petra; Horváth, Ernő; Farsang, Attila; Hedley, Susan J; Palya, Vilmos; Bakács, Tibor; Kovesdi, Imre

    2015-01-01

    Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro

    2012-09-01

    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  8. Relationship between morphological and amplified fragment length ...

    African Journals Online (AJOL)

    Relationship between morphological and amplified fragment length polymorphism (AFLP) marker based genetic distance with heterosis in hot pepper (Capsicum annuum L.) SL Krishnamurthy, A Mohan Rao, K Madhavi Reddy, S Ramesh, Shailaja Hittalmani, Rao M. Gopinath ...

  9. Do rivers and human-induced habitat fragmentation affect genetic diversity and population structure of the European ground squirrel at the edge of its Pannonian range?

    Czech Academy of Sciences Publication Activity Database

    Ćosić, N.; Říčanová, Štěpánka; Bryja, Josef; Penezić, A.; Ćirović, D.

    2013-01-01

    Roč. 14, č. 2 (2013), s. 345-354 ISSN 1566-0621 R&D Projects: GA MŠk LC06073 Grant - others:GA AV ČR(CZ) KJB601410816; European Science Foundation(XE) ConGen SV/2159 Institutional support: RVO:68081766 Keywords : Souslik * Barriers * Genetic structure * Gene flow * Microsatellites Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.846, year: 2013

  10. Serum Bactericidal Antibody Responses of Adults Immunized with the MenB-4C Vaccine against Genetically Diverse Serogroup B Meningococci

    Science.gov (United States)

    Giuntini, Serena; Lujan, Eduardo; Gibani, Malick M.; Dold, Christina; Rollier, Christine S.; Pollard, Andrew J.

    2016-01-01

    ABSTRACT MenB-4C is a meningococcal vaccine for the prevention of serogroup B disease. The vaccine contains factor H binding protein (FHbp) and three other antigens that can elicit serum bactericidal antibodies (SBA). For vaccine licensure, efficacy was inferred from the SBA responses against three antigen-specific indicator strains. The relation between those results and broad protection against circulating genetically diverse strains is not known. Twenty adults were immunized with two doses of MenB-4C given 1 to 2 months apart. SBA activity against 3 reference strains and 15 serogroup B test strains (6 from college outbreaks) was measured. Compared to the preimmunization titers, 70%, 95%, and 95% of subjects had ≥4-fold increases in the titers of anti-PorA P1.4, anti-NadA, and anti-FHbp antibodies against the reference strains, respectively. In contrast, only 25 to 45% of the subjects had ≥4-fold increases in responses to 10 of the 15 test strains, including 8 that expressed one to three of the antigens in the vaccine. At 1 month, the majority of subjects with <4-fold titer increases had serum titers of ≥1:4, which are considered sufficient for protection. However, the titers against four strains declined to <1:4 by 4 to 6 months in one-third to greater than 50% of the subjects tested. Clinically relevant isolates are often more resistant to SBA than the indicator strains used to measure antigen-specific SBA. A working model is that the percentage of subjects with titers of ≥1:4 at 1 month postimmunization correlates with short-term protection against that strain, whereas the percentage of subjects with ≥4-fold titer increases represents a more robust response. (The protocol used at the Oxford Vaccine Group has been registered at ClinicalTrials.gov under registration no. NCT02398396.) PMID:27847367

  11. Primary alveolar echinococcosis: course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis.

    Science.gov (United States)

    Matsumoto, Jun; Kouguchi, Hirokazu; Oku, Yuzaburo; Yagi, Kinpei

    2010-09-01

    We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host-parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.

  12. Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

    Science.gov (United States)

    Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

    2017-11-01

    The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  14. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

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

  15. Genetic Characterization of Campylobacter Jejuni and C. coli Isolated From Broilers Using flaA PCR-Restriction Fragment Length Polymorphism Method in Shiraz, Southern Iran

    OpenAIRE

    Khoshbakht, Rahem; Tabatabaei, Mohammad; Hosseinzadeh, Saeid; Shirzad Aski, Hesamaddin; Seifi, Saeed

    2015-01-01

    Background: Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders. Objectives: The aim of t...

  16. Genome-wide assessment of population structure and genetic diversity and development of a core germplasm set for sweet potato based on specific length amplified fragment (SLAF) sequencing.

    Science.gov (United States)

    Su, Wenjin; Wang, Lianjun; Lei, Jian; Chai, Shasha; Liu, Yi; Yang, Yuanyuan; Yang, Xinsun; Jiao, Chunhai

    2017-01-01

    Sweet potato, Ipomoea batatas (L.) Lam., is an important food crop that is cultivated worldwide. However, no genome-wide assessment of the genetic diversity of sweet potato has been reported to date. In the present study, the population structure and genetic diversity of 197 sweet potato accessions most of which were from China were assessed using 62,363 SNPs. A model-based structure analysis divided the accessions into three groups: group 1, group 2 and group 3. The genetic relationships among the accessions were evaluated using a phylogenetic tree, which clustered all the accessions into three major groups. A principal component analysis (PCA) showed that the accessions were distributed according to their population structure. The mean genetic distance among accessions ranged from 0.290 for group 1 to 0.311 for group 3, and the mean polymorphic information content (PIC) ranged from 0.232 for group 1 to 0.251 for group 3. The mean minor allele frequency (MAF) ranged from 0.207 for group 1 to 0.222 for group 3. Analysis of molecular variance (AMOVA) showed that the maximum diversity was within accessions (89.569%). Using CoreHunter software, a core set of 39 accessions was obtained, which accounted for approximately 19.8% of the total collection. The core germplasm set of sweet potato developed will be a valuable resource for future sweet potato improvement strategies.

  17. CHARACTERIZATION OF A SYNTHETIC 37-RESIDUE FRAGMENT OF A MONOCLONAL-ANTIBODY AGAINST HERPES-VIRUS BY CAPILLARY ELECTROPHORESIS ELECTROSPRAY (IONSPRAY) MASS-SPECTROMETRY AND CF-252 PLASMA DESORPTION MASS-SPECTROMETRY

    NARCIS (Netherlands)

    KOSTIAINEN, R; LASONDER, E; BLOEMHOFF, W; VANVEELEN, PA; WELLING, GW; BRUINS, AP

    A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass

  18. Bone marrow transplantation from genetically HLA-nonidentical donors in children with fatal inherited disorders excluding severe combined immunodeficiencies: use of two monoclonal antibodies to prevent graft rejection.

    Science.gov (United States)

    Jabado, N; Le Deist, F; Cant, A; De Graeff-Meeders, E R; Fasth, A; Morgan, G; Vellodi, A; Hale, G; Bujan, W; Thomas, C; Cavazzana-Calvo, M; Wijdenes, J; Fischer, A

    1996-09-01

    For children with life-threatening inborn errors of metabolism without a matched related bone marrow donor, transplantation from an HLA genetically nonidentical donor is the only therapeutic option. To reduce the high risk of graft rejection in this setting without increasing the conditioning regimen, a protocol based on the infusion of an antiadhesion antibody directed against the CD11a (leukocyte function-associated antigen 1 [LFA-1]) molecule was performed by the European Bone Marrow Transplantation-European Society for Immunodeficiency group with promising results. To optimize engraftment, and thereby survival, further, the additional blockade of a second important leukocyte adhesion and signalization pathway mediated by the CD2 and LFA-3 interaction was attempted in a multicenter protocol conducted by the European Bone Marrow Transplantation-European Society for Immunodeficiency group. Results of this study (ie, engraftment and survival) were compared with a historical control group that received the anti-LFA-1 antibody alone. Factors that may have affected engraftment and survival were also considered in this study. Forty-four children with inborn errors, including inherited immunodeficiencies (excluding severe combined immunodeficiencies), Chédiak-Higashi syndrome, familial hemophagocytic lymphohistiocytosis, and malignant osteopetrosis, received bone marrow from HLA-nonidentical related donors or from HLA-identical unrelated donors at 13 European centers between August 1990 and June 1993. Bone marrow was depleted of T cells by use of either erythrocyte (E) rosetting or monoclonal antibodies (MoAbs) to prevent graft-versus-host disease. The conditioning regimen consisted of busulfan and cyclophosphamide for all patients plus etoposide for patients with osteopetrosis, familial hemophagocytic lymphohistiocytosis, and Chédiak-Higashi syndrome. Infusions of MoAbs specific for the CD11a and the CD2 molecules were started 4 and 3 days, respectively, before and

  19. Genetics

    Science.gov (United States)

    ... Likelihood of getting certain diseases Mental abilities Natural talents An abnormal trait (anomaly) that is passed down ... one of them has a genetic disorder. Information Human beings have cells with 46 chromosomes . These consist ...

  20. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  1. Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: allozymes and mitochondrial DNA restriction fragment length polymorphisms.

    Science.gov (United States)

    Narang, S K; Klein, T A; Perera, O P; Lima, J B; Tang, A T

    1993-02-01

    Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations of Anopheles albitarsis in Brazil. Two sympatric species, An. deaneorum and An. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (for Had-1, Pgi-1, Pep-1, Mpi-1, and Idh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species of An. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes. An. marajoara form CM had a higher variability (mean heterozygosity, H = 0.22, and percentage of polymorphic loci, P = 66.7) than did form IG and form MA (H = 0.08 in both, and P = 25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates of F statistics, and genetic similarities, we propose that An. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1) An. deaneorum (currently one taxon) and (2) the An. marajoara species complex, which further consists of at least three cryptic forms, marajoara form MA, marajoara form IG, and marajoara form CM.

  2. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  3. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  4. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Science.gov (United States)

    Flego, Michela; Ascione, Alessandro; Zamboni, Silvia; Dupuis, Maria L; Imperiale, Valentina; Cianfriglia, Maurizio

    2007-01-01

    Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP). Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease. PMID:17605808

  5. Genetics

    International Nuclear Information System (INIS)

    Hubitschek, H.E.

    1975-01-01

    Progress is reported on the following research projects: genetic effects of high LET radiations; genetic regulation, alteration, and repair; chromosome replication and the division cycle of Escherichia coli; effects of radioisotope decay in the DNA of microorganisms; initiation and termination of DNA replication in Bacillus subtilis; mutagenesis in mouse myeloma cells; lethal and mutagenic effects of near-uv radiation; effect of 8-methoxypsoralen on photodynamic lethality and mutagenicity in Escherichia coli; DNA repair of the lethal effects of far-uv; and near uv irradiation of bacterial cells

  6. Genetics

    DEFF Research Database (Denmark)

    Christensen, Kaare; McGue, Matt

    2016-01-01

    The sequenced genomes of individuals aged ≥80 years, who were highly educated, self-referred volunteers and with no self-reported chronic diseases were compared to young controls. In these data, healthy ageing is a distinct phenotype from exceptional longevity and genetic factors that protect...

  7. Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants.

    Science.gov (United States)

    Rönnelid, Johan; Hansson, Monika; Mathsson-Alm, Linda; Cornillet, Martin; Reed, Evan; Jakobsson, Per-Johan; Alfredsson, Lars; Holmdahl, Rikard; Skriner, Karl; Serre, Guy; Lundberg, Karin; Klareskog, Lars

    2018-02-01

    The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients. We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array. The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset. Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  8. Universal elements of fragmentation

    International Nuclear Information System (INIS)

    Yanovsky, V. V.; Tur, A. V.; Kuklina, O. V.

    2010-01-01

    A fragmentation theory is proposed that explains the universal asymptotic behavior of the fragment-size distribution in the large-size range, based on simple physical principles. The basic principles of the theory are the total mass conservation in a fragmentation process and a balance condition for the energy expended in increasing the surface of fragments during their breakup. A flux-based approach is used that makes it possible to supplement the basic principles and develop a minimal theory of fragmentation. Such a supplementary principle is that of decreasing fragment-volume flux with increasing energy expended in fragmentation. It is shown that the behavior of the decreasing flux is directly related to the form of a power-law fragment-size distribution. The minimal theory is used to find universal asymptotic fragment-size distributions and to develop a natural physical classification of fragmentation models. A more general, nonlinear theory of strong fragmentation is also developed. It is demonstrated that solutions to a nonlinear kinetic equation consistent with both basic principles approach a universal asymptotic size distribution. Agreement between the predicted asymptotic fragment-size distributions and experimental observations is discussed.

  9. Poly(I:C) adjuvant strongly enhances parasite-inhibitory antibodies and Th1 response against Plasmodium falciparum merozoite surface protein-1 (42-kDa fragment) in BALB/c mice.

    Science.gov (United States)

    Mehrizi, Akram Abouie; Rezvani, Niloufar; Zakeri, Sedigheh; Gholami, Atefeh; Babaeekhou, Laleh

    2018-04-01

    Malaria vaccine development has been confronted with various challenges such as poor immunogenicity of malaria vaccine candidate antigens, which is considered as the main challenge. However, this problem can be managed using appropriate formulations of antigens and adjuvants. Poly(I:C) is a potent Th1 inducer and a human compatible adjuvant capable of stimulating both B- and T-cell immunity. Plasmodium falciparum merozoite surface protein 1 42 (PfMSP-1 42 ) is a promising vaccine candidate for blood stage of malaria that has faced several difficulties in clinical trials, mainly due to improper adjuvants. Therefore, in the current study, poly(I:C), as a potent Th1 inducer adjuvant, was evaluated to improve the immunogenicity of recombinant PfMSP-1 42 , when compared to CFA/IFA, as reference adjuvant. Poly(I:C) produced high level and titers of anti-PfMSP-1 42 IgG antibodies in which was comparable to CFA/IFA adjuvant. In addition, PfMSP-1 42 formulated with poly(I:C) elicited a higher ratio of IFN-γ/IL-4 (23.9) and IgG2a/IgG1 (3.77) with more persistent, higher avidity, and titer of IgG2a relative to CFA/IFA, indicating a potent Th1 immune response. Poly(I:C) could also help to induce anti-PfMSP-1 42 antibodies with higher growth-inhibitory activity than CFA/IFA. Altogether, the results of the current study demonstrated that poly(I:C) is a potent adjuvant that can be appropriate for being used in PfMSP-1 42 -based vaccine formulations.

  10. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

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

  11. Fission fragment angular momentum

    International Nuclear Information System (INIS)

    Frenne, D. De

    1991-01-01

    Most of the energy released in fission is converted into translational kinetic energy of the fragments. The remaining excitation energy will be distributed among neutrons and gammas. An important parameter characterizing the scission configuration is the primary angular momentum of the nascent fragments. Neutron emission is not expected to decrease the spin of the fragments by more than one unit of angular momentum and is as such of less importance in the determination of the initial fragment spins. Gamma emission is a suitable tool in studying initial fragment spins because the emission time, number, energy, and multipolarity of the gammas strongly depend on the value of the primary angular momentum. The main conclusions of experiments on gamma emission were that the initial angular momentum of the fragments is large compared to the ground state spin and oriented perpendicular to the fission axis. Most of the recent information concerning initial fragment spin distributions comes from the measurement of isomeric ratios for isomeric pairs produced in fission. Although in nearly every mass chain isomers are known, only a small number are suitable for initial fission fragment spin studies. Yield and half-life considerations strongly limit the number of candidates. This has the advantage that the behavior of a specific isomeric pair can be investigated for a number of fissioning systems at different excitation energies of the fragments and fissioning nuclei. Because most of the recent information on primary angular momenta comes from measurements of isomeric ratios, the global deexcitation process of the fragments and the calculation of the initial fragment spin distribution from measured isomeric ratios are discussed here. The most important results on primary angular momentum determinations are reviewed and some theoretical approaches are given. 45 refs., 7 figs., 2 tabs

  12. Radioimmunotherapy with Tenarad, a {sup 131}I-labelled antibody fragment targeting the extra-domain A1 of tenascin-C, in patients with refractory Hodgkin's lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Aloj, Luigi [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Medicina Nucleare, Napoli (Italy); D' Ambrosio, Laura; Aurilio, Michela; Morisco, Anna; Caraco' , Corradina; Di Gennaro, Francesca; Lastoria, Secondo [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa Medicina Nucleare, Napoli (Italy); Frigeri, Ferdinando; Capobianco, Gaetana; Pinto, Antonio [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Ematologia Oncologica, Napoli (Italy); Giovannoni, Leonardo; Menssen, Hans D. [Philogen, SpA, Siena (Italy); Neri, Dario [Institute of Pharmaceutical Sciences, ETH, Zurich (Switzerland)

    2014-05-15

    The extra-domain A1 of tenascin-C (TC-A1) is highly expressed in the extracellular matrix of tumours and on newly formed blood vessels and is thus a valuable target for radionuclide therapy. Tenarad is a fully human miniantibody or small immunoprotein (SIP, molecular weight 80 kDa) labelled with {sup 131}I that is derived from a TC-A1-binding antibody. Previous phase I/II studies with a similar compound ({sup 131}I-L19SIP) used for radioimmunotherapy (RIT) have shown preliminary efficacy in a variety of cancer types. In this ongoing phase I/II trial, Tenarad was administered to patients with recurrent Hodgkin's lymphoma (HL) refractory to conventional treatments. Eight patients (four men, four women; age range 19 - 41) were enrolled between April 2010 and March 2011. All patients had received a median of three previous lines of chemotherapy (range three to six) and seven had also undergone autologous stem cell transplantation (ASCT) or bone marrow transplantation. In addition, seven patients received external beam radiation. All patients had nodal disease, constitutional B symptoms and some showed extranodal disease in skeletal bone (four patients), lung (three), liver (two) and spleen (one). Baseline assessments included whole-body FDG PET with contrast-enhanced CT and diagnostic Tenarad planar and SPECT studies. Patients were considered eligible to receive a therapeutic dose of Tenarad (2.05 GBq/m{sup 2}) if tumour uptake was more than four times higher than that of muscle. All patients were eligible and received the therapeutic dose of Tenarad. Only one patient developed grade 4 thrombocytopenia and leucocytopenia, requiring hospitalization and therapeutic intervention. All other patients had haematological toxicity of grade 3 or lower, which resolved spontaneously. At the first response assessment (4 - 6 weeks after therapy), one patient showed a complete response, one showed a partial response (PR) and five had disease stabilization (SD). Five patients

  13. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  14. Genetic characterization by amplified fragment length polymorphism ...

    African Journals Online (AJOL)

    Mariela Vázquez Calderón, Javier O Mijangos-Cortés, Manuel JL Zavala, L Felipe Sánchez Teyer, Adriana M Quiroz, Matilde Margarita G Ortiz, Amilcar Contreras M Fernando, Espadas G Francisco, Gabriela Fuentes Ortiz, Jorge M Santamaría ...

  15. Fragments of protein A eluted during protein A affinity chromatography.

    Science.gov (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert

    2007-09-07

    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  16. The future of antibodies as cancer drugs.

    Science.gov (United States)

    Reichert, Janice M; Dhimolea, Eugen

    2012-09-01

    Targeted therapeutics such as monoclonal antibodies (mAbs) have proven successful as cancer drugs. To profile products that could be marketed in the future, we examined the current commercial clinical pipeline of mAb candidates for cancer. Our analysis revealed trends toward development of a variety of noncanonical mAbs, including antibody-drug conjugates (ADCs), bispecific antibodies, engineered antibodies and antibody fragments and/or domains. We found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets and mechanisms of action (MOA). Although well-validated targets, such as epidermal growth factor receptor (EGFR) and CD20, continue to provide opportunities for companies, we found notable trends toward targeting less-well-validated antigens and exploration of innovative MOA such as the generation of anticancer immune responses or recruitment of cytotoxic T cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor.

    Directory of Open Access Journals (Sweden)

    Clémence Bernard

    2016-05-01

    Full Text Available During postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells. In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these "transfer" sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity. We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system.

  18. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Profile of sequential determinants in tissue polypeptide antigen BrCN:B fragment.

    Science.gov (United States)

    Chersi, A; Camera, M; Trinca, M L; Castelli, M

    1989-02-15

    A synthetic approach has been applied to determine the profile of sequential determinants of one immunodominant region of Tissue Polypeptide Antigen (TPA). Five overlapping peptides, covering 30 of the 32 amino acid residues of this fragment, were chemically synthesized, and their antibody-binding activities for rabbit anti-TPA antibodies determined by enzyme-linked immunoadsorbant assays. Anti-TPA reacted with two overlapping fragments at the COOH-terminal end of the fragment, but not with peptides that include Arg 15 considered as essential for the antigenicity of the whole fragment. This might suggest that this critical residue is involved in the formation of a complex conformational determinant.

  20. Do monoclonal antibodies recognize linear sequential determinants?

    Science.gov (United States)

    Camera, M; Muratti, E; Trinca, M L; Chersi, A

    1988-01-01

    A group of 19 anti-class II monoclonal antibodies produced in different laboratories were tested in ELISA for their ability to bind to a panel of synthetic peptides selected from HLA-DQ alpha and beta chains. No one of the antibodies tested was found to react with the synthetic fragments, thus confirming the common finding that MoAbs generally fail to recognize fragments of the native antigen. The possibility that this result might be partly due to the procedure used for screening hybridoma supernatants is discussed.

  1. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Glaessner, H.

    1986-01-01

    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

  2. Embedded Fragments Registry (EFR)

    Data.gov (United States)

    Department of Veterans Affairs — In 2009, the Department of Defense estimated that approximately 40,000 service members who served in OEF/OIF may have embedded fragment wounds as the result of small...

  3. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  4. Fragmented Work Stories

    DEFF Research Database (Denmark)

    Humle, Didde Maria; Reff Pedersen, Anne

    2015-01-01

    , edited and performed by the storyteller in an ongoing process allowing tensions, discontinuities and editing between failures and achievements, between dreams and work realities and between home and work life. We argue that by including different types of fragmentation, we offer a new type......Following a strand of narrative studies pointing to the living conditions of storytelling and the micro-level implications of working within fragmented narrative perspectives, this article contributes to narrative research on work stories by focusing on how meaning is created from fragmented...... stories. We argue that meaning by story making is not always created by coherence and causality; meaning is created by different types of fragmentation: discontinuities, tensions and editing. The objective of this article is to develop and advance antenarrative practice analysis of work stories...

  5. Fragmentation Main Model

    Data.gov (United States)

    Earth Data Analysis Center, University of New Mexico — The fragmentation model combines patch size and patch continuity with diversity of vegetation types per patch and rarity of vegetation types per patch. A patch was...

  6. Physics of projectile fragments

    International Nuclear Information System (INIS)

    Minamisono, Tadanori

    1982-01-01

    This is a study report on the polarization phenomena of the projectile fragments produced by heavy ion reactions, and the beta decay of fragments. The experimental project by using heavy ions with the energy from 50 MeV/amu to 250 MeV/amu was designed. Construction of an angle-dispersion spectrograph for projectile fragments was proposed. This is a two-stage spectrograph. The first stage is a QQDQQ type separator, and the second stage is QDQD type. Estimation shows that Co-66 may be separated from the nuclei with mass of 65 and 67. The orientation of fragments can be measured by detecting beta-ray. The apparatus consists of a uniform field magnet, an energy absorber, a stopper, a RF coil and a beta-ray hodoscope. This system can be used for not only this purpose but also for the measurement of hyperfine structure. (Kato, T.)

  7. Fragment Impact Toolkit (FIT)

    Energy Technology Data Exchange (ETDEWEB)

    Shevitz, Daniel Wolf [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Key, Brian P. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Garcia, Daniel B. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-09-05

    The Fragment Impact Toolkit (FIT) is a software package used for probabilistic consequence evaluation of fragmenting sources. The typical use case for FIT is to simulate an exploding shell and evaluate the consequence on nearby objects. FIT is written in the programming language Python and is designed as a collection of interacting software modules. Each module has a function that interacts with the other modules to produce desired results.

  8. Intracellular Targeting of CEA Results in Th1-Type Antibody Responses Following Intradermal Genetic Vaccination by a Needle-Free Jet Injection Device

    Directory of Open Access Journals (Sweden)

    Susanne Johansson

    2007-01-01

    Full Text Available The route and method of immunization, as well as the cellular localization of the antigen, can influence the generation of an immune response. In general, intramuscular immunization results in Th1 responses, whereas intradermal delivery of DNA by gene gun immunization often results in more Th2 responses. Here we investigate how altering the cellular localization of the tumor antigen CEA (carcinoembryonic antigen affects the quality and amplitude of DNA vaccine-induced antibody responses in mice following intradermal delivery of DNA by a needle-free jet injection device (Biojector. CEA was expressed either in a membrane-bound form (wild-type CEA or in two truncated forms (CEA6 and CEA66 with cytoplasmic localization, where CEA66 was fused to a promiscuous T-helper epitope from tetanus toxin. Repeated intradermal immunization of BALB/c mice with DNA encoding wild-type CEA produced high antibody titers of a mixed IgG1/IgG2a ratio. In contrast, utilizing the DNA construct that resulted in intracellular targeting of CEA led to a reduced capacity to induce CEA-specific antibodies, but instead induced a Th1-biased immune response.

  9. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

  10. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  11. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  12. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  13. A Study on Campylobacter jejuni and Campylobacter coli through Commercial Broiler Production Chains in Thailand: Antimicrobial Resistance, the Characterization of DNA Gyrase Subunit A Mutation, and Genetic Diversity by Flagellin A Gene Restriction Fragment Length Polymorphism.

    Science.gov (United States)

    Thomrongsuwannakij, Thotsapol; Blackall, Patrick J; Chansiripornchai, Niwat

    2017-06-01

    chain reaction-restriction fragment length polymorphism of the flagellin A gene (flaA-RFLP) to determine their genetic relationships. Ten distinct clusters were recognized by flaA-RFLP typing. The results showed that horizontal transmission was the major route of Campylobacter transmission in this study. In conclusion, the emergence of MDR and high resistance rates to several antimicrobials are major concerns identified in this study. The prudent use of these agents and active surveillance of resistance at the farm level are essential steps to reduce the public health risks identified in this work.

  14. Hybrid IgG4/IgG4 Fc antibodies form upon 'Fab-arm' exchange as demonstrated by SDS-PAGE or size-exclusion chromatography

    NARCIS (Netherlands)

    Rispens, Theo; den Bleker, Tamara H.; Aalberse, Rob C.

    2010-01-01

    Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab + Fc) with a molecular weight of ca. 100 kDa. These antibodies can

  15. Cross-Neutralizing Antibodies in HIV-1 Individuals Infected by Subtypes B, F1, C or the B/Bbr Variant in Relation to the Genetics and Biochemical Characteristics of the env Gene.

    Directory of Open Access Journals (Sweden)

    Dalziza Victalina de Almeida

    Full Text Available Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (p<0.005, however neither was there a correlation verified between the variable region length and neutralization potency, nor between PNLG and HIV-1 subtypes. The enrichment of W at top of V3 loop in weak neutralizing response viruses and the P in viruses with higher neutralization susceptibility was statistically significant (p = 0.013. Some other signatures sites were associated to HIV-1 subtype-specific F1 and B/Bbr samples might influence in the distinct neutralizing response. These results indicate that a single amino acid substitution may lead to a distinct conformational exposure or load in the association domain of the trimer of gp120 and interfere with the induction power of the neutralizing response, which affects the sensitivity of the neutralizing antibody and has significant implications for vaccine design.

  16. Cross-Neutralizing Antibodies in HIV-1 Individuals Infected by Subtypes B, F1, C or the B/Bbr Variant in Relation to the Genetics and Biochemical Characteristics of the env Gene.

    Science.gov (United States)

    de Almeida, Dalziza Victalina; Macieira, Karine Venegas; Grinsztejn, Beatriz Gilda Jegerhorn; Veloso Dos Santos, Valdiléa Gonçalves; Guimarães, Monick Lindenmeyer

    2016-01-01

    Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs) and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (pHIV-1 subtypes. The enrichment of W at top of V3 loop in weak neutralizing response viruses and the P in viruses with higher neutralization susceptibility was statistically significant (p = 0.013). Some other signatures sites were associated to HIV-1 subtype-specific F1 and B/Bbr samples might influence in the distinct neutralizing response. These results indicate that a single amino acid substitution may lead to a distinct conformational exposure or load in the association domain of the trimer of gp120 and interfere with the induction power of the neutralizing response, which affects the sensitivity of the neutralizing antibody and has significant implications for vaccine design.

  17. Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity.

    Science.gov (United States)

    Fernández-Ulibarri, Inés; Hammer, Katharina; Arndt, Michaela A E; Kaufmann, Johanna K; Dorer, Dominik; Engelhardt, Sarah; Kontermann, Roland E; Hess, Jochen; Allgayer, Heike; Krauss, Jürgen; Nettelbeck, Dirk M

    2015-05-01

    Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof. © 2014 UICC.

  18. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

    2016-01-01

    fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...... small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271(+), as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer...

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

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

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

  20. Functionally fused antibodies--a novel adjuvant fusion system

    DEFF Research Database (Denmark)

    Larsen, Martin; Jensen, Kim Bak; Christensen, Peter Astrup

    2008-01-01

    Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radio...

  1. Fragmentation of kidney stones

    International Nuclear Information System (INIS)

    Kovacs, K.; Kun, F.; Vertse, T.

    2005-01-01

    Complete text of publication follows. Fragmentation, i.e. the breaking of particulate materials into smaller pieces is abundant in nature and underlies several industrial processes, which attracted a continuous interest in scientific and engineering research over the past decades. In industrial applications, fragmentation processes are mostly used for the comminution of ores in various types of mills. Kidney stone is a well known human dis- ease which embitters the life of many people (in a country like the USA about 10 6 cases are registered yearly). In order to extract large kidney stones (diameter ≥ 1 cm) from the human body without operation, one of the most efficient treatment is the fragmentation of kidney stones by the so-called extracorporal shock wave lithography method: a shock wave penetrating the human body is generated by an electric pulse. The repeated application of the shock wave gradually fragments the stones into pieces of size ≤ 2 mm which then leave the body through the urine system. Recently, a novel type of lithographic method has been suggested by using widely focused shock waves which fragment the stones by a squeezing mechanism. Laboratory experiments showed that the widely focused squeezing waves achieve a higher fragmentation efficiency than the frequently used shock waves of sharp focus. Based on this method a novel medical treatment can be introduced which is less demanding for the patients. Before the application of the method in the clinical practice a detailed understanding of the fragmentation mechanism of kidney stones due to shock waves is required. Since analytic theoretical methods have serious limitations in this field, we develop a realistic model of the mechanical behavior of kidney stones and a simulation code which makes possible to study the mechanism of breakup under various external conditions. Computer simulations in two dimensions have revealed a peculiar way of crack formation, i.e. the crack which finally breaks

  2. Genetic structure of a bird-dispersed tropical tree (Dendropanax arboreus in a fragmented landscape in Mexico Estructura genética de un árbol tropical dispersado por aves (Dendropanax arboreus en un paisaje fragmentado en México

    Directory of Open Access Journals (Sweden)

    Elsa M. Figueroa-Esquivel

    2010-12-01

    Full Text Available We analyzed the genetic structure of the tropical tree Dendropanax arboreus (Araliaceae in relation to habitat fragmentation. Genetic variation, structure, and genetic differentiation among populations from Los Tuxtlas tropical rainforest were estimated using ISSRs as molecular markers. DNA from 219 individuals belonging to 9 populations was amplified with 4 primers yielding a total of 75 loci. Adults and juveniles from each population were analyzed to assess the genetic diversity and structure pre and post-fragmentation, respectively. Dendropanax arboreus showed high levels of genetic diversity (h = 0.253 and significant but low genetic differentiation among populations ( or = 0.062. A hierarchical analysis of the gvenetic structure showed that 91.5% of the genetic variation is attributable to individual differences within populations. The average Nei's genetic distance among populations was low (D = 0.034 and genetic distance among pairs of populations increased with geographic distance separating them. Because genetic diversity is similar between adult and juvenile trees at all but 2 populations, we suggest that seed dispersal prevented genetic differentiation and maintains genetic connectivity among fragments and continuous forest populations. Juvenile populations showed a higher genetic differentiation ( or = 0.15 than adult trees, indicating a role of genetic drift via reduced population size.Se analizó la estructura genética del árbol tropical Dendropanax arboreus (Araliaceae en relación con la fragmentación del hábitat en la selva tropical de Los Tuxtlas, Veracruz, México. La variación, estructura y diferenciación genética entre poblaciones del bosque continuo y de fragmentos se estimó usando ISSR como marcador molecular. El ADN de 219 individuos de 9 poblaciones se amplificó para 4 primers (75 loci. Se analizaron las poblaciones de árboles adultos y juveniles en cada sitio, representando la diversidad y estructura gen

  3. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  4. Optimal Resource Allocation to Survival and Reproduction in Parasitic Wasps Foraging in Fragmented Habitats

    OpenAIRE

    Wajnberg, Eric; Coquillard, Patrick; Vet, Louise E. M.; Hoffmeister, Thomas

    2012-01-01

    International audience; Expansion and intensification of human land use represents the major cause of habitat fragmentation. Such fragmentation can have dramatic consequences on species richness and trophic interactions within food webs. Although the associated ecological consequences have been studied by several authors, the evolutionary effects on interacting species have received little research attention. Using a genetic algorithm, we quantified how habitat fragmentation and environmental...

  5. Fragments of the Past

    OpenAIRE

    Peter Szende; Annie Holcombe

    2016-01-01

    With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  6. Picking Up (On) Fragments

    NARCIS (Netherlands)

    Ellis, Phil

    2015-01-01

    abstractThis article discusses the implications for archival and media archaeological research and reenactment artwork relating to a recent arts practice project: reenacttv: 30 lines / 60 seconds. It proposes that archival material is unstable but has traces and fragments that are full of creative

  7. Fragments of Time

    DEFF Research Database (Denmark)

    Christiansen, Steen Ledet

    Time travel films necessarily fragment linear narratives, as scenes are revisited with differences from the first time we saw it. Popular films such as Back to the Future mine comedy from these visitations, but there are many different approaches. One extreme is Chris Marker's La Jetée - a film...

  8. Wildlife habitat fragmentation.

    Science.gov (United States)

    John. Lehmkuhl

    2005-01-01

    A primary issue in forest wildlife management is habitat fragmentation and its effects on viability, which is the "bottom line" for plant and animal species of conservation concern. Population viability is the likelihood that a population will be able to maintain itself (remain viable) over a long period of time-usually 100 years or more. Though it is true...

  9. Fragments of the Past

    Directory of Open Access Journals (Sweden)

    Peter Szende

    2016-10-01

    Full Text Available With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  10. Stone fragmentation by ultrasound

    Indian Academy of Sciences (India)

    Some delicate nerves and fibres in the surrounding areas of the stones present in the kidney are also damaged by high ultrasonic intensity used in such systems. In the present work, enhancement of the kidney stone fragmentation by using ultrasound is studied. The cavitation bubbles are found to implode faster, with more ...

  11. Synthesis of arabinoxylan fragments

    DEFF Research Database (Denmark)

    Underlin, Emilie Nørmølle; Böhm, Maximilian F.; Madsen, Robert

    , or production of commercial chemicals which are mainly obtained from fossil fuels today.The arbinoxylan fragments have a backbone of β-1,4-linked xylans with α-L-arabinose units attached at specific positions. The synthesis ultilises an efficient synthetic route, where all the xylan units can be derived from D...

  12. Genetic diversity and population structure of endangered Aquilaria ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 94; Issue 4. Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation ... Keywords. agarwood; conservation; home gardens; genetic diversity; population genetic structure; amplified fragment length polymorphism.

  13. Bglbrick strategy for the construction of single domain antibody fusions

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2017-12-01

    Full Text Available Single domain antibodies, recombinantly expressed variable domains derived from camelid heavy chain antibodies, are often expressed as multimers for detection and therapeutic applications. Constructs in which several single domain antibodies are genetically fused serially, as well as those in which single domain antibodies are genetically linked with domains that naturally form multimers, yield improvement in apparent binding affinity due to avidity. Here, using a single domain antibody that binds envelope protein from the Dengue virus, we demonstrated the construction of single domain antibody dimers using the Bglbrick cloning strategy. Constructing single domain antibodies and multimerization domains as Bglbrick parts enables the easy mixing and matching of parts. The dimeric constructs provided enhanced fluorescent signal in assays for detection of Dengue virus like particles over the monomeric single domain antibody.

  14. Construction of phage antibody repertoires from the blood of West Nile virus-infected donors.

    Science.gov (United States)

    Throsby, Mark; de Kruif, John

    2009-01-01

    A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a repertoire that can be used in panning procedures to identify many unique monoclonal antibodies.

  15. VHH Antibodies: Reagents for Mycotoxin Detection in Food Products

    Directory of Open Access Journals (Sweden)

    Jia Wang

    2018-02-01

    Full Text Available Mycotoxins are the toxic secondary metabolites produced by fungi and they are a worldwide public health concern. A VHH antibody (or nanobody is the smallest antigen binding entity and is produced by heavy chain only antibodies. Compared with conventional antibodies, VHH antibodies overcome many pitfalls typically encountered in clinical therapeutics and immunodiagnostics. Likewise, VHH antibodies are particularly useful for monitoring mycotoxins in food and feedstuffs, as they are easily genetic engineered and have superior stability. In this review, we summarize the efforts to produce anti-mycotoxins VHH antibodies and associated assays, presenting VHH as a potential tool in mycotoxin analysis.

  16. Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment

    OpenAIRE

    Syvänen, Stina; Edén, Desireé; Sehlin, Dag

    2017-01-01

    Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to d...

  17. Subcloning of DNA fragments.

    Science.gov (United States)

    Struhl, K

    2001-05-01

    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.