WorldWideScience

Sample records for camelid single-domain antibody

  1. 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 a

  2. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

    Science.gov (United States)

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-05-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.

  3. Isolation and optimization of camelid single-domain antibodies:Dirk Saerens’work on nanobodies

    Institute of Scientific and Technical Information of China (English)

    Dirk; Saerens

    2010-01-01

    It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.

  4. Fungal Glucosylceramide-Specific Camelid Single Domain Antibodies Are Characterized by Broad Spectrum Antifungal Activity

    Directory of Open Access Journals (Sweden)

    Barbara De Coninck

    2017-06-01

    Full Text Available Chemical crop protection is widely used to control plant diseases. However, the adverse effects of pesticide use on human health and environment, resistance development and the impact of regulatory requirements on the crop protection market urges the agrochemical industry to explore innovative and alternative approaches. In that context, we demonstrate here the potential of camelid single domain antibodies (VHHs generated against fungal glucosylceramides (fGlcCer, important pathogenicity factors. To this end, llamas were immunized with purified fGlcCer and a mixture of mycelium and spores of the fungus Botrytis cinerea, one of the most important plant pathogenic fungi. The llama immune repertoire was subsequently cloned in a phage display vector to generate a library with a diversity of at least 108 different clones. This library was incubated with fGlcCer to identify phages that bind to fGlcCer, and VHHs that specifically bound fGlcCer but not mammalian or plant-derived GlcCer were selected. They were shown to inhibit the growth of B. cinerea in vitro, with VHH 41D01 having the highest antifungal activity. Moreover, VHH 41D01 could reduce disease symptoms induced by B. cinerea when sprayed on tomato leaves. Based on all these data, anti-fGlcCer VHHs show the potential to be used as an alternative approach to combat fungal plant diseases.

  5. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-06-01

    Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

  7. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  8. Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

    Science.gov (United States)

    Alturki, Norah A; Henry, Kevin A; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

  9. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

    Science.gov (United States)

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick

    2014-05-21

    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

  10. Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies.

    Directory of Open Access Journals (Sweden)

    Laura Ratier

    Full Text Available The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS, a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98% by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

  11. Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

    Science.gov (United States)

    Tian, Baomin; Wong, Wah Yau; Hegmann, Elda; Gaspar, Kim; Kumar, Praveen; Chao, Heman

    2015-06-17

    A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

  12. Single-domain antibodies for biomedical applications.

    Science.gov (United States)

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

  13. 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.

  14. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    Science.gov (United States)

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  15. Sequence Tolerance of a Highly Stable Single Domain Antibody: Comparison of Computational and Experimental Profiles

    Science.gov (United States)

    2016-09-09

    popular construct of the heavy chain is single-domain antibody (sdAb) chains derived from camelids. The interest in sdAbs lies in their biotechnological ...thermal stable lama single domain antibody specific for Staphylococcus aureus enterotoxin B. BMC Biotechnology 11. 3. Zabetakis D, Anderson GP, Bayya N...neighbors from a Dali search of the single-chain conformers of the assembly. (a) Conformers of A3 where the color green denotes the A chain and blue

  16. Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies.

    Science.gov (United States)

    Lipovsek, Dasa; Lippow, Shaun M; Hackel, Benjamin J; Gregson, Melissa W; Cheng, Paul; Kapila, Atul; Wittrup, K Dane

    2007-05-11

    The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from

  17. Thermal stability and refolding capability of shark derived single domain antibodies.

    Science.gov (United States)

    Liu, Jinny L; Zabetakis, Dan; Brown, Jazmine C; Anderson, George P; Goldman, Ellen R

    2014-06-01

    Single-domain antibodies (sdAb) from camelids and sharks represent the smallest immunoglobulin-based functional binding domains, and are known for their thermal stability and ability to refold after denaturation. Whereas target-binding sdAb have been derived from both immunized and naïve sharks and camelids, the stability of camelid-derived sdAb have been evaluated much more extensively. To address this disparity we characterized 20 sdAb derived from spiny dogfish shark and smooth dogfish shark in terms of their protein production, melting temperature and ability to refold after heat denaturation. Using the same expression system and protocol as we follow to produce camelid sdAb, production of the shark sdAb was quite poor, often resulting in less than a tenth of the typical yield for camelid sdAb. We measured the melting temperature of each of the sdAb. Similar to camelid sdAb, the shark-derived sdAb, showed a range of melting temperature values from 42°C to 77°C. Also similar to what has been observed in camelids, the sdAb from both shark species showed a range of ability to refold after heat denaturation. This work demonstrated that although shark sdAb can possess high melting temperatures and refolding ability, no clear advantage over sdAb derived from camelids in terms of thermostability and renaturation was obtained.

  18. Ricin Detection Using Phage Displayed Single Domain Antibodies

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2009-01-01

    Full Text Available Phage-displayed single domain antibodies (sdAb were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb. Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (~16 kDa, while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping.

  19. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

    Science.gov (United States)

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; Adriouch, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei; Serreze, David V; Goldbaum, Fernando A; Haag, Friedrich; Koch-Nolte, Friedrich

    2009-08-01

    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

  20. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

    Science.gov (United States)

    Anderson, George P.; Teichler, Daniel D.; Zabetakis, Dan; Shriver-Lake, Lisa C.; Liu, Jinny L.; Lonsdale, Stephen G.; Goodchild, Sarah A.; Goldman, Ellen R.

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  1. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

    Science.gov (United States)

    Anderson, George P; Teichler, Daniel D; Zabetakis, Dan; Shriver-Lake, Lisa C; Liu, Jinny L; Lonsdale, Stephen G; Goodchild, Sarah A; Goldman, Ellen R

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

  2. Using llama derived single domain antibodies to target botulinum neurotoxins

    Science.gov (United States)

    Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

    2010-04-01

    Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

  3. Development and Testing of Recombinant Single Domain Antibodies

    Science.gov (United States)

    2010-12-31

    single domain antibodies from a naïve shark library. Molecular Immunology. 44:1786-1794. Sherwood, L.J., Osborn, L., Carrion , R., Patterson, J., Hayhurst...for viral diagnostics in hot climates 9. Sherwood, L.J., Osborn, L, Carrion , R., Patterson, J.P and Hayhurst, A. April 22, 2007 Rapid assembly of...and Austin Life Sciences Association, New Bruanfels, TX. 11. Sherwood, L.J., Osborn, L., Carrion , R., Patterson, J.P. and Hayhurst, A. May 14, 2007

  4. Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection.

    Science.gov (United States)

    Turner, Kendrick B; Zabetakis, Dan; Goldman, Ellen R; Anderson, George P

    2014-03-01

    Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5 °C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0 °C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ∼ 4.1 °C of the 6.5 °C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable.

  5. Improving the biophysical properties of anti-ricin single-domain antibodies

    Directory of Open Access Journals (Sweden)

    Kendrick B. Turner

    2015-06-01

    Full Text Available Single-domain antibodies (sdAbs derived from heavy-chain only antibodies produced in camelids are attractive immunoreagents due to their small size, high affinity, and ability to refold and retain binding activity after denaturation. It has been observed that some sdAbs, however, exhibit undesirable properties including reduced solubility when subjected to heating or upon long-term storage at production-relevant concentrations, which can limit their usefulness. Using a multi-step, rational design approach that included consensus-sequence driven sequence repairs, the alteration of net protein charge, and the introduction of non-native disulfide bonds, augmented solubility and increased melting temperatures were achieved. The improved sdAbs tolerated storage in solution at high concentration (10 mg/mL and were able to withstand multiple cycles of heating to high temperature (70 °C. This work demonstrates a pathway for improving the biophysical characteristics of sdAbs which is essential for expanding their utility for both diagnostic as well as therapeutic applications.

  6. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    Directory of Open Access Journals (Sweden)

    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  7. The new research and application progress in heavy chain antibody and its genetic engineering single domain antibody%重链抗体及其基因工程单域抗体的研究和应用进展

    Institute of Scientific and Technical Information of China (English)

    蔡家麟; 王颖; 潘欣(通讯作者)

    2013-01-01

      In recent years, researchers found unusual antibodies composed only of heavy chains from camelids and sharks. These peculiar heavy chain antibodies (hcAbs) lack light chains but stil remain dedicated variable domain with the antigen-binding site. Recombinant single-domain antibody is the smalest intact antigen-binding fragment derived from heavy-chain antibodies. The advantageous features of single domain antibody reagents derived from these hcAbs include their smal dimension, high apparent stability, high expression. As expectations, high-affinity, improved solubility without any sign of aggregation single-domain antibodies can be widely used in basic research and improved diagnostic field.%  近年来在骆驼和护士鲨等动物的体内发现了一种重链抗体。重链抗体和普通的抗体相比缺少轻链,仅有可变区,但依然保留了抗原结合能力。通过基因工程改造重链抗体形成的单域抗体不仅保留了分子量小、物理稳定性高、容易表达等特性优点,而且亲和力高、不易聚集,被广泛应用于科研和临床诊断。

  8. Exploitation of the transcytotic pathway in polarised cells by camelid single domain antibodies

    NARCIS (Netherlands)

    Emmerson, C.D.

    2013-01-01

    Delivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls wh

  9. Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides

    NARCIS (Netherlands)

    Harmsen, M.M.; Fijten, H.P.D.

    2012-01-01

    We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to

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

    Science.gov (United States)

    Pereira, Soraya S; Moreira-Dill, Leandro S; Morais, Michelle S S; Prado, Nidiane D R; Barros, Marcos L; Koishi, Andrea C; Mazarrotto, Giovanny A C A; Gonçalves, Giselle M; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Pereira da Silva, Luiz H; Duarte dos Santos, Claudia N; Fernandes, Carla F C; Stabeli, Rodrigo G

    2014-01-01

    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 hantavirus

  11. 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

  12. Pentavalent single-domain antibodies reduce Campylobacter jejuni motility and colonization in chickens.

    Directory of Open Access Journals (Sweden)

    Ali Riazi

    Full Text Available Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.

  13. Pentavalent single-domain antibodies reduce Campylobacter jejuni motility and colonization in chickens.

    Science.gov (United States)

    Riazi, Ali; Strong, Philippa C R; Coleman, Russell; Chen, Wangxue; Hirama, Tomoko; van Faassen, Henk; Henry, Matthew; Logan, Susan M; Szymanski, Christine M; Mackenzie, Roger; Ghahroudi, Mehdi Arbabi

    2013-01-01

    Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.

  14. Crystal structure of a shark single-domain antibody V region in complex with lysozyme.

    Science.gov (United States)

    Stanfield, Robyn L; Dooley, Helen; Flajnik, Martin F; Wilson, Ian A

    2004-09-17

    Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies.

  15. Analysis of the binding loops configuration and surface adaptation of different crystallized single-domain antibodies in response to various antigens.

    Science.gov (United States)

    Al Qaraghuli, Mohammed M; Ferro, Valerie A

    2017-04-01

    Monoclonal antibodies have revolutionized the biomedical field through their ubiquitous utilization in different diagnostics and therapeutic applications. Despite this widespread use, their large size and structural complexity have limited their versatility in specific applications. The antibody variable region that is responsible for binding antigen is embodied within domains that can be rescued individually as single-domain antibody (sdAb) fragments. Because of the unique characteristics of sdAbs, such as low molecular weight, high physicochemical stability, and the ability to bind antigens inaccessible to conventional antibodies, they represent a viable alternative to full-length antibodies. Consequently, 149 crystal structures of sdAbs, originating from human (VH), camelids (VHH), or sharks (VNAR), were retrieved from the Protein Data Bank, and their structures were compared. The 3 types of sdAbs displayed complementarity determining regions (CDRs) with different lengths and configurations. CDR3 of the VHH and VNAR domains were dominated by pleated and extended orientations, respectively. Although VNAR showed the smallest average molecular weight and molecular surface area compared with VHH and VH antibodies. However, the solvent accessible surface area measurements of the 3 tested sdAbs types were very similar. All the antihapten VHH antibodies showed pleated CDR3, which were sufficient to create a binding pocket to accommodate haptens (methotrexate and azo dyes) in terms of shape and electrostatic potential. The sdAbs that recognized lysozyme showed more diversity in their CDR3 orientation to enable them to recognize various topographies of lysozyme. Subsequently, the three sdAb classes were different in size and surface area and have shown distinguishable ability to optimize their CDR length and orientation to recognize different antigen classes.

  16. VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry.

    Science.gov (United States)

    Häsler, Julien; Flajnik, Martin F; Williams, Gareth; Walsh, Frank S; Rutkowski, J Lynn

    2016-07-01

    B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders.

  17. The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

    Science.gov (United States)

    Bauer, Georg; Motz, Manfred

    2016-11-01

    Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy.

  18. Generation and isolation of target-specific single-domain antibodies from shark immune repertoires.

    Science.gov (United States)

    Müller, Mischa Roland; O'Dwyer, Ronan; Kovaleva, Marina; Rudkin, Fiona; Dooley, Helen; Barelle, Caroline Jane

    2012-01-01

    The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).

  19. Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

    Science.gov (United States)

    Rossey, Iebe; Gilman, Morgan S. A.; Kabeche, Stephanie C.; Sedeyn, Koen; Wrapp, Daniel; Kanekiyo, Masaru; Chen, Man; Mas, Vicente; Spitaels, Jan; Melero, José A.; Graham, Barney S.; Schepens, Bert; McLellan, Jason S.; Saelens, Xavier

    2017-01-01

    Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV. PMID:28194013

  20. Dimerisation strategies for shark IgNAR single domain antibody fragments.

    Science.gov (United States)

    Simmons, David P; Abregu, Fiona A; Krishnan, Usha V; Proll, David F; Streltsov, Victor A; Doughty, Larissa; Hattarki, Meghan K; Nuttall, Stewart D

    2006-08-31

    Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.

  1. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  2. Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy

    Science.gov (United States)

    Broekgaarden, M.; van Vught, R.; Oliveira, S.; Roovers, R. C.; van Bergen En Henegouwen, P. M. P.; Pieters, R. J.; van Gulik, T. M.; Breukink, E.; Heger, M.

    2016-03-01

    Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested. Electronic supplementary information (ESI) available: Materials and methods. See DOI: 10.1039/c6nr00014b

  3. Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditions.

    Science.gov (United States)

    Doña, Vanina; Urrutia, Mariela; Bayardo, Mariela; Alzogaray, Vanina; Goldbaum, Fernando Alberto; Chirdo, Fernando G

    2010-01-27

    Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

  4. Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast

    NARCIS (Netherlands)

    Harmsen, M.M.; Smits, C.B.; Geus, de B.

    2002-01-01

    We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with

  5. Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Setten, van M.C.

    2005-01-01

    The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin

  6. i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

    Science.gov (United States)

    Griffiths, Katherine; Dolezal, Olan; Cao, Benjamin; Nilsson, Susan K; See, Heng B; Pfleger, Kevin D G; Roche, Michael; Gorry, Paul R; Pow, Andrew; Viduka, Katerina; Lim, Kevin; Lu, Bernadine G C; Chang, Denison H C; Murray-Rust, Thomas; Kvansakul, Marc; Perugini, Matthew A; Dogovski, Con; Doerflinger, Marcel; Zhang, Yuan; Parisi, Kathy; Casey, Joanne L; Nuttall, Stewart D; Foley, Michael

    2016-06-10

    CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

  7. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  8. Direct injection of functional single-domain antibodies from E. coli into human cells.

    Directory of Open Access Journals (Sweden)

    Ana Blanco-Toribio

    Full Text Available Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS. The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 10⁵-10⁶ molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies into human cells for analytical and therapeutic purposes.

  9. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Science.gov (United States)

    Walper, Scott A; Anderson, George P; Brozozog Lee, P Audrey; Glaven, Richard H; Liu, Jinny L; Bernstein, Rachel D; Zabetakis, Dan; Johnson, Linwood; Czarnecki, Jill M; Goldman, Ellen R

    2012-01-01

    Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  10. Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

    Science.gov (United States)

    Glukhov, S.; Berestovoy, M.; Chames, P.; Baty, D.; Nabiev, I.; Sukhanova, A.

    2017-01-01

    This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or "nanobodies") conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD-sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis.

  11. Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

    Institute of Scientific and Technical Information of China (English)

    Zuanning Yuan; Minge Du; Yiwen Chen; Fei Dou

    2013-01-01

    Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli-gomers and constructed a naïve human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe-cifical y recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked immunosorbent assay demonstrated this antibody bound specifical y to human amyloid-beta 42 te-tramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc-cessful y constructed a human phage display library and screened a single-domain antibody that specifical y recognized amyloid-beta 42 oligomers.

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

    Science.gov (United States)

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

    2015-02-24

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

  13. Unveiling a Drift Resistant Cryptotope within Marburgvirus Nucleoprotein Recognized by Llama Single-Domain Antibodies

    Directory of Open Access Journals (Sweden)

    John Anthony Garza

    2017-10-01

    Full Text Available Marburg virus (MARV is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH specific for nucleoprotein (NP, each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or “cryptic” until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should

  14. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  15. Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti.

    Science.gov (United States)

    Lyashchenko, K P; Greenwald, R; Esfandiari, J; Meylan, M; Burri, I Hengrave; Zanolari, P

    2007-12-15

    Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

  16. Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.

    Directory of Open Access Journals (Sweden)

    Valencio Salema

    Full Text Available Screening of antibody (Ab libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM. In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs from camelids (nanobodies or VHH on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS. We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC. We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM(EHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

  17. 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 capac...... to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs....

  18. Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.

    NARCIS (Netherlands)

    Gorlani, A.; Hulsik, D.L.; Adams, H.; Vriend, G.; Hermans, P.; Verrips, T.

    2012-01-01

    Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that

  19. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D. (UIUC); (Scripps); (UCSF)

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  20. A camelid anti-PrP antibody abrogates PrP replication in prion-permissive neuroblastoma cell lines.

    Directory of Open Access Journals (Sweden)

    Daryl Rhys Jones

    Full Text Available The development of antibodies effective in crossing the blood brain barrier (BBB, capable of accessing the cytosol of affected cells and with higher affinity for PrP(Sc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the "proof-of-principle" for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrP(C. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrP(Sc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.

  1. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

    Directory of Open Access Journals (Sweden)

    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  2. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    Science.gov (United States)

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  3. Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

    Science.gov (United States)

    Griffin, Laura M; Snowden, James R; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Baker, Terry S

    2014-03-01

    Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

  4. Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

    Science.gov (United States)

    Rozan, Caroline; Cornillon, Amélie; Pétiard, Corinne; Chartier, Martine; Behar, Ghislaine; Boix, Charlotte; Kerfelec, Brigitte; Robert, Bruno; Pèlegrin, André; Chames, Patrick; Teillaud, Jean-Luc; Baty, Daniel

    2013-08-01

    Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.

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

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

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

  6. Engineered single-domain antibodies with high protease resistance and thermal stability.

    Directory of Open Access Journals (Sweden)

    Greg Hussack

    Full Text Available The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(HHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(HHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(HH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(HHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(HH trypsin resistance was similar to that of wild-type V(HHs, although the trypsin resistance of one V(HH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(HH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(HH stability at low pH and impart

  7. Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.

    Science.gov (United States)

    Stanfield, Robyn L; Dooley, Helen; Verdino, Petra; Flajnik, Martin F; Wilson, Ian A

    2007-03-23

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  8. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  9. Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA formulation.

    Directory of Open Access Journals (Sweden)

    Laura J Sherwood

    Full Text Available BACKGROUND: Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP. Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. METHODS AND FINDINGS: In parallel we selected panels of llama single domain antibodies (sdAb from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. CONCLUSIONS: The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal

  10. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  11. Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2.

    Directory of Open Access Journals (Sweden)

    Xiangjing Fu

    Full Text Available Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6 cfu/3 µg and 2×10(9 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2 Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

  12. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

    Science.gov (United States)

    Tillib, S V; Privezentseva, M E; Ivanova, T I; Vasilev, L F; Efimov, G A; Gursky, Y G; Georgiev, G P; Goldman, I L; Sadchikova, E R

    2014-02-15

    Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.

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

    Science.gov (United States)

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

    2007-03-01

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

  14. Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

    Science.gov (United States)

    Sukhanova, Alyona; Even-Desrumeaux, Klervi; Millot, Jean-Marc; Chames, Patrick; Baty, Daniel; Artemyev, Mikhail; Oleinikov, Vladimir; Cohen, Jacques H. M.; Nabiev, Igor

    2012-03-01

    Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

  15. Single domain antibody coated gold nanoparticles as enhancer for Clostridium difficile toxin detection by electrochemical impedance immunosensors

    Science.gov (United States)

    Zhu, Zanzan; Shi, Lianfa; Feng, Hanping; Zhou, H. Susan

    2016-01-01

    This work presents a sandwich-type electrochemical impedance immunosensor for detecting Clostridium difficile toxin A (TcdA) and toxin B (TcdB). Single domain antibody conjugated gold nanoparticles were applied to amplify the detection signal. Gold nanoparticles (Au NPs) were characterized by transmission electron microscopy and UV–vis spectra. The electron transfer resistance (Ret) of the working electrode surface was used as a parameter in the measurement of the biosensor. With the increase of the concentration of toxins from 1 pg/mL to 100 pg/mL, a linear relationship was observed between the relative electron transfer resistance and toxin concentration. In addition, the detection signal was enhanced due to the amplification effect. The limit of detection for TcdA and TcdB was found to be 0.61 pg/mL and 0.60 pg/mL respectively at a signal-to-noise ratio of 3 (S/N = 3). This method is simple, fast and ultrasensitive, thus possesses a great potential for clinical applications in the future. PMID:25460611

  16. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    Institute of Scientific and Technical Information of China (English)

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴

    1997-01-01

    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  17. Camelid cardiology.

    Science.gov (United States)

    Margiocco, Marco L; Scansen, Brian A; Bonagura, John D

    2009-07-01

    Cardiovascular disorders, although not thoroughly described in the literature, are frequently diagnosed in South American camelids, causing morbidity, mortality, and loss of production. Definitive confirmation concerning the heritability of cardiac defects in these species is lacking; however, this potential exists and should be taken into account when counseling breeders and owners. This article describes the diagnosis and treatment of cardiovascular diseases in llamas and alpacas and reviews the most recent literature. Unique aspects of the cardiovascular physiology in these species are also reviewed.

  18. 单域抗体与抗感染免疫%Research progress of single-domain antibodies in infection immunity

    Institute of Scientific and Technical Information of China (English)

    夏丽洁; 张富春

    2012-01-01

    With the development of molecular biology techniques, single domain antibodies as tumor targeting agents for cancer therapy has become one of the not research areas. Single-domain antibodies are essential tools for experimental research and medical applications. And it has a broad prospective as therapeutic agents in regulating immune function, neutralizing the toxin and targeting pathogens of anti-infectious activity.%随着分子生物学技术的发展及抗体研究的不断深入,单域抗体(single domain antibodie,sdAb)的研究已成为肿瘤靶向治疗研究领域的热点之一.作为实验研究和治疗应用的重要工具与制剂,单域抗体在调节免疫功能,中和毒素和抗微生物感染等方面具有广阔的应用前景.

  19. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

    Energy Technology Data Exchange (ETDEWEB)

    Fanning, Sean W.; Horn, James R. (NIU)

    2014-03-05

    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

  20. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    DEFF Research Database (Denmark)

    Álvarez-Cienfuegos, Ana; Alanes, Natalia Nuñez del Prado; Compte, Marta

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we p...

  1. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    Energy Technology Data Exchange (ETDEWEB)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  2. Induction of adhesion-inhibitory antibodies against placental Plasmodium falciparum parasites by using single domains of VAR2CSA

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Pinto, Vera V; Resende, Mafalda

    2009-01-01

    between a parasite protein expressed on erythrocytes named variant surface antigen 2-chondroitin sulfate A (VAR2CSA) and CSA on syncytiotrophoblasts. VAR2CSA is a large polymorphic protein consisting of six Duffy binding-like (DBL), domains and with current constraints on recombinant protein production...... which induce antibodies that inhibit CSA binding of different parasite strains. In this study, we produced a large panel of VAR2CSA proteins and raised antibodies against these antigens. We show that antibodies against the DBL4 domain effectively inhibit parasite binding. As the inhibition...... was not limited to homologous parasite strains, it seems feasible to base a protective malaria vaccine on a single VAR2CSA DBL domain....

  3. Engineering of ultra-small diagnostic nanoprobes through oriented conjugation of single-domain antibodies and quantum dots

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Alyona Sukhanova, Klervi Even-Desrumeaux, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov & Igor Nabiev ### Abstract Nanoparticle-based biodetection commonly employs monoclonal antibodies (mAbs) for targeting. Although several types of conjugates have been used for biomarker labeling, the large size of mAbs limits the number of ligands per nanoparticle, impedes their intratumoral distribution, and limits intracellular penetration. Furthermore, the condi...

  4. Engineering of ultra-small diagnostic nanoprobes through oriented conjugation of single-domain antibodies and quantum dots

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Alyona Sukhanova, Klervi Even-Desrumeaux, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov & Igor Nabiev ### Abstract Nanoparticle-based biodetection commonly employs monoclonal antibodies (mAbs) for targeting. Although several types of conjugates have been used for biomarker labeling, the large size of mAbs limits the number of ligands per nanoparticle, impedes their intratumoral distribution, and limits intracellular penetration. Furthermore, the condi...

  5. Single domain antibodies with VH hallmarks are positively selected during panning of llama (Lama glama) naïve libraries.

    Science.gov (United States)

    Monegal, Ana; Olichon, Aurelien; Bery, Nicolas; Filleron, Thomas; Favre, Gilles; de Marco, Ario

    2012-01-01

    Independent variable domains with VH hallmarks have been repeatedly identified in immune and pre-immune VHH libraries. In some cases, stable independent VH domains have been also isolated in mouse and human recombinant antibody repertoires. However, we have come to realize that VHs were selected with a higher efficiency than VHHs during biopanning of a pre-immune (VHH) library. The biochemical and biophysical comparison did not indicate a presence of any feature that would favor the VH binders during the selection process. In contrast, selected VHHs seemed to be more stable than the VHs, ruling out the existence of a thermodynamically - favored VH sub-class. Therefore, we reasoned that a certain degree of thermodynamic instability may be beneficial for both displaying and expression of VH(H)s when the Sec-pathway is used for their secretion to avoid the cytoplasmic trapping of fast-folding polypeptides. Indeed, VHHs, but not VHs, were accumulated at higher concentrations when expressed fused to the dsbA leader peptide, a sequence that drives the linked polypeptides to the co-translational SRP secretion machinery. These data suggest that the thermodynamically favored VHHs can be lost during biopanning, as previously observed for DARPins and in contrast to the recombinant antibodies in scFv format.

  6. Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

    Science.gov (United States)

    Schneeweis, Lumelle A; Obenauer-Kutner, Linda; Kaur, Parminder; Yamniuk, Aaron P; Tamura, James; Jaffe, Neil; O'Mara, Brian W; Lindsay, Stuart; Doyle, Michael; Bryson, James

    2015-12-01

    Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

  7. Novel nanoscale bacteriophage-based single-domain antibodies for the therapy of systemic infection caused by Candida albicans.

    Science.gov (United States)

    Dong, Shuai; Shi, Hongxi; Cao, Donghui; Wang, Yicun; Zhang, Xintong; Li, Yan; Gao, Xiang; Wang, Li

    2016-08-25

    Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C. albicans. The in vivo efficacies were assessed by mortality rates, fungal burden and histological examination. Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the scFv-phages relative to the control groups. In order to investigate the immune response provoked by scFv-phages, three kinds of cytokines (Th1, Th2 and Th17 types) were measured and a clear immune response was observed. These findings suggest that anti-rSap2 scFv-phages have potential in the therapy of systemic infection caused by C. albicans.

  8. Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

    Institute of Scientific and Technical Information of China (English)

    MIAO QingFang; SHANG BoYang; OUYANG ZhiGang; LIU XiaoYun; ZHEN YongSu

    2007-01-01

    Type Ⅳ collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size compared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type Ⅳ collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay,VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice.Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.

  9. Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.

  10. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120.

    Science.gov (United States)

    Matz, Julie; Kessler, Pascal; Bouchet, Jérôme; Combes, Olivier; Ramos, Oscar Henrique Pereira; Barin, Francis; Baty, Daniel; Martin, Loïc; Benichou, Serge; Chames, Patrick

    2013-01-01

    Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

  11. Prevention of oculopharyngeal muscular dystrophy-associated aggregation of nuclear polyA-binding protein with a single-domain intracellular antibody.

    Science.gov (United States)

    Verheesen, Peter; de Kluijver, Anna; van Koningsbruggen, Silvana; de Brij, Marjolein; de Haard, Hans J; van Ommen, Gert-Jan B; van der Maarel, Silvère M; Verrips, C Theo

    2006-01-01

    Oculopharyngeal muscular dystrophy (OPMD) belongs to the group of protein aggregation disorders and is caused by extensions of the N-terminal polyalanine stretch of the nuclear polyA-binding protein 1 (PABPN1). The presence of PABPN1-containing intranuclear aggregates in skeletal muscle is unique for OPMD and is also observed in transgenic mouse and cell models for OPMD. These models consistently support a direct role for the protein aggregation in OPMD pathogenesis. We have isolated and characterized a diverse panel of single-domain antibody reagents (VHH), recognizing different epitopes in PABPN1. The antibody reagents specifically detect endogenous PABPN1 in cell lysates on western blot and label PABPN1 in cultured cells and muscle sections. When expressed intracellularly as intrabodies in a cellular model for OPMD, aggregation of PABPN1 was prevented in a dose-dependent manner. More importantly yet, these intrabodies could also reduce the presence of already existing aggregates. Given the domain specificity of VHH-mediated aggregation interference, this approach at least allows the definition of the nucleation kernel in aggregation-prone proteins, thus facilitating etiological insight into this and other protein aggregation disorders, and ultimately, it may well provide useful therapeutic agents.

  12. Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation, migration, invasion and angiogenesis of pancreatic cancer cells.

    Science.gov (United States)

    Cheng, Tsai-Mu; Murad, Yanal M; Chang, Chia-Ching; Yang, Ming-Chi; Baral, Toya Nath; Cowan, Aaron; Tseng, Shin-Hua; Wong, Andrew; Mackenzie, Roger; Shieh, Dar-Bin; Zhang, Jianbing

    2014-03-01

    Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC50s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5μM, 8μM and 12nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method.

    Science.gov (United States)

    Haqqani, Arsalan S; Caram-Salas, Nadia; Ding, Wen; Brunette, Eric; Delaney, Christie E; Baumann, Ewa; Boileau, Eve; Stanimirovic, Danica

    2013-05-06

    FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.

  14. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    Science.gov (United States)

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  15. Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hairul Bahara, Nur Hidayah; Chin, Siang Tean; Choong, Yee Siew; Lim, Theam Soon

    2016-01-01

    The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

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

    Science.gov (United States)

    Harmsen, Michiel M; Seago, Julian; Perez, Eva; Charleston, Bryan; Eblé, Phaedra L; Dekker, Aldo

    2017-01-01

    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.

  17. Natural and Man-made Antibody Repertories for Antibody Discovery

    Directory of Open Access Journals (Sweden)

    Juan C eAlmagro

    2012-11-01

    Full Text Available Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of human, mice and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity and composition of a repertoire impact the antibody discovery process.

  18. A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

    Science.gov (United States)

    Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

    2015-03-06

    Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

  19. [Advances in the study of natural small molecular antibody].

    Science.gov (United States)

    Zhu, Lei; Zhang, Da-peng

    2012-10-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.

  20. Natural and man-made V-gene repertoires for antibody discovery.

    Science.gov (United States)

    Finlay, William J J; Almagro, Juan C

    2012-01-01

    Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process.

  1. Single domain antibodies in tissue engineering

    NARCIS (Netherlands)

    Rodrigues, Emilie Dooms

    2014-01-01

    The aim of this thesis is to demonstrate the potential of VHH in tissue engineering applications, with a focus on bone and cartilage tissue regeneration. After a general introduction to this thesis in chapter 1, the selection of VHH targeting growth factors is described in chapter 2. VHH were select

  2. Humanized-Single Domain Antibodies (VH/VHH that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Wanpen Chaicumpa

    2012-07-01

    Full Text Available Naja kaouthia (monocled cobra venom contains many isoforms of secreted phospholipase A2 (sPLA2. The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3 produced humanized-VHH, while another clone (P3-7 produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis, if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  3. A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection.

    Science.gov (United States)

    Vrentas, Catherine E; Moayeri, Mahtab; Keefer, Andrea B; Greaney, Allison J; Tremblay, Jacqueline; O'Mard, Danielle; Leppla, Stephen H; Shoemaker, Charles B

    2016-10-07

    Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LFN, EFN) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Accurate quantitation for in vitro refolding of single domain antibody fragments expressed as inclusion bodies by referring the concomitant expression of a soluble form in the periplasms of Escherichia coli.

    Science.gov (United States)

    Noguchi, Tomoaki; Nishida, Yuichi; Takizawa, Keiji; Cui, Yue; Tsutsumi, Koki; Hamada, Takashi; Nishi, Yoshisuke

    2017-03-01

    Single domain antibody fragments from two species, a camel VHH (PM1) and a shark VNAR (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm. In order to verify the refolding efficiencies from inclusion bodies correctly, proteins purified from periplasmic soluble fractions were used as reference samples. These samples showed far-UV spectra of a typical β-sheet-dominant structure in circular dichroism (CD) spectroscopy and so did the refolded samples as well. As the maximal magnitude of ellipticity in millidegrees (θmax) observed at a given wave length was proportional to the concentrations of the respective reference samples, we could draw linear regression lines for the magnitudes vs. sample concentrations. By using these lines, we measured the concentrations for the refolded PM1 and A6 samples purified from solubilized cytoplasmic insoluble fractions. The refolding efficiency of PM1 was almost 50% following TCDR and 40% and 30% following OHDR and OCDR, respectively, whereas the value of A6 was around 30% following TCDR, and out of bound for quantitation following the other two recipes. The ELISA curves, which were derived from the refolded samples, coincided better with those obtained from the reference samples after converting the values from the protein-concentrations at recovery to the ones of refolded proteins using recovery ratios, indicating that such a correction gives better results for the accurate measure of the ELISA curves than those without correction. Our method require constructing a dual expression system, expressed both in

  5. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

    Science.gov (United States)

    Barrera, Daniel J; Rosenberg, Julian N; Chiu, Joanna G; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T; Shoemaker, Charles B; Oyler, George A; Mayfield, Stephen P

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

  6. Screening Anti-LPS VHH from Llama Single Domain Antibody Li-brary and Preparation of Anti-LPS Nanobody Pentamer%抗脂多糖纳米抗体的筛选及其五聚体的制备

    Institute of Scientific and Technical Information of China (English)

    武文; 李胜华; 张伟京; 王炳奇

    2013-01-01

    目的::筛选抗脂多糖(LPS)纳米单域抗体,并制备抗LPS纳米抗体五聚体。方法:以LPS为抗原,从驼源天然单域重链抗体库中筛选抗LPS纳米抗体,利用分子克隆技术将抗LPS单域抗体基因组装入志贺杆菌样毒素B亚基蛋白结构域(VTB)的五聚体特异性表达载体中进行可溶性表达,并用ELISA法鉴定所获抗体的抗原结合活性和特异性。结果:获得抗LPS纳米单域抗体及LPS纳米抗体五聚体;经鉴定,LPS纳米抗体五聚体的抗原结合活性优于抗LPS单域抗体。结论:利用驼源天然单域重链抗体库制备了抗LPS纳米单域抗体及抗LPS纳米抗体五聚体,为脓毒血症的分子诊断、预后判断及寻找生物治疗新靶点奠定了基础。%Objective: To obtain anti-LPS VHH and prepare anti-LPS nanobody pentamer. Methods: Using phage display screening technology, we selected anti-LPS single domain antibody(sdAb, or nanobody) from a phage display library of llama single domain antibody, and then cloned the anti-LPS single-domain antibody gene into specific expression vector pVTB1 with molecular cloning technology, and expressed anti-LPS pentamer effi-ciently in E.coli. The affinity of different type of anti-LPS nanobodies were detected by ELISA. Results: We ob-tained anti-LPS single domain antibody and anti-LPS antibody pentamer, and proved anti-LPS nanobody pentamer is extremely enhance the specificity and affinity strength of anti-LPS antibody. Conclusion: The selected anti-LPS antibody pentamer is a leading candidate for therapies against LPS-mediated sepsis.

  7. High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Andrew V Oleinikov

    2009-04-01

    Full Text Available Plasmodium falciparum-infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLbetaC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLbetaC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2betaC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2betaC2(PF11_0521 best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLbetaC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLbetaC2 domain. DBL2betaC2(PF11_0521 binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521 and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

  8. Research Progress on Immunological Therapy Application of Single Domain Antibodies Derived from Camel%骆驼来源单域抗体在免疫治疗中的研究进展

    Institute of Scientific and Technical Information of China (English)

    苏幼红; 李江伟

    2010-01-01

    近几年多种单克隆抗体药物已成功上市并用于治疗人类多种疾病,但因单克隆抗体的复杂结构和高昂的生产成本,限制了其在临床上的广泛应用.随着生物技术的进步,使抗体朝着小型化的基因工程单域抗体发展.20世纪末,在骆驼科动物(camelidae)和护士鲨(nurse shark)体内发现一种天然缺失轻链的重链抗体(heavy chain antibody, HCAb),这种特殊抗体的抗原结合位点仅由单一结构域构成.该结构域在骆驼中称为VHH,经基因重组修饰易于进行工程表达,产物即称为VHH单域抗体,具有分子量小,可溶性好,稳定性高及组织穿透力强等优势,在调节免疫功能方面的应用前景更加广阔.就目前国际上对骆驼来源的VHH单域抗体在免疫治疗应用研究的进展情况进行综述,为基因工程单域抗体改造提供新思路.

  9. Thermomagnetic Stability in Pseudo Single Domain Grains

    Science.gov (United States)

    Nagy, Lesleis; Williams, Wyn; Muxworthy, Adrian; Fabian, Karl; Conbhuí, Pádraig Ó.

    2016-04-01

    The reliability of paleomagnetic remanences are well understood for fine grains of magnetite that are single-domain (SD, uniformly magnetized). In particular Néel's theory [1] outlined the thermal energies required to block and unblock magnetic remanences. This lead to determination of thermal stability for magnetization in fine grains as outlined in Pullaiah et. al. [2] and a comprehensive understanding of SD paleomagnetic recordings. It has been known for some time that single domain magnetite is possible only in the grain size range 30 - 80nm, which may only account for a small fraction of the grain size distribution in any rock sample. Indeed rocks are often dominated by grains in the pseudo single domain (PSD) size range, at approximately 80 - 1000nm. Toward the top end of this range multi-domain features begin to dominate. In order to determine thermomagnetic stability in PSD grains we need to identify the energy barriers between all possible pairs of local energy minima (LEM) domain states as a function of both temperature and grain size. We have attempted to do this using the nudged elastic band (NEB) method [3] which searches for minimum energy paths between any given pair of LEM states. Using this technique we have determined, for the first time, complete thermomagnetic stability curves for PSD magnetite. The work presented is at a preliminary stage. However it can be shown that PSD grains of magnetite with simple geometries (e.g. cubes or cuboctahedra) have very few low energy transition paths and the stability is likely to be similar to that observed for SD grains (as expected form experimental observations). The results will provide a basis for a much more rigorous understanding of the fidelity of paleomagnetic signals in assemblages of PSD grains and their ability to retain ancient recordings of the geomagnetic field. References: [1] Néel, Louis. "Théorie du traînage magnétique des ferromagnétiques en grains fins avec applications aux terres

  10. Fluid therapy in small ruminants and camelids.

    Science.gov (United States)

    Jones, Meredyth; Navarre, Christine

    2014-07-01

    Body water, electrolytes, and acid-base balance are important considerations in the evaluation and treatment of small ruminants and camelids with any disease process, with restoration of these a priority as adjunctive therapy. The goals of fluid therapy should be to maintain cardiac output and tissue perfusion, and to correct acid-base and electrolyte abnormalities. Hypoglycemia, hyperkalemia, and acidosis are the most life-threatening abnormalities, and require most immediate correction.

  11. Magnetically Multiplexed Heating of Single Domain Nanoparticles

    CERN Document Server

    Christiansen, Michael G; Anikeeva, Polina

    2014-01-01

    Selective hysteretic heating of multiple collocated sets of single domain magnetic nanoparticles (SDMNPs) by alternating magnetic fields (AMFs) may offer a useful tool for biomedical applications. The possibility of magnetothermal multiplexing has not yet been realized, in part due to prevalent use of linear response theory to model SDMNP heating in AMFs. Predictive successes of dynamic hysteresis (DH), a more generalized model for heat dissipation by SDMNPs, are observed experimentally with detailed calorimetry measurements performed at varied AMF amplitudes and frequencies. The DH model suggests that specific driving conditions play an underappreciated role in determining optimal material selection strategies for high heat dissipation. Motivated by this observation, magnetothermal multiplexing is theoretically predicted and empirically demonstrated for the first time by selecting SDMNPs with properties that suggest optimal hysteretic heat dissipation at dissimilar AMF driving conditions. This form of multip...

  12. Hepatitis B from diagnosis towards prophylactic single domain antibodies

    NARCIS (Netherlands)

    van Roosmalen, M.H.

    2012-01-01

    Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants wit

  13. Hepatitis B from diagnosis towards prophylactic single domain antibodies

    NARCIS (Netherlands)

    van Roosmalen, M.H.

    2012-01-01

    Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants

  14. A general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition.

    Science.gov (United States)

    Maaß, Alexander; Heiseler, Tim; Maaß, Franziska; Fritz, Janine; Hofmeyer, Thomas; Glotzbach, Bernhard; Becker, Stefan; Kolmar, Harald

    2014-02-01

    We report here a generally applicable method for the selective covalent attachment of a reporter molecule to a replicating entity that allows one to obtain specific binders from a single round of library screening. We show that selective biotinylation of phage particles displaying a binder to any given target can be achieved by application of a coupled enzyme reaction on the surface of the target-binding phage particles that includes a peroxidase, an oxidase and a catalase. Due to the covalent linkage of biotin together with the tight and stable interaction of biotin with streptavidin, very stringent wash conditions for removal of nonspecific binders can be applied. The method termed (3)CARD (triple catalytic reporter deposition) was successfully applied to single-round screening of a phage display library of camelid single-domain antibodies against three different target proteins.

  15. Adenoviral targeting using genetically incorporated camelid single variable domains.

    Science.gov (United States)

    Kaliberov, Sergey A; Kaliberova, Lyudmila N; Buggio, Maurizio; Tremblay, Jacqueline M; Shoemaker, Charles B; Curiel, David T

    2014-08-01

    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.

  16. Induction of superovulation in South American camelids.

    Science.gov (United States)

    Ratto, Marcelo H; Silva, Mauricio E; Huanca, Wilfredo; Huanca, Teodosio; Adams, Gregg P

    2013-01-10

    The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular

  17. Camelid antivenom development and potential in vivo neutralization of Hottentotta saulcyi scorpion venom.

    Science.gov (United States)

    Darvish, Maryam; Ebrahimi, Soltan Ahmad; Shahbazzadeh, Delavar; Bagheri, Kamran-Pooshang; Behdani, Mahdi; Shokrgozar, Mohammad Ali

    2016-04-01

    Scorpion envenoming is a serious health problem which can cause a variety of clinical toxic effects. Of the many scorpion species native to Iran, Hottentotta saulcyi is important because its venom can produce toxic effects in man. Nowadays, antivenom derived from hyper immune horses is the only effective treatment for sever scorpion stings. Current limitations of immunotherapy urgently require an efficient alternative with high safety, target affinity and more promising venom neutralizing capability. Recently, heavy chain-only antibodies (HC-Abs) found naturally in camelid serum met the above mentioned advantages. In this study, immuno-reactivities of polyclonal antibodies were tested after successful immunization of camel using H. saulcyi scorpion crude venom. The lethal potency of scorpion venom in C57BL/6 mice injected intraperitoneally was determined to be 2.7 mg/kg. These results were followed by the efficient neutralization of lethal activity of H. saulcyi scorpion venom by injection of antivenom and purified IgG fractions into mice intraperitonelly or intravenously, respectively. HC-Ab camelid antivenom could be considered as a useful serotherapeutics instead of present treatment for scorpion envenomation.

  18. Parasitic diversity found in coprolites of camelids during the Holocene.

    Science.gov (United States)

    Taglioretti, Verónica; Fugassa, Martín Horacio; Sardella, Norma Haydée

    2015-07-01

    Knowledge of parasitic infections to which fauna was exposed in the past provides information on the geographical origin of some parasites, on the possible dispersal routes and for archaeological fauna on the potential zoonotic risk that human and animal populations could be exposed. The aim of the present study was to examine the gastrointestinal parasite present in camelid coprolites collected from the archaeological site Cerro Casa de Piedra, cave 7 (CCP7), Patagonia, Argentina. Coprolites were collected from different stratified sequences dating from the Pleistocene-Holocene transition to the late Holocene. Paleoparasitological examination revealed the presence of eggs of Trichostrongylidae attributed to Lamanema chavezi or Nematodirus lamae, eggs of three unidentified capillariids, Strongylus-type eggs and oocysts of Eimeria macusaniensis. These parasites affected camelids living in the studied area since the Pleistocene-Holocene transition, about 10,000 years ago. Gastrointestinal parasite fauna of patagonian camelids did not vary significatively from Pleistocene-Holocene transition to late Holocene, although environmental conditions fluctuated greatly throughout this period, as indicative of the strength and the stability of these associations over time. In this study, the zoonotic and biogeography importance of parasites of camelids are also discussed.

  19. Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Kaczmarek, Jakub; Skottrup, Peter Durand

    2015-01-01

    , increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence...... for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure–function relationships....

  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 a

  1. Oleander intoxication in New World camelids: 12 cases (1995-2006).

    Science.gov (United States)

    Kozikowski, Tania A; Magdesian, K Gary; Puschner, Birgit

    2009-08-01

    To characterize the clinical and clinicopathologic effects and evaluate outcome associated with oleander toxicosis in New World camelids. Retrospective case series. 11 llamas and 1 alpaca. Medical records from a veterinary medical teaching hospital from January 1, 1995, to December 31, 2006, were reviewed. Records of all New World camelids that had detectable amounts of oleandrin in samples of serum, urine, or gastrointestinal fluid were included in the study. Descriptive statistics were used to evaluate the history, physical examination findings, clinicopathologic data, and outcome of affected camelids. 11 llamas and 1 alpaca met the inclusion criteria of the study. Either oleander plants were present where the camelids resided (n = 7) or oleander plant material was identified in the hay fed to the camelids (5). One llama was dead on arrival at the hospital, and another was euthanized upon admission because of financial concerns. Of the 10 treated camelids, 9 had evidence of acute renal failure, 7 had gastrointestinal signs, and 4 had cardiac dysrhythmias on initial evaluation. The overall mortality rate was 25%, but the mortality rate for the 10 camelids that were medically treated was 10%. In New World camelids, oleander intoxication was associated with a triad of clinical effects (ie, renal, gastrointestinal, and cardiovascular dysfunction). Oleander intoxication often represented a herd problem but carried a fair to good prognosis if treated promptly. Oleander toxicosis should be considered a differential diagnosis in sick camelids.

  2. Biochemical adaptation of camelids during periods where feed is withheld

    Directory of Open Access Journals (Sweden)

    J. Wensvoort

    2001-03-01

    Full Text Available Biochemical changes during fasting or the withholding of feed for 5 day were studied in serum of camelids (dromedary camel, llama and ruminants (sheep, steers. Camels maintained low levels of 13-hydroxybutyrate (BHB and high levels of glucose but showed some increased levels of non-esterified fatty acid (NEFA and urea when fasting. Sheep and steers showed a rise in serum BHB and much higher increases of NEFA than camels and llamas. Sheep showed decreased serum glucose. The llama showed some increase in BHB but NEFA was lower than the other three species. The results indicate that camelids have a unique ability to control lipolytic and gluconeogenic activity to prevent or postpone the state of ketosis. Understanding and manipulation of these metabolic mechanisms in cattle and sheep could have great benefit to the livestock industry.

  3. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

  4. Biomechanical analysis of the camelid cervical intervertebral disc

    Directory of Open Access Journals (Sweden)

    Dean K. Stolworthy

    2015-01-01

    Full Text Available Chronic low back pain (LBP is a prevalent global problem, which is often correlated with degenerative disc disease. The development and use of good, relevant animal models of the spine may improve treatment options for this condition. While no animal model is capable of reproducing the exact biology, anatomy, and biomechanics of the human spine, the quality of a particular animal model increases with the number of shared characteristics that are relevant to the human condition. The purpose of this study was to investigate the camelid (specifically, alpaca and llama cervical spine as a model of the human lumbar spine. Cervical spines were obtained from four alpacas and four llamas and individual segments were used for segmental flexibility/biomechanics and/or morphology/anatomy studies. Qualitative and quantitative data were compared for the alpaca and llama cervical spines, and human lumbar specimens in addition to other published large animal data. Results indicate that a camelid cervical intervertebral disc (IVD closely approximates the human lumbar disc with regard to size, spinal posture, and biomechanical flexibility. Specifically, compared with the human lumbar disc, the alpaca and llama cervical disc size are approximately 62%, 83%, and 75% with regard to area, depth, and width, respectively, and the disc flexibility is approximately 133%, 173%, and 254%, with regard to range of motion (ROM in axial-rotation, flexion-extension, and lateral-bending, respectively. These results, combined with the clinical report of disc degeneration in the llama lower cervical spine, suggest that the camelid cervical spine is potentially well suited for use as an animal model in biomechanical studies of the human lumbar spine.

  5. Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling

    Science.gov (United States)

    Adams, Ralph; Burnley, Rebecca J.; Valenzano, Chiara R.; Qureshi, Omar; Doyle, Carl; Lumb, Simon; del Carmen Lopez, Maria; Griffin, Robert; McMillan, David; Taylor, Richard D.; Meier, Chris; Mori, Prashant; Griffin, Laura M.; Wernery, Ulrich; Kinne, Jörg; Rapecki, Stephen; Baker, Terry S.; Lawson, Alastair D. G.; Wright, Michael; Ettorre, Anna

    2017-01-01

    Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions. PMID:28134246

  6. Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile.

    Science.gov (United States)

    Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N; Klassen, John S; Ng, Kenneth K S

    2014-01-24

    Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.

  7. Antibody

    Science.gov (United States)

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

  8. Development and application of camelid molecular cytogenetic tools.

    Science.gov (United States)

    Avila, Felipe; Das, Pranab J; Kutzler, Michelle; Owens, Elaine; Perelman, Polina; Rubes, Jiri; Hornak, Miroslav; Johnson, Warren E; Raudsepp, Terje

    2014-01-01

    Cytogenetic chromosome maps offer molecular tools for genome analysis and clinical cytogenetics and are of particular importance for species with difficult karyotypes, such as camelids (2n = 74). Building on the available human-camel zoo-fluorescence in situ hybridization (FISH) data, we developed the first cytogenetic map for the alpaca (Lama pacos, LPA) genome by isolating and identifying 151 alpaca bacterial artificial chromosome (BAC) clones corresponding to 44 specific genes. The genes were mapped by FISH to 31 alpaca autosomes and the sex chromosomes; 11 chromosomes had 2 markers, which were ordered by dual-color FISH. The STS gene mapped to Xpter/Ypter, demarcating the pseudoautosomal region, whereas no markers were assigned to chromosomes 14, 21, 22, 28, and 36. The chromosome-specific markers were applied in clinical cytogenetics to identify LPA20, the major histocompatibility complex (MHC)-carrying chromosome, as a part of an autosomal translocation in a sterile male llama (Lama glama, LGL; 2n = 73,XY). FISH with LPAX BACs and LPA36 paints, as well as comparative genomic hybridization, were also used to investigate the origin of the minute chromosome, an abnormally small LPA36 in infertile female alpacas. This collection of cytogenetically mapped markers represents a new tool for camelid clinical cytogenetics and has applications for the improvement of the alpaca genome map and sequence assembly.

  9. Designing magnetic superlattices that are composed of single domain nanomagnets

    Directory of Open Access Journals (Sweden)

    Derek M. Forrester

    2014-07-01

    Full Text Available Background: The complex nature of the magnetic interactions between any number of nanosized elements of a magnetic superlattice can be described by the generic behavior that is presented here. The hysteresis characteristics of interacting elliptical nanomagnets are described by a quasi-static method that identifies the critical boundaries between magnetic phases. A full dynamical analysis is conducted in complement to this and the deviations from the quasi-static analysis are highlighted. Each phase is defined by the configuration of the magnetic moments of the chain of single domain nanomagnets and correspondingly the existence of parallel, anti-parallel and canting average magnetization states.Results: We give examples of the phase diagrams in terms of anisotropy and coupling strength for two, three and four magnetic layers. Each phase diagrams character is defined by the shape of the magnetic hysteresis profile for a system in an applied magnetic field. We present the analytical solutions that enable one to define the “phase” boundaries between the emergence of spin-flop, anti-parallel and parallel configurations. The shape of the hysteresis profile is a function of the coupling strength between the nanomagnets and examples are given of how it dictates a systems magnetic response. Many different paths between metastable states can exist and this can lead to instabilities and fluctuations in the magnetization.Conclusion: With these phase diagrams one can find the most stable magnetic configurations against perturbations so as to create magnetic devices. On the other hand, one may require a magnetic system that can easily be switched between phases, and so one can use the information herein to design superlattices of the required shape and character by choosing parameters close to the phase boundaries. This work will be useful when designing future spintronic devices, especially those manipulating the properties of CoFeB compounds.

  10. `Anomalous' magnetic fabrics of dikes in the stable single domain/superparamagnetic threshold

    Science.gov (United States)

    Soriano, Carles; Beamud, Elisabet; Garcés, Miguel; Ort, Michael H.

    2016-02-01

    `Anomalous' magnetic fabrics in dikes that appear to indicate flow into the wall confound many workers. Here, we present extensive magnetic data on five dikes from Tenerife, Canary Islands, and use these to interpret the causes of the anomalous fabrics. Comparison of the anisotropy of magnetic susceptibility (AMS) and anhysteretic magnetization (AARM) results show that, in some cases, the anomalous fabrics are caused by single-domain grains, which produce AMS fabrics perpendicular to the grain elongation, whereas AARM fabrics are parallel. To check this, hysteresis experiments were used to characterize the domain state. These show most are mixtures of pseudo-single-domain or single-domain plus multi-domain particles, but many have wasp-waisted hysteresis loops, likely indicating mixed populations of stable single-domain and superparamagnetic grains. First-order reversal curves were used to better characterize this and show mixtures of stable single-domain and superparamagnetic grains dominate the magnetic signal. Magnetic particles at the stable single-domain/superparamagnetic threshold are unstable at timespans relevant to the analytical techniques, so they produce complicated results. This suggests that anomalous AMS fabrics in dikes cannot simply be attributed to elongated stable single-domain particles and that mixtures of the different grain types can produce hybrid fabrics, in which the fabrics are neither perpendicular or parallel to the dike plane, that are difficult to interpret without extensive magnetic analysis.

  11. Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, George P. [Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States); Glaven, Richard H. [Nova Research, Inc., 1900 Elkin Street, Suite 230, Alexandria, VA 22308 (United States); Algar, W. Russ [Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States); College of Science, George Mason University, Fairfax, VA 22030 (United States); Susumu, Kimihiro [Optical Sciences Division, Code 5600, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States); Sotera Defense Solutions, Annapolis Junction, MD 20701 (United States); Stewart, Michael H. [Optical Sciences Division, Code 5600, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States); Medintz, Igor L. [Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States); Goldman, Ellen R., E-mail: ellen.goldman@nrl.navy.mil [Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375 (United States)

    2013-07-05

    Graphical abstract: -- Highlights: •Anti-ricin single domain antibodies (sdAb) were self-assembled on quantum dots (QDs). •Conjugates were prepared using dihydrolipoic acid-capped CdSe–ZnS core–shell QDs. •The sdAb–QD conjugates functioned in fluoroimmunoassays for ricin detection. •The conjugates provided signal amplification in surface plasmon resonance assays. •Conjugates provided sensitive detection compared to unconjugated sdAb reporters. -- Abstract: The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.

  12. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

    Science.gov (United States)

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-02-13

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

  13. Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

    Science.gov (United States)

    2012-08-01

    cell lines to produce hybridomas, and the arduous isolation by phage display technology often leads to reduced binding affinity. One method that has...with agitation and then plated on lauria broth (Thermo-Fisher Scientific) with appropriate antibiotic (30 µg/mL kanamycin). The plates were incubated

  14. Measuring Single-Domain Antibody Interactions with Epitopes in Jet Fuel Using Microscale Thermophoresis

    Science.gov (United States)

    2015-01-01

    Flanders Institute for Biotechnology ). Data analysis for both MST and SPR was performed using Prism 5 (GraphPad Software) and errors presented are...one can imagine testing in bleach, sea water, alkaline vents, and many others. CONCLUSIONS We have demonstrated that nanobodies are capable of

  15. [The occurrence of "Candidatus Mycoplasma haemolamae" infections in clinically asymptomatic South American Camelids in Austria].

    Science.gov (United States)

    Franz, Sonja; Spergser, Joachim; Schwendenwein, Ilse; Stanitznig, Anna; Lambacher, Bianca; Tichy, Alexander; Wittek, Thomas

    2016-01-01

    Reports of CMhl infections in South American Camelids in Europe are only available from the United Kingdom and Switzerland. Knowing that CMhl infections can lead to severe disease resulting in death if combined with other diseases or stress, it was the aim of this study to assess prevalence data from camelids in Austria. In comparison to the previous studies a representative number of camelids was investigated nationwide. Data were assessed due to differences in geographical region, age, sex, species, and origin. A relatively high prevalence of 25.8% was recorded. CMhl was detected significantly more often in alpacas (Vicunja pacos) than in llamas (Lama glama) and more frequently in animals younger than 2 years. Additionally regional differences have been observed, which might be due to climatic differences and/or variations in insect vectors. In this study apperantly clinical healthy animals were shown to be infected with CMhl. Camelids infected with CMhl are a pathogen reservoir. The results of this study indicate different risk levels of infection between llamas and alpacas and between younger and older animals. The data presented underline the necessity of further studies on CMhlI infections in South American Camelids.

  16. Searching for single domain magnetite in the "pseudo-single- domain" sedimentary haystack: Implications of biogenic magnetite preservation for sediment magnetism and relative paleointensity determinations

    NARCIS (Netherlands)

    Roberts, A.P.; Chang, L.; Heslop, D.; Florindo, F.; Larrasoaña, J.C.

    2012-01-01

    Magnetic hysteresis measurements of sediments have resulted in widespread reporting of “pseudo-single-domain”-like magnetic properties. In contrast, the ideal single domain (SD) properties that would be expected to be responsible for high quality paleomagnetic records are rare. Determining whether S

  17. Semen preservation and artificial insemination in domesticated South American camelids.

    Science.gov (United States)

    Bravo, P Walter; Alarcon, V; Baca, L; Cuba, Y; Ordoñez, C; Salinas, J; Tito, F

    2013-01-10

    Semen preservation and artificial insemination in South American camelids are reviewed giving emphasis to work done in Peru and by the authors. Reports on semen evaluation and the preservation process indicate that semen of alpacas and llamas can be manipulated by making it liquid first. Collagenase appears to be the best enzyme to eliminate viscosity. Tris buffer solution maintains a higher motility than egg-yolk citrate, phosphate buffered saline (PBS), Triladyl, and Merck-I extenders. Cooling of semen took 1h after collected, and equilibrated with 7% glycerol presented a better motility and spermatozoa survival at 1, 7, 15 and 30days after being slowly frozen in 0.25mL plastic straws. Trials of artificial insemination with freshly diluted semen and frozen-thawed semen are encouraging and needs to be tested extensively under field conditions. Recently, fertility rates varied from 3 to 67%. Semen preservation and most important, artificial insemination appear to be a reality, and could be used to improve the genetic quality of alpacas and llamas.

  18. Antibody discovery: sourcing of monoclonal antibody variable domains.

    Science.gov (United States)

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  19. A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

    Science.gov (United States)

    Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk

    2014-12-01

    The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

  20. First complete mitochondrial genome data from ancient South American camelids - The mystery of the chilihueques from Isla Mocha (Chile).

    Science.gov (United States)

    Westbury, Michael; Prost, Stefan; Seelenfreund, Andrea; Ramírez, José-Miguel; Matisoo-Smith, Elizabeth A; Knapp, Michael

    2016-12-08

    In South American societies, domesticated camelids were of great cultural importance and subject to trade and translocation. South American camelids were even found on remote and hard to reach islands, emphasizing their importance to historic and pre-historic South American populations. Isla Mocha, a volcanic island 35 km offshore of Central-South Chile, is an example of such an island. When Dutch and Spanish explorers reached the island in the early 17th century, they found that domesticated camelids called "chilihueque" played a major role in the island's society. The origin and taxonomy of these enigmatic camelids is unclear and controversial. This study aims to resolve this controversy through genetic analyses of Isla Mocha camelid remains dating from pre-Columbian to early historic times. A recent archaeological excavation of site P21-3 on Isla Mocha yielded a number of camelid remains. Three complete mitochondrial genomes were successfully recovered and analysed. Phylogenetic analyses suggest that "chilihueque" was a local term for a domesticated guanaco. Results from phylogeographic analyses are consistent with Isla Mocha camelids being sourced from Southern Chilean guanaco populations. Our data highlights the capability of ancient DNA to answer questions about extinct populations which includes species identity, potential translocation events and origins of founding individuals.

  1. Advances in the study of natural small molecular antibody%天然小分子抗体研究进展

    Institute of Scientific and Technical Information of China (English)

    朱磊; 张大鹏

    2012-01-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called VNAr fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.%与传统意义上的由两条重链和两条轻链组成的单体抗体不同,天然小分子抗体是指在自然界中存在的一些奇特的小分子抗体,它们分子量比较小,有的仅由重链组成,有的仅由轻链组成.在骆驼体内发现的仅由重链组成的抗体可以发挥正常的生物学功能,克隆其可变区即获得具有抗原结合能力的纳米抗体(Nanobody).鲨鱼体内也存在一种由重链同源二聚体组成的非典型抗体IgNAR.另外,在人体内也存在游离的轻链抗体(FLC),在疾病的发生诊断中具有重要意义.本文综述了Nanobody、IgNAR及FLC的结构特点与作用机制.

  2. Polarization dependence of the spin-density-wave excitations in single-domain chromium

    Energy Technology Data Exchange (ETDEWEB)

    Boeni, P. [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Roessli, B. [Institut Max von Laue - Paul Langevin, 75 - Paris (France); Sternlieb, B.J. [Brookhaven (United States); Lorenzo, E. [Centre National de la Recherche Scientifique (CNRS), 38 - Grenoble (France); Werner, S.A. [Missouri (United States)

    1997-09-01

    A polarized neutron scattering experiment has been performed with a single-Q, single domain sample of chromium in a magnetic field of 4 T. It is confirmed that the longitudinal fluctuations are enhanced for small energy transfers and that the spin wave modes with {delta}S parallel to Q and {delta}S perpendicular to Q are similar. (author) 2 figs., 1 tab., 2 refs.

  3. Prepared GdBa2 Cu3Oy Single Domain Superconducting Bulks in Air

    Institute of Scientific and Technical Information of China (English)

    Zheng Minghui; Xiao Ling; Ren Hongtao; Jiao Yulei; Xu Bin; Fang Yuan

    2004-01-01

    The preparation of the single domain GBCO bulk with 25 mm in diameter in air was reported. In order to avoid the Gd ions substituted the Ba sites, different BaCuO2 contents was added into the precursor powders. Tc of the samples achieved 94.3 K. The magnetization hysteresis loops of the samples were measured at 77 K. It is shown that the irreverswas obtained.

  4. New method for introducing nanometer flux pinning centers into single domain YBCO bulk superconductors

    Energy Technology Data Exchange (ETDEWEB)

    Yang, W.M., E-mail: yangwm@snnu.edu.cn; Wang, Miao

    2013-10-15

    Highlights: • Single domain YBCO bulks with Bi{sub 2}O{sub 3} additions fabricated by TSIG process. • Nanoscale Y{sub 2}Ba{sub 4}CuBiOx(YBi2411) particles introduced by Bi{sub 2}O{sub 3} additions. • The YBi2411 particles are about 150 nm, can act as effective flux pinning centers. • The optimal addition of Bi{sub 2}O{sub 3} is 0.7wt% to achieve higher levitation force. • The result is helpful to improve the quality of REBCO bulk superconductors. -- Abstract: Single domain YBCO superconductors with different additions of Bi{sub 2}O{sub 3} have been fabricated by top seeded infiltration and growth process (TSIG). The effect of Bi{sub 2}O{sub 3} additions on the growth morphology, microstructure and levitation force of the YBCO bulk superconductor has been investigated. The results indicate that single domain YBCO superconductors can be fabricated with the additions of Bi{sub 2}O{sub 3} less than 2 wt%; Bi{sub 2}O{sub 3} can be reacted with Y{sub 2}BaCuO{sub 5} and liquid phase and finally form Y{sub 2}Ba{sub 4}CuBiO{sub x}(YBi2411) nanoscale particles; the size of the YBi2411 particles is about 100 nm, which can act as effective flux pinning centers. It is also found that the levitation force of single domain YBCO bulks is increasing from 13 N to 34 N and decreasing to 11 N with the increasing of Bi{sub 2}O{sub 3} addition from 0.1 wt% to 0.7 wt% and 2 wt%. This result is helpful for us to improve the physical properties of REBCO bulk superconductors.

  5. Pathology of Haemonchus contortus in New World camelids in the southeastern United States: a retrospective review.

    Science.gov (United States)

    Edwards, Erin E; Garner, Bridget C; Williamson, Lisa H; Storey, Bob E; Sakamoto, Kaori

    2016-03-01

    Most small ruminant farms in tropical climates are plagued by Haemonchus contortus, a hematophagous, abomasal parasite. Heavy burdens of this parasite can cause anemia, hypoproteinemia, weight loss, and mortality in susceptible animals. Haemonchus contortus is becoming a major health concern in New World camelids as well, namely llamas (Llama glama) and alpacas (Vicugna pacos), yet little research has been conducted regarding its prevalence or pathology in these species. Herein, we present a retrospective review of llamas and alpacas that were admitted to The University of Georgia Veterinary Teaching Hospital and Athens Diagnostic Laboratory between the years 2002 and 2013. Antemortem fecal egg count (FEC) estimates performed on 30 alpacas were negatively correlated with hematocrit, hemoglobin, and red blood cell count. Total protein was not significantly correlated with FEC. On postmortem examination, 55 of 198 camelids, including 2 from the aforementioned antemortem review, were infected with H. contortus, with llamas (42.6%) having a significantly higher infection rate than alpacas (22.2%). In 15.7% of the total cases, the parasite was the major cause of death. Common gross lesions included peritoneal, thoracic, and pericardial effusions, visceral pallor, subcutaneous edema, and serous atrophy of fat. Histologic lesions included centrilobular hepatic necrosis, hepatic atrophy, lymphoplasmacytic inflammation of the mucosa of the third gastric compartment (C3), extramedullary hematopoiesis in both the liver and spleen, and the presence of nematodes in C3. Our study emphasizes the importance of H. contortus diagnosis and herd monitoring in New World camelids, particularly llamas.

  6. Diagnosis of tuberculosis in camelids: old problems, current solutions and future challenges.

    Science.gov (United States)

    Alvarez, J; Bezos, J; Juan, L de; Vordermeier, M; Rodriguez, S; Fernandez-de-Mera, I G; Mateos, A; Domínguez, L

    2012-02-01

    In spite of great efforts for its control and eradication, tuberculosis remains one of the most important zoonosis worldwide. Its causative agents, the members of the Mycobacterium tuberculosis complex, have a wide host range that complicates the epidemiology of this disease. Among susceptible species to these pathogens, camelids from the New World (llama, alpaca and vicuña) and Old World (Bactrian camel and dromedary) are acquiring an increasing importance in several European countries because of its growing number and could act as reservoirs of the disease for livestock and humans in their natural habitat. In addition, tuberculosis caused by a number of M. tuberculosis complex members is a life-threatening disease in these animal species. Although tuberculosis has been known to affect camelids for a long time, ante-mortem diagnosis is still challenging because of the lack of standardized diagnostic techniques and the limited sensitivity and specificity of the most widely applied tests. However, in recent years, several techniques that can at least partially overcome these limitations have been developed. This paper reviews the results and advances achieved in tuberculosis diagnosis in camelids in the last decade as well as the progresses on ongoing investigations, with special attention to the remaining challenges that still have to be faced to assure the availability of reliable tools for the detection of tuberculosis-infected animals and herds.

  7. Macroscopic Quantum Phenomena and Topological Phase Interference Effects in Single-Domain Magnets

    Institute of Scientific and Technical Information of China (English)

    L(U) Rong; ZHU Jialin

    2001-01-01

    The tunneling of macroscopic object is one of the most fascinating phenomena in condensed matter physics.During the last decade,the problem of quantum tunneling of magnetization in nanometer-scale magnets has attracted a great deal of theoretical and experimental interest.A review of recent theoretical research of the macroscopic quantum phenomena in nanometer-scale single-domain magnets is presented in this paper.It includes macroscopic quantum tunneling (MQT) and coherence (MQC) in single-domain magnetic particles,the topological phase interference or spin-parity effects,and tunneling of magnetization in an arbitrarily directed magnetic field.The general formulas are shown to evaluate the tunneling rate and the tunneling level splitting for single-domain AFM particles.A nontrivial generalization of Kramers degeneracy for double-well system is provided to coherently spin tunneling for spin systems with m-fold rotational symmetry.The effects induced by the external magnetic field have been studied,where the field is along the easy,medium,hard axis,or arbitrary direction.

  8. Identification and isolation of stimulator of interferon genes (STING): an innate immune sensory and adaptor gene from camelids.

    Science.gov (United States)

    Premraj, A; Aleyas, A G; Nautiyal, B; Rasool, T J

    2013-10-01

    The mechanism by which type I interferon-mediated antiviral response is mounted by hosts against invading pathogen is an intriguing one. Of late, an endoplasmic reticulum transmembrane protein encoded by a gene called stimulator of interferon genes (STING) is implicated in the innate signalling pathways and has been identified and cloned in few mammalian species including human, mouse and pig. In this article, we report the identification of STING from three different species of a highly conserved family of mammals - the camelids. cDNAs encoding the STING of Old World camels - dromedary camel (Camelus dromedarius) and bactrian camel (Camelus bactrianus) and a New World camel - llama (Llama glama) were amplified using conserved primers and RACE. The complete STING cDNA of dromedary camel is 2171 bp long with a 706-bp 5' untranslated regions (UTR), an 1137-bp open reading frame (ORF) and a 328-bp 3' UTR. Sequence and phylogenetic analysis of the ORF of STING from these three camelids indicate high level of similarity among camelids and conservation of critical amino acid residues across different species. Quantitative real-time PCR analysis revealed high levels of STING mRNA expression in blood, spleen, lymph node and lung. The identification of camelid STING will help in better understanding of the role of this molecule in the innate immunity of the camelids and other mammals.

  9. Self-templated synthesis of single-crystal and single-domain ferroelectric nanoplates

    KAUST Repository

    Chao, Chunying

    2012-08-15

    Free-standing single-crystal PbTiO 3 nanoplates (see picture) were synthesized by a facile hydrothermal method. A "self-templated" crystal growth is presumed to lead to the formation of the PbTiO 3 nanoplates, which have ferroelectric single-domain structures, whose polarization areas can be manipulated by writing and reading. The nanoplates are also effective catalysts for the oxidation of carbon monoxide. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. What does it mean to be pseudo single domain? Demystifying the PSD state

    Science.gov (United States)

    Lascu, I.; Harrison, R. J.; Einsle, J. F.; Ball, M.

    2016-12-01

    Until recently, non-interacting stable single domain grains were thought to be the sole reliable paleomagnetic recorders. However most natural samples contain so-called "non-ideal" paleomagnetic recorders, which are either interacting single domain particles, or magnetic grains larger than single domain grains, but smaller than proper multi domain grains, which are poor paleomagnetic recorders. The grain size range for these recorders, which for magnetite comprises grains from 100 nm to a few μm in size, is known as the pseudo single domain (PSD) state. Natural samples containing abundant PSD grains have been shown time and again to reliably record thermomagnetic remanent magnetizations that are stable over billions of years. Here we attempt to shed new light on the PSD state by investigating obsidian varieties found at Glass Butte, Oregon, which present the opportunity to study simple cases of magnetic grains encapsulated in volcanic glass. We do this by combining rock magnetism, scanning electron microscopy (SEM) nanotomography, and finite-element micromagnetic modeling. Using rock magnetism we have identified PSD signatures in these samples via their fingerprint in first-order reversal curve (FORC) diagrams. Tomographic reconstructions obtained by stacking SEM images acquired via sequential milling through sample volumes of a few tens of cubic μm reveal the presence of abundant grains that span the PSD grain size interval. These grains have a variety of shapes, from simple ellipsoidal particles, to more complex morphologies attained through the coalescence of neighboring grains during crystallization, to intricate "rolling snowball" morphologies in larger grains that contain appendices formed as a result of particle growth in a dynamic environment as the flowing lava cooled. Micromagnetic modeling of the simplest morphologies reveals that these grains are in single vortex states, with the remanence controlled by irregularities in grain morphology. Coalesced

  11. Effect of maghemization on the magnetic properties of nonstoichiometric pseudo-single-domain magnetite particles

    DEFF Research Database (Denmark)

    Almeida, Trevor P.; Muxworthy, Adrian R.; Kasama, Takeshi

    2015-01-01

    The effect of maghemization on the magnetic properties of magnetite (Fe3O4) grains in the pseudo-single-domain (PSD) size range is investigated as a function of annealing temperature. X-ray diffraction and transmission electron microscopy confirm the precursor grains as Fe3O4 ranging from 150......-energy loss spectroscopy reveal slightly oxidized Fe3O4 grains, heated to 140°C, exhibit higher oxygen content at the surface. Off-axis electron holography allows for construction of magnetic induction maps of individual Fe3O4 and γ-Fe2O3 grains, revealing their PSD (vortex) nature, which is supported...

  12. Semi-analytical Single-domain Modeling of Magnetoresistive Multilayer Thin Films

    Science.gov (United States)

    Oti, John O.

    1997-03-01

    Sub-micrometer magnetoresistive (MR) multilayer thin films show great promise as active elements of future-generation magnetic recording read heads and sensors, and non-volatile magnetic random access memory (MRAM) devices. In very small films the transition lengths of internal Neel magnetic domain walls films may become larger than the dimensions of the films, thereby rendering the films incapable of sustaining domain walls. The films then display a predominant single-domain behavior. This greatly simplifies the mathematical modeling of very small MR multilayers. Simulated size dependence properties of the MR behavior of spin-valve read heads and MRAMs, using a semi-analytical single-domain model [1,2], are presented. The model forms part of a personal computer based semi-analytical micromagnetics design tool we have developed. The program utilizes a convenient graphical windowing interface that facilitates the design and analysis of the system, and enables the 3-D rendering of the interacting films and animation of their magnetization reversals. A real-time demonstration of the program will be given as part of the presentation. [1] C. E. Johnson, J. Appl. Phys. 33, 2515 (1962). [2] J. O. Oti, J. Appl. Phys. 79, 6386 (1996).

  13. Elastic, dielectric and piezoelectric characterization of single domain PIN-PMN-PT: Mn crystals.

    Science.gov (United States)

    Huo, Xiaoqing; Zhang, Shujun; Liu, Gang; Zhang, Rui; Luo, Jun; Sahul, Raffi; Cao, Wenwu; Shrout, Thomas R

    2012-12-15

    Mn modified 0.26Pb(In(1/2)Nb(1/2))O(3)-0.42Pb(Mg(1/3)Nb(2/3))O(3)-0.32PbTiO(3) (PIN-PMN-PT:Mn) single crystals with orthorhombic perovskite crystal structure were polarized along [011] direction, resulting in the single domain state "1O." The complete set of material constants was determined using the combined resonance and ultrasonic methods. The thickness shear piezoelectric coefficient d(15) and electromechanical coupling factor k(15) were found to be on the order of 3100 pC/N and 94%, respectively, much higher than longitudinal d(33) ∼ 270 pC/N and k(33) ∼ 70%. Using the single domain data, the rotated value of d(33)* along [001] direction was found to be 1230 pC/N, in agreement with the experimentally determined d(33) value of 1370 pC/N, conferring extrinsic contributions being about 10%, which was also confirmed using the Rayleigh analysis. In addition, the mechanical quality factors Q(m) were evaluated for different "1O" vibration modes, where the longitudinal Q(m) was found to be ∼1200, much higher than the value for "4O" crystals, ∼300.

  14. Fabrication and properties of single domain GdBCO superconducting rings by a buffer aided Gd+011 TSIG method

    Science.gov (United States)

    Yang, P. T.; Yang, W. M.; Chen, J. L.

    2017-08-01

    Single domain REBCO bulk superconductors can be fabricated by both the top-seeded melt texture growth process and the top-seeded infiltration and growth (TSIG) process. The buffer pellet technique used in these two methods has been demonstrated to act as an efficient seed and to aid the growth of the REBCO bulk superconductors. In this paper, we combined the Gd+011 TSIG method and buffer pellet technique together to fabricate single domain REBCO superconducting rings without any machining and obvious shrinkage of the final ring, which shows a satisfactory trapped field and very good magnetic shielding at the same time. It is found that (1) a single domain GdBCO ring, with inner diameter of 10 mm and outer diameter of 32 mm, has been fabricated by this method successfully. (2) The trapped field (0.32 T, 77 K) and maximum levitation force (80.1 N, 77 K) of the single domain GdBCO ring are nearly the same as the trapped field (0.33 T, 77 K) and maximum levitation force (80.2 N, 77 K) of the single domain GdBCO bulk with the same diameter and thickness, but the uniformity of trapped field is much better for the single domain GdBCO ring. (3) The single domain GdBCO ring shows very good magnetic shielding property, the magnetic flux density shielded by the superconducting ring is up to 0.40 T at 77 K, which is much higher than that of reported samples. The results indicate that the buffer aided Gd+011 TSIG method can provide an important way to fabricate high quality single domain REBCO superconducting rings directly.

  15. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    Science.gov (United States)

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  16. Antithyroglobulin antibody

    Science.gov (United States)

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

  17. Epitaxial growth of a single-domain hexagonal boron nitride monolayer.

    Science.gov (United States)

    Orlando, Fabrizio; Lacovig, Paolo; Omiciuolo, Luca; Apostol, Nicoleta G; Larciprete, Rosanna; Baraldi, Alessandro; Lizzit, Silvano

    2014-12-23

    We investigate the structure of epitaxially grown hexagonal boron nitride (h-BN) on Ir(111) by chemical vapor deposition of borazine. Using photoelectron diffraction spectroscopy, we unambiguously show that a single-domain h-BN monolayer can be synthesized by a cyclic dose of high-purity borazine onto the metal substrate at room temperature followed by annealing at T=1270 K, this method giving rise to a diffraction pattern with 3-fold symmetry. In contrast, high-temperature borazine deposition (T=1070 K) results in a h-BN monolayer formed by domains with opposite orientation and characterized by a 6-fold symmetric diffraction pattern. We identify the thermal energy and the binding energy difference between fcc and hcp seeds as key parameters in controlling the alignment of the growing h-BN clusters during the first stage of the growth, and we further propose structural models for the h-BN monolayer on the Ir(111) surface.

  18. The generation and selection of single-domain, v region libraries from nurse sharks.

    Science.gov (United States)

    Flajnik, Martin F; Dooley, Helen

    2009-01-01

    The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display.

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

    Science.gov (United States)

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

    2007-01-01

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

  20. Single-domain chemical, thermochemical and thermal remanences in a basaltic rock

    CERN Document Server

    Draeger, U; Poidras, T; Riisager, J; Draeger, Ulrike; Pr\\'{e}vot, Michel; Poidras, Thierry; Riisager, Janna

    2005-01-01

    Tiny basaltic samples containing finely grained titanomagnetite with Curie temperature less than 100 C were heated in air in weak field (25 to 100microT) at temperatures between 400 and 560 C for times as long as 32 hours. Oxyexsolution of titanomagnetite resulted in the crystallization of interacting single domain particles with Curie point close to 540 C and the concomitant development of one of two types chemical remanence, depending upon thermal treatment: isothermal chemical remanence (CRM) or thermochemical remanence (TCRM), the latter acquired under the combined effects of chemical change and temperature decrease. CRM and TCRM acquired under various conditions were subjected to Thellier-type experiments. The CRM/TRM ratio is found to be less than one and increases rapidly with acquisition temperature (0.35 at 400 C, 0.65 at 450 C, and 0.90 at 500 C). Thus, very large underestimate of geomagnetic field paleostrength can occur when a natural CRM is not recognized as such and is believed to be a natural T...

  1. Direct visualization of the thermomagnetic behavior of pseudo–single-domain magnetite particles

    Science.gov (United States)

    Almeida, Trevor P.; Muxworthy, Adrian R.; Kovács, András; Williams, Wyn; Brown, Paul D.; Dunin-Borkowski, Rafal E.

    2016-01-01

    The study of the paleomagnetic signal recorded by rocks allows scientists to understand Earth’s past magnetic field and the formation of the geodynamo. The magnetic recording fidelity of this signal is dependent on the magnetic domain state it adopts. The most prevalent example found in nature is the pseudo–single-domain (PSD) structure, yet its recording fidelity is poorly understood. Here, the thermoremanent behavior of PSD magnetite (Fe3O4) particles, which dominate the magnetic signatures of many rock lithologies, is investigated using electron holography. This study provides spatially resolved magnetic information from individual Fe3O4 grains as a function of temperature, which has been previously inaccessible. A small exemplar Fe3O4 grain (~150 nm) exhibits dynamic movement of its magnetic vortex structure above 400°C, recovering its original state upon cooling, whereas a larger exemplar Fe3O4 grain (~250 nm) is shown to retain its vortex state on heating to 550°C, close to the Curie temperature of 580°C. Hence, we demonstrate that Fe3O4 grains containing vortex structures are indeed reliable recorders of paleodirectional and paleointensity information, and the presence of PSD magnetic signals does not preclude the successful recovery of paleomagnetic signals. PMID:27152353

  2. Direct visualization of the thermomagnetic behavior of pseudo-single-domain magnetite particles.

    Science.gov (United States)

    Almeida, Trevor P; Muxworthy, Adrian R; Kovács, András; Williams, Wyn; Brown, Paul D; Dunin-Borkowski, Rafal E

    2016-04-01

    The study of the paleomagnetic signal recorded by rocks allows scientists to understand Earth's past magnetic field and the formation of the geodynamo. The magnetic recording fidelity of this signal is dependent on the magnetic domain state it adopts. The most prevalent example found in nature is the pseudo-single-domain (PSD) structure, yet its recording fidelity is poorly understood. Here, the thermoremanent behavior of PSD magnetite (Fe3O4) particles, which dominate the magnetic signatures of many rock lithologies, is investigated using electron holography. This study provides spatially resolved magnetic information from individual Fe3O4 grains as a function of temperature, which has been previously inaccessible. A small exemplar Fe3O4 grain (~150 nm) exhibits dynamic movement of its magnetic vortex structure above 400°C, recovering its original state upon cooling, whereas a larger exemplar Fe3O4 grain (~250 nm) is shown to retain its vortex state on heating to 550°C, close to the Curie temperature of 580°C. Hence, we demonstrate that Fe3O4 grains containing vortex structures are indeed reliable recorders of paleodirectional and paleointensity information, and the presence of PSD magnetic signals does not preclude the successful recovery of paleomagnetic signals.

  3. Thermochemical remanence in single-domain particle assemblages can lead to paleointensity overestimates

    Science.gov (United States)

    Fabian, K.

    2009-04-01

    Single-domain (SD) particle ensembles are the best studied systems in rock magnetism. Yet, even for this simple system, a complete theoretical treatment of the acquisition of thermochemical remanent magnetization (TCRM) is missing. Based on the SD theory of chemical-remanent magnetization (CRM), it is generally taken for granted that TCRM acquisition is less efficient than acquisition of a thermoremanent magnetization (TRM), and that consequently the paleofield intensity is inevitably underestimated when a TCRM is interpreted as a TRM. Here it is shown that this conclusion holds only when a continuous chemical change (e.g. grain growth) takes place during remanence blocking. In cases where an initial TRM, at a later time and at lower temperature, is modified by chemical processes, the final TCRM can result in considerable paleofield overestimation when interpreted as a TRM. This is demonstrated for two different scenarios. First, partial grain dissolution after TRM acquisition and second, low-temperature oxidation. These mechanisms are not unlikely to occur in nature, and numerical experiments indicate that both would lead to perfectly straight Arai plots, which make such SD TCRMs indistinguishable from true TRMs in the Thellier experiment.

  4. The Role of Interactions in Systems of Single Domain Ferrimagnetic Iron Oxide Nanoparticles

    Directory of Open Access Journals (Sweden)

    Silvio Dutz

    2012-05-01

    Full Text Available Magnetic nanoparticles are interesting materials for a lot of medical and technical applications. A less experimentally investigated question is the influence of particle packing density on magnetic properties due to magnetic interactions between single particles. For this, magnetic nanoparticles of iron oxides prepared as fine dry powder by laser deposition are investigated with respect to their structural and magnetic properties as function of packing density. The particles are nearly spherically shaped single crystals in the magnetic single domain size range with a mean diameter of 21 nm occasionally exhibiting spinel growth facets. Powders of these particles are diluted by nonmagnetic silicon oxide particles in a range of volume concentrations from 0.2 % up to 68 % of the bulk density of magnetite. The concentration dependence of remanence, coercivity and hysteresis losses is determined by measurements of minor loops in a vibrating sample magnetometer. Results which are discussed in the frame of present theoretical models may be understood in terms of the cubic anisotropy of magnetite distorted by a small uniaxial shape contribution.

  5. Deterministic switching of a magnetoelastic single-domain nano-ellipse using bending

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Cheng-Yen; Sepulveda, Abdon; Keller, Scott; Carman, Gregory P. [Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, California 90095 (United States)

    2016-03-21

    In this paper, a fully coupled analytical model between elastodynamics with micromagnetics is used to study the switching energies using voltage induced mechanical bending of a magnetoelastic bit. The bit consists of a single domain magnetoelastic nano-ellipse deposited on a thin film piezoelectric thin film (500 nm) attached to a thick substrate (0.5 mm) with patterned electrodes underneath the nano-dot. A voltage applied to the electrodes produces out of plane deformation with bending moments induced in the magnetoelastic bit modifying the magnetic anisotropy. To minimize the energy, two design stages are used. In the first stage, the geometry and bias field (H{sub b}) of the bit are optimized to minimize the strain energy required to rotate between two stable states. In the second stage, the bit's geometry is fixed, and the electrode position and control mechanism is optimized. The electrical energy input is about 200 (aJ) which is approximately two orders of magnitude lower than spin transfer torque approaches.

  6. Day plots of mixtures of superparamagnetic, single-domain, pseudosingle-domain, and multidomain magnetites

    Science.gov (United States)

    Dunlop, David J.; Carter-Stiglitz, Brian

    2006-12-01

    We test how well a few hysteresis parameters (saturation remanence Mrs, coercive force Hc and remanent coercivity Hcr) serve to determine the proportions of end-members in binary mixtures. Our end-members are six magnetites whose grain sizes are within the superparamagnetic (SP), stable single-domain (SD, three samples), pseudo-SD (PSD), and multidomain (MD) ranges (Carter-Stiglitz et al., 2001). The three SD magnetites have contrasting origins and properties: (1) bacterial magnetite crystals of a single size and coercivity, arranged in chains; (2) natural volcanic magnetites with a narrow distribution of coercivities; and (3) synthetic magnetites precipitated in glass, with a broader coercivity distribution. Our parameter mixing theory assumes linear magnetization curves of the end-members between zero field and the largest coercive force Hc (that of the SD phase, if present). Similarly remanent hysteresis curves should be linear up to the maximum remanent coercive force Hcr. Three of our mixtures (SP plus bacterial SD, PSD plus bacterial SD, MD plus volcanic SD) had acceptable agreement between predicted and measured dependences of Hc, Hcr and the curve of Mrs/Ms versus. Hcr/Hc (Day plot) on end-member concentrations. A nonlinear approximation to remanent hysteresis curves gave a reasonable fit to MD plus glass SD results. In this case, Hcr/Hc for the most MD-rich mixture is larger than Hcr/Hc of either end-member. Such behavior is characteristic of bimodal mixtures in which Hcr is largely determined by the hard (SD) phase and Hc by the soft (MD) phase. The only mixture that could not be modeled by linear or nonlinear parameter theory was MD plus bacterial SD. The bacterial SD hysteresis loop descends almost vertically at -Hc because of the extremely narrow range of particle sizes and coercivities. In general, linear and nonlinear parameter mixing models are adequate if only an approximate fit to real data is needed. An inversion method using complete

  7. Acoustic-Wave-Induced Magnetization Switching of Magnetostrictive Nanomagnets from Single-Domain to Nonvolatile Vortex States

    Science.gov (United States)

    Sampath, Vimal; D'Souza, Noel; Bhattacharya, Dhritiman; Atkinson, Gary M.; Bandyopadhyay, Supriyo; Atulasimha, Jayasimha

    2016-09-01

    We report manipulation of the magnetic states of elliptical cobalt magnetostrictive nanomagnets (of nominal dimensions ~ 340 nm x 270 nm x 12 nm) delineated on bulk 128{\\deg} Y-cut lithium niobate with Surface Acoustic Waves (SAWs) launched from interdigitated electrodes. Isolated nanomagnets that are initially magnetized to a single domain state with magnetization pointing along the major axis of the ellipse are driven into a vortex state by surface acoustic waves that modulate the stress anisotropy of these nanomagnets. The nanomagnets remain in the vortex state until they are reset by a strong magnetic field to the initial single domain state, making the vortex state non-volatile. This phenomenon is modeled and explained using a micromagnetic framework and could lead to the development of extremely energy efficient magnetization switching methodologies.

  8. Poly(acrylic acid)-directed synthesis of colloidally stable single domain magnetite nanoparticles via partial oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Altan, Cem L. [Department of Chemical Engineering, Yeditepe University, Istanbul 34755 (Turkey); Laboratory of Materials and Interface Chemistry & Soft Matter cryoTEM Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven 5600 MB (Netherlands); Gurten, Berna [Department of Chemical Engineering, Yeditepe University, Istanbul 34755 (Turkey); Sadza, Roel [Laboratory of Materials and Interface Chemistry & Soft Matter cryoTEM Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven 5600 MB (Netherlands); Yenigul, Elcin [Department of Chemical Engineering, Yeditepe University, Istanbul 34755 (Turkey); Sommerdijk, Nico A.J.M., E-mail: n.sommerdijk@tue.nl [Laboratory of Materials and Interface Chemistry & Soft Matter cryoTEM Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven 5600 MB (Netherlands); Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven 5600 MB (Netherlands); Bucak, Seyda, E-mail: seyda@yeditepe.edu.tr [Department of Chemical Engineering, Yeditepe University, Istanbul 34755 (Turkey)

    2016-10-15

    Octahedral, single domain magnetite nanoparticles with average size of ~55 nm were synthesized through oxidative aging of a ferrous hydroxide (Fe(OH){sub 2}) precursor at high pH in water. The synthesis was also carried out in the presence of the hydrophilic polymer poly(acrylic acid). Presence of the polymer changed the particle morphology from octahedral to spherical while average size decreased to 40–50 nm. Although these particles have a tendency to precipitate due to their high magnetic moment, dispersions of these particles were obtained in the presence of this particular polymer which made the particles stable in water for several days making them suitable for various biotechnological applications such as cell separation owing to their low toxicity. - Highlights: • Stable, single domain magnetite nanoparticles are synthesized via partial oxidation. • Particles are readily stabilized in water by a biocompatible polymer. • Steric barrier is essential for the stabilization of large magnetite nanoparticles.

  9. Remarkable Electromechanical Coupling in the 2–2 Composite Based on Single-domain PMN–0.33PT Crystal

    Directory of Open Access Journals (Sweden)

    Vitaly Yu. TOPOLOV

    2009-10-01

    Full Text Available A novel parallel-connected 2–2 single-domain 0.67Pb(Mg1/3Nb2/3O3 – 0.33PbTiO3 crystal / polymer composite with various orientations of polarization vectors of the components is proposed to analyze behavior of electromechanical coupling factors k*3j and k*k where j = 1, 2 and 3. It is shown that the combination of the highly piezo-active relaxor-ferroelectric single-domain component and the piezoelectric polymer provides considerable values of k*k (min k*k » –0.8 and max k*k» 0.7 and |k*33| (about 0.9. The active role of the polarization orientation effect and the composite structure in attaining the high performance is emphasized in this work. A strong correlation between k*k and the hydrostatic piezoelectric coefficient is first revealed near min k*k and max k*k of the 2–2 composite. Some advantages concerned with the presence of the single-domain component in the 2–2 composite are discussed in connection with the large values of k*3j and k*k as well as with the considerable anisotropy of k*3j.

  10. Torque undergone by assemblies of single-domain magnetic nanoparticles submitted to a rotating magnetic field

    Science.gov (United States)

    Carrey, J.; Hallali, N.

    2016-11-01

    In the last 10 years, it has been shown in various types of experiments that it is possible to induce biological effects in cells using the torque generated by magnetic nanoparticles submitted to an alternating or a rotating magnetic field. In biological systems, particles are generally found under the form of assemblies because they accumulate at the cell membrane, are internalized inside lysosomes, or are synthesized under the form of beads containing several particles. The torque undergone by assemblies of single-domain magnetic nanoparticles has not been addressed theoretically so far and is the subject of the present article. The results shown in the present article have been obtained using kinetic Monte Carlo simulations, in which thermal activation is taken into account, so the torque undergone by ferromagnetic and superparamagnetic nanoparticles could both be simulated. The first system under study is a single ferromagnetic particle with its easy axis in the plane of the rotating magnetic field. Then, elements adding complexity to the problem are introduced progressively and the properties of the resulting system presented and analyzed: random anisotropy axes, thermal activation, assemblies, and finally magnetic interactions. The most complex studied systems are particularly relevant for applications and are assemblies of interacting superparamagnetic nanoparticles with randomly oriented anisotropy axes. Whenever it is possible, analytical equations describing the torque properties are provided, as well as their domain of validity. Although the properties of an assembly naturally derive from those of single particles, it is shown here that several of them were unexpected and are particularly interesting with regard to the maximization of torque amplitude in biological applications. In particular, it is shown that, in a given range of parameters, the torque of an assembly increases dramatically in the direction perpendicular to the plane of the rotating

  11. The history of Old World camelids in the light of molecular genetics.

    Science.gov (United States)

    Burger, Pamela Anna

    2016-06-01

    Old World camels have come into the focus as sustainable livestock species, unique in their morphological and physiological characteristics and capable of providing vital products even under extreme environmental conditions. The evolutionary history of dromedary and Bactrian camels traces back to the middle Eocene (around 40 million years ago, mya), when the ancestors of Camelus emerged on the North American continent. While the genetic status of the two domestic species has long been established, the wild two-humped camel has only recently been recognized as a separate species, Camelus ferus, based on molecular genetic data. The demographic history established from genome drafts of Old World camels shows the independent development of the three species over the last 100,000 years with severe bottlenecks occurring during the last glacial period and in the recent past. Ongoing studies involve the immune system, relevant production traits, and the global population structure and domestication of Old World camels. Based on the now available whole genome drafts, specific metabolic pathways have been described shedding new light on the camels' ability to adapt to desert environments. These new data will also be at the origin for genome-wide association studies to link economically relevant phenotypes to genotypes and to conserve the diverse genetic resources in Old World camelids.

  12. Surgical repair of femoral fractures in New World camelids: five cases (1996-2003).

    Science.gov (United States)

    Shoemaker, R W; Wilson, D G

    2007-04-01

    Five New World camelids were admitted to the Western College of Veterinary Medicine between 1996 and 2003 for evaluation of femoral fractures. There were three alpacas and two llamas. Four of the animals were female and three were less than 3 months of age. Fracture configurations consisted of distal physeal fractures (three), a comminuted diaphyseal/metaphyseal fracture, and a transverse diaphyseal fracture. Fractures were diagnosed with a combination of physical examination and radiographs in all cases. All five fractures were repaired with internal fixation and three animals were discharged from the hospital with fractures that healed. One cria underwent successful internal fixation but died from pulmonary oedema during recovery from anaesthesia. Postoperative complications were rare and limited to inadequate fracture stability in one alpaca and prolonged recovery to weight bearing in another. One llama with a comminuted metaphyseal fracture, repaired with a 4.5 mm dynamic compression plate, subsequently had catastrophic failure of the bone 17 days after surgery. Overall the clients were pleased with the outcome of discharged animals. Although femoral fractures are considered rare, they pose a unique opportunity for the large animal veterinarian to successfully achieve fracture union with the aid of internal fixation.

  13. Bluetongue disease and seroprevalence in South American camelids from the northwestern region of the United States.

    Science.gov (United States)

    Allen, Andrew J; Stanton, James B; Evermann, James F; Fry, Lindsay M; Ackerman, Melissa G; Barrington, George M

    2015-03-01

    In late summer/early fall of 2013, 2 South American camelids from central Washington were diagnosed with fatal bluetongue viral disease, an event which is rarely reported. A 9-year-old intact male llama (Lama glama), with a 1-day history of anorexia, recumbency, and dyspnea before death. Abundant foam discharged from the mouth and nostrils, and the lungs were severely edematous on postmortem examination. Histologically, there was abundant intra-alveolar edema with fibrin. Hemorrhage and edema disrupted several other organs. Bluetongue viral RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and serotype 11 was identified by sequencing a segment of the VP2 outer capsid gene. Approximately 1 month later, at a site 150 miles north of the index case, a 2-year-old female alpaca with similar, acutely progressive clinical signs was reported. A postmortem examination was performed, and histologic lesions from the alpaca were similar to those of the llama, and again serotype 11 was detected by PCR. The occurrence of bluetongue viral infection and disease is described in the context of seasonal Bluetongue virus activity within the northwestern United States and southwestern Canada.

  14. Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock.

    Science.gov (United States)

    Bojalil, Rafael; Mata-González, María Teresa; Sánchez-Muñoz, Fausto; Yee, Yepci; Argueta, Iván; Bolaños, Lucía; Amezcua-Guerra, Luis Manuel; Camacho-Villegas, Tanya Amanda; Sánchez-Castrejón, Edna; García-Ubbelohde, Walter Jakob; Licea-Navarro, Alexei Fedorovish; Márquez-Velasco, Ricardo; Paniagua-Solís, Jorge Fernando

    2013-04-02

    at 24 h only in the anti-TNF VNAR group. Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.

  15. Prospección serológica del virus parainfluenza 3 en camélidos sudamericanos en Chile A serological survey of parainfluenza-3 virus in South American camelids from Chile

    Directory of Open Access Journals (Sweden)

    CP Cepeda

    2011-01-01

    Full Text Available En un total de 370 sueros obtenidos de 87 alpacas (Lama pacos, 88 llamas (Llama glama, 89 guanacos (Lama guanicoe y 106 vicuñas (Vicugna vicugna se investigó la presencia de anticuerpos para el virus parainfluenza 3 (VPI-3. La prueba de inhibición de la hemoaglutinación mostró que 91 (24,6% de los camélidos sudamericanos (CSA distribuidos en 18 (20,7% alpacas, 36 (40,9% llamas, 27 (18,9% vicuñas y 10 (11,2% guanacos tenían anticuerpos. El rango de los títulos de las muestras positivas estuvo entre 8 y 256 con una media geométrica de 31. Los resultados establecen que los CSA en Chile están infectados con VPI-3.A total of 370 sera collected from 87 alpacas (Lama pacos, 88 llamas (Lama glama, 89 guanacos (Lama guanicoe and 106 vicunas (Vicugna vicugna were investigated for the presence of antibodies to parainfluenza 3 virus (PIV-3. Hemagglutination inhibition test indicated that a total of 91 (24.6% South American camelids (SAC had PIV-3 antibodies, including 18 (20.7% alpacas, 36 (40.9% llamas, 27 (18.9% vicunas and 10 (11.2% guanacos. Titers of the positive samples ranged from 8 to 256 with a geometric mean of 31. These results establish that SAC in Chile are infected with PIV-3.

  16. Transition from Superparamagnetism to Ferromagnetic Single-Domain in a Heisenberg Model for Nano-cluster Magnetic System

    Institute of Scientific and Technical Information of China (English)

    SHAO Yuan-Zhi; HE Zhen-Hui

    2000-01-01

    By considering a magnetic system of an ensemble of nanometer clusters without any external magnetic field, the Monte Carlo simulation of the Heisenberg model is used to investigate the transition from a superparamagnetism to a ferromagnetic single-domain state, which is caused by the growth of the clusters. We studied the variation of the dynamic growth exponent P and the transition critical size of the cluster Nc against the reduced temperature t and the uniaxial anisotropy constant A. We found that the growth exponent maximized at a certain temperature,other than being taken as a universal constant as some researchers suggested previously.

  17. Generation of Small Single Domain Nanobody Binders for Sensitive Detection of Testosterone by Electrochemical Impedance Spectroscopy.

    Science.gov (United States)

    Li, Guanghui; Zhu, Min; Ma, Lu; Yan, Junrong; Lu, Xiaoling; Shen, Yanfei; Wan, Yakun

    2016-06-01

    A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases. Herein, a simple and sensitive immunosensor based on electrochemical impedance spectroscopy (EIS) and Nbs was successfully developed for the determination of testosterone. We successfully isolated the antitestosterone Nbs from an immune phage display library. Moreover, one of the Nbs was biotinylated according to in vivo BirA system, which showed the highest production yield and the most stable case. Further, the EIS immunosensor was set up for testosterone detection by applying the biotinylated antitestosterone Nb. As a result, the biosensor exhibited a linear working range from 0.05 to 5 ng mL(-1) with a detection limit of 0.045 ng mL(-1). In addition, the proposed immunosensor was successfully applied in determining testosterone in serum samples. In conclusion, the proposed immunosensor revealed high specificity of testosterone detection and showed as a potential approach for sensitive and accurate diagnosis of testosterone.

  18. Molecular characterization and antibiotic resistance of Enterococcus species from gut microbiota of Chilean Altiplano camelids

    Directory of Open Access Journals (Sweden)

    Katheryne Guerrero-Olmos

    2014-10-01

    Full Text Available Background: Enterococcus is one of the major human pathogens able to acquire multiple antibiotic-resistant markers as well as virulence factors which also colonize remote ecosystems, including wild animals. In this work, we characterized the Enterococcus population colonizing the gut of Chilean Altiplano camelids without foreign human contact. Material and methods: Rectal swabs from 40 llamas and 10 alpacas were seeded in M-Enterococcus agar, and we selected a total of 57 isolates. Species identification was performed by biochemical classical tests, semi-automated WIDER system, mass spectrometry analysis by MALDI-TOF (matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer, and, finally, nucleotide sequence of internal fragments of the 16S rRNA, rpoB, pheS, and aac(6-I genes. Genetic diversity was measured by pulsed field gel electrophoresis (PFGE-SmaI, whereas the antibiotic susceptibility was determined by the WIDER system. Carriage of virulence factors was explored by polymerase chain reaction (PCR. Results: Our results demonstrated that the most prevalent specie was Enterococcus hirae (82%, followed by other non–Enterococcus faecalis and non–Enterococcus faecium species. Some discrepancies were detected among the identification methods used, and the most reliable were the rpoB, pheS, and aac(6-I nucleotide sequencing. Selected isolates exhibited susceptibility to almost all studied antibiotics, and virulence factors were not detected by PCR. Finally, some predominant clones were characterized by PFGE into a diverse genetic background. Conclusion: Enterococcus species from the Chilean camelids’ gut microbiota were different from those adapted to humans, and they remained free of antibiotic resistance mechanisms as well as virulence factors.

  19. [Dry matter intake of South American camelids and its effects on the composition of feed rations].

    Science.gov (United States)

    Stölzl, Anna Maria; Lambertz, Christian; Moors, Eva; Stiehl, Jennifer; Gauly, Matthias

    2014-01-01

    The number of South American camelids (SAC) is increasing in Germany since decades. Due to a lack of scientifically based publications the knowledge about feeding SACs is still poor. Therefore, the aim of this study was to estimate the dry matter intake (DMI) of SACs as a basis for calculations of feed rations. Previous studies proposed a DMI of up to 3% of the body weight (BW) (Vaughan und Gauly, 2011). In the present study, eight llamas (Llama glama) were allocated to two groups of four animals each. The two groups were fed with hay of different qualities over a total period often weeks, which was divided into two runs of five weeks each. During the first run, group 1 was fed with hay 1 (15.1% crude protein; 8.5% crude ash; 3.1% crude fat; 52.6% NDF per kg DM) and group 2 with hay 2 (6.6% crude protein; 6.2% crude ash; 2.1% crude fat; 64.3% NDF per kg DM). After five weeks the groups were changed and group 1 received hay 2 and group 2 received hay 1. BW was measured at the start and end of each run (week zero, five and ten). The hay quality affected the DMI, but the animals did not compensate a lower feed quality with an increased DMI. The total DMI was 1.26% and 0.89% of the BW for hay 1 and hay 2, respectively, which was lower than expected in both groups. In conclusion, calculations of feed rations for SACs should be adjusted to the present findings of a lower DMI capability.

  20. Efficacy of anthelmintics on South American camelid (llama and alpaca) farms in Georgia.

    Science.gov (United States)

    Gillespie, Rose-Ann M; Williamson, Lisa H; Terrill, Thomas H; Kaplan, Ray M

    2010-08-27

    The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern United States, and infection with gastrointestinal nematodes (GIN) is a major health concern in this region. There is widespread resistance to anthelmintic remedies in small ruminants (sheep and goats), but a paucity of information on llamas and alpacas. Anthelmintic resistance was evaluated on three SAC farms (two llama; one alpaca) in Georgia in the southern United States using fecal egg count reduction (FECR) tests. For each farm, animals were randomly assigned to 1 of 5 treatment groups based on initial fecal egg count (FEC) and number of animals available (2-5 groups, n=9-11 per treatment). Ivermectin (IVM, subcutaneous injection; 0.3mg/kg body weight (BW)) and a control group were tested on an alpaca farm, and fenbendazole (FBZ, oral; 10mg/kg BW; two farms), moxidectin (MOX oral; 0.2mg/kg BW; two farms), and levamisole (LEV, oral; 8 mg/kg BW; one farm) were added for the llama farms. Anthelmintic efficacy was determined by comparing FEC of treatment and control animals 14 days post-treatment, with resistance evaluated using the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Based upon these guidelines, there was GIN resistance to IVM in both llamas and alpacas in Georgia and to FBZ on both llama farms where this drug was tested. There was MOX resistance on one llama farm using the FECR test, while there was no resistance to LEV detected in this study. These data demonstrate a serious emerging problem in the United States of llama and alpaca GIN resistant to drugs from two of the three major anthelmintic classes.

  1. Trapped field of YBCO single-domain samples using pulse magnetization from 77K to 20K

    Energy Technology Data Exchange (ETDEWEB)

    Chaud, X [CNRS/CRETA, 25 avenue des martyrs, 38042 Grenoble Cedex 09 (France); Haanappel, E [LNCMP, 143 avenue de Rangueil, 31400 Toulouse (France); Noudem, J G; Horvath, D [CRISMAT/ENSICAEN, 6 bd Marechal Juin, 14050 Caen (France)], E-mail: xavier.chaud@grenoble.cnrs.fr

    2008-02-01

    ReBCO single-domain bulk superconductors have been shown to trap significant magnetic field at 77K and below. They can advantageously replace permanent magnets in cryogenic motors; more power in a smaller volume can be achieved. But practically, their magnetization has to be performed in situ. Usually it implies the use of pulse magnetization which is severe for the samples. This technique generates heat and stress on the superconductors. The magnetic-flux-trapping capabilities of YBCO single-domain samples were explored using the pulse-field facilities at the LNCMP (National Pulsed Magnetic Field Laboratory) at Toulouse, France. The flux dynamic was monitored during magnetic pulses by measuring the surface induction with a Hall probe on top of the samples at different temperatures from 77K to 20K. The samples were 16 mm in diameter and about 10 mm in height. The best one trapped 400 mT at 77K and 2.5T at 20K. The trapped field increases almost linearly down to 40K. The magnetic pulse is seen to generate heat. The temperature rise increases with decreasing temperature dwell because of lower heat capacity. The achieved trapped field is a compromise between the temperature rise and the applied field, and depends greatly of the magnetization history.

  2. Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

    NARCIS (Netherlands)

    Houwelingen, van A.M.M.L.; Saeger, de T.; Rusanova, T.; Waalwijk, C.; Beekwilder, M.J.

    2008-01-01

    To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin o

  3. 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.

    2005-01-01

    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 single-domain

  4. Palynological analysis of camelid coprolites: seasonality in the use of the site Cerro Casa de Piedra 7 (Santa Cruz, Argentina)

    Science.gov (United States)

    Velázquez, Nadia Jimena; Burry, Lidia Susana; Fugassa, Martín Horacio; Civalero, María Teresa; Aschero, Carlos Alberto

    2014-01-01

    Palynological, palaeoparasitological and paleobotanical studies of coprolites found in archaeological sites from Perito Moreno National Park (47°57‧S72°05‧W) yielded information on diet, palaeoenvironment and health. These studies allowed adding evidence to the reconstruction of life history of the hunter-gatherers that inhabited Patagonia during the Holocene. We examined the season of the year when camelid Lama guanicoe coprolites (5400 ± 64 yr 14C BP to 9640 ± 190 yr 14C BP) were deposited at Cerro Casa de Piedra 7 (site CCP7). The study used palynological evidence and comparison with pollen spectra of modern feces collected during summer, fall, winter and spring of 2010. The dominant types were: pollen of Nothofagus, Empetrum rubrum, Asteraceae subfam. Asteroideae, Nassauvia, Caryophyllaceae and Poaceae; fern spores; remains of Eimeria macusaniensis; and plant remains of Poaceae, Festuca pallescens, Stipa speciosa, Armeria maritima, Gaultheria mucronata and E. rubrum. Pollen spectra of modern and fossil feces were used for multivariate analysis. Coprolites associated to fall and winter modern feces. These results and those obtained from pollen concentration values and the presence of pollen types indicators of seasonality, allowed the determination of summer, fall and winter coprolites. However, caution must be taken with the seasonality results of coprolites dated earlier than 9000 years BP since the environmental conditions differed from now. The site was probably a camelid shelter during the unfavorable seasons.

  5. Morphofunctional structure of the lingual papillae in three species of South American Camelids: Alpaca, guanaco, and llama.

    Science.gov (United States)

    Erdoğan, Serkan; Villar Arias, Silvia; Pérez, William

    2016-02-01

    The aim of this study was to compare the anatomical and functional characteristics of the lingual papilla among the Camelidae. For this purpose, tongues of alpaca, guanaco, and llama were used. Numerous long and thin filiform papillae were located in the median groove and none were detected on the rest of the dorsal surface of the lingual apex in alpaca. Secondary papillae originated from the base of some filiform papillae on the ventral surface of alpaca tongue. The bases of some filiform papillae of the lateral surface of the lingual apex were inserted into conspicuous grooves in guanaco and tips of filiform papillae on the dorsal surface of the lingual body were ended by bifurcated apex. On the dorsal surface of the lingual apex of llama, there were no filiform papillae but there were numerous filiform papillae on both the lateral margins of the ventral surface of the lingual apex. Fungiform papillae were distributed randomly on dorsal lingual surface and ventral margins of the tongues of all camelid species. Lenticular papillae were located on the lingual torus and varied in size and topographical distribution for each species. Circumvallate papillae had irregular surfaces in llama and alpaca, and smooth surface in guanaco. In conclusion, llama and alpaca tongues were more similar to each other, and tongues of all camelid species displayed more similarities to those of Bactrian and dromedary camels in comparison with other herbivores and ruminants.

  6. Influence of the Inverse Faraday Effect on Switching and Oscillations of Magnetization in Single-Domain Nanoparticles

    Directory of Open Access Journals (Sweden)

    A.L. Daniluyk

    2012-03-01

    Full Text Available We have performed a numerical simulation of magnetization switching and oscillations in a ferromagnetic single-domain particle in the disk form under the influence of nanosecond laser pulses with linear and circular polarization. The analysis has shown that the interaction of laser pulses with a ferromagnetic nanodisk leads to change in the direction of its magnetization. This process is accompanied by magnetization oscillations with duration from units to tens of nanoseconds. As it follows from the obtained results, the main cause of magnetization switching is the reduction of magnetic anisotropy energy at heating of the structure by laser. The field of the inverse Faraday effect can lead to an increase in frequency and amplitude of this oscillations. We have performed a numerical simulation of magnetization switching and oscillations in a ferromagnetic single-domain particle in the disk form under the influence of nanosecond laser pulses with linear and circular polarization. The analysis has shown that the interaction of laser pulses with a ferromagnetic nanodisk leads to change in the direction of its magnetization. This process is accompanied by magnetization oscillations with duration from units to tens of nanoseconds. As it follows from the obtained results, the main cause of magnetization switching is the reduction of magnetic anisotropy energy at heating of the structure by laser. The field of the inverse Faraday effect can lead to an increase in frequency and amplitude of this oscillations. We have performed a numerical simulation of magnetization switching and oscillations in a ferromagnetic single-domain particle in the disk form under the influence of nanosecond laser pulses with linear and circular polarization. The analysis has shown that the interaction of laser pulses with a ferromagnetic nanodisk leads to change in the direction of its magnetization. This process is accompanied by magnetization oscillations with duration from

  7. Optimizing selection of large animals for antibody production by screening immune response to standard vaccines.

    Science.gov (United States)

    Thompson, Mary K; Fridy, Peter C; Keegan, Sarah; Chait, Brian T; Fenyö, David; Rout, Michael P

    2016-03-01

    Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.

  8. Pastoralism in northern Peru during pre-Hispanic times: insights from the Mochica Period (100-800 AD) based on stable isotopic analysis of domestic camelids.

    Science.gov (United States)

    Dufour, Elise; Goepfert, Nicolas; Gutiérrez Léon, Belkys; Chauchat, Claude; Jordán, Régulo Franco; Vásquez Sánchez, Segundo

    2014-01-01

    Llama (Lama glama) and alpaca (Vicugna pacos) are the only large domesticated animals indigenous to the Americas. Pastoralism occupies a fundamental economic, social and religious role in Andean life. Today, camelid livestock are confined to the ecozone of the puna (above 3,500 masl), while their presence on the Pacific coast during pre-Hispanic times is attested by archaeological skeletal remains. This study aims to document herding practices on the northern Peruvian coast during the Early Intermediate Period (200 BC-600 AD) by gaining insights into diet, location of breeding and mobility of archaeological camelids from the funerary and ritual contexts of two Mochica sites, Uhle Platform in Huacas de Moche and El Brujo. The three first early years and the long-term life histories of the animals were documented by the combined bulk analysis of bone collagen (δ(13)C col and δ(15)N col) and bone structural carbonate (δ(13)C bone and δ(18)O bone) and the serial analysis of structural carbonate of molar tooth enamel (δ(13)C enamel and δ(18)O enamel). Mochica camelids were bred in the low and/or middle valleys, unlike their modern counterparts, who are restricted to highland puna C3 pastures. Archaeological camelids had diverse and complex life histories, usually with substantial maize foddering. An ontogenetic switch in diet and possible residential mobility during the course of life were identified for some specimens. Although the inference of geographic origin from δ(18)O bone and δ(18)O enamel values was limited because of the lack of understanding of the influence of environmental and biological factors, tooth enamel analysis has great potential for exploring camelid herding practices and Andean pastoralism. Our study suggested that Mochica herders adapted their practices to the difficult lowland environment and that herding practices were varied and not restricted to breeding at higher altitudes. The role of maize in different aspects of the economic life

  9. Poly(acrylic acid)-directed synthesis of colloidally stable single domain magnetite nanoparticles via partial oxidation

    Science.gov (United States)

    Altan, Cem L.; Gurten, Berna; Sadza, Roel; Yenigul, Elcin; Sommerdijk, Nico A. J. M.; Bucak, Seyda

    2016-10-01

    Octahedral, single domain magnetite nanoparticles with average size of ~55 nm were synthesized through oxidative aging of a ferrous hydroxide (Fe(OH)2) precursor at high pH in water. The synthesis was also carried out in the presence of the hydrophilic polymer poly(acrylic acid). Presence of the polymer changed the particle morphology from octahedral to spherical while average size decreased to 40-50 nm. Although these particles have a tendency to precipitate due to their high magnetic moment, dispersions of these particles were obtained in the presence of this particular polymer which made the particles stable in water for several days making them suitable for various biotechnological applications such as cell separation owing to their low toxicity.

  10. Single-domain shape anisotropy in near-macroscopic Ni80Fe20 thin-film rectangles

    Science.gov (United States)

    Li, Yi; Lu, Yiran; Bailey, W. E.

    2013-05-01

    Shape anisotropy provides a simple mechanism to adjust the local bias field in patterned structures. It is well known that for ellipsoidal particles elements, domain formation is thought to limit the effectiveness of shape anisotropy. In our work, we show that very soft lithographically patterned Ni80Fe20 films with control of induced magnetic anisotropy can exhibit shape anisotropy fields in agreement with single-domain models, for both hysteresis loop measurements at low field and ferromagnetic resonance measurements at high field. We show the superiority of the fluxmetric form over the magnetometric form of anisotropy estimate for thin films with control dimensions from 10 μm to 150 μm and in-plane aspect ratios above 10.

  11. Strain Effects of the Structural Characteristics of Ferroelectric Transition in Single-Domain Epitaxial BiFeO3 Films

    Institute of Scientific and Technical Information of China (English)

    LIU Yang; PENG Xing-Ping

    2011-01-01

    Structural characteristics of phase transition in single-domain epitaxial BiFeOz films are studied by the Landau-Devonshire theory. It is predicted that remanent polarization shows strong strain dependence for different temperatures while spontaneous polarization is almost independent of strain over a wide temperature (0-500 °C). We also obtain the thickness dependence of the c-axis lattice parameter and Curie temperature, and make a comparison between the polarization rotation angle and the angle attributed to the structural evolution in epitaxial (001)p BiFeOa h'lms grown on SrTiO3 substrates. The theoretical results are in agreement with recent experimental and theoretical data. Our calculations show that the clamping effect should also be taken into account in order to depict the mechanism of the polarization rotation completely.%@@ Structural characteristics of phase transition in single-domain epitaxial BiFeO films are studied by the Landau- Devonshire theory.It is predicted that remanent polarization shows strong strain dependence for different tem- peratures while spontaneous polarization is almost independent of strain over a wide temperature (0-500 ℃).We also obtain the thickness dependence of the c-axis lattice parameter and Curie temperature, and make a compari- son between the polarization rotation angle and the angle attributed to the structural evolution in epitaxial (001) BiFeO films grown on SrTiO substrates.The theoretical results are in agreement with recent experimental and theoretical data.Our calculations show that the damping effect should also be taken into account in order to depict the mechanism of the poIarization rotation completely.

  12. Experimental Study on Mixtures of Superparamgnetic and Single-Domain Magnetite with Respect to Day-Dunlop Plots

    Science.gov (United States)

    Hirt, A. M.; Kumari, M.; Senn, N.; Uebe, R.; Schueler, D.

    2013-12-01

    Day-Dunlop plots are widely used as a diagnostic of domain state: superparamagnetic (SP), stable single-domain (SD), pseudo-single-domain (PSD), multi-domain (MD) and mixture of these. A n earlier experimental study on mixtures of SP and SD magnetite showed that ratios of saturation remanent magnetization to saturation magnetization and the coercivity of remanence to coercivity are lower than the expected theoretical mixing trend (Dunlop & Carter-Stiglitz, J. Geophys. Res., 111, B12S09). This study reexamines Day-Dunlop plots with experimentally determined mixtures of SD and SP magnetite grains. The starting materials that are used in this study are biogenic magnetite with SD and SP particle size, and a commercial ferrofluid. The SD-biogenic magnetite is from magnetotactic bacteria that are arranged in chains. The average magnetosome size is 45 mm; however, there is also a minor amount of SP material among the magnetosomes. The SP component consists either of: 1) SP-biogenic magnetite, which occurs as isolated particles within the bacteria with an average size of 18 nm, or 2) Chemicell ferrofluid, magnetite with 10 nm particle size and hydrodynamic diameter of 45 nm. The magnetic properties of the pure materials were first characterized by measurements of hysteresis, acquisition of an isothermal remanent magnetization, and first-order reversal curves. The magnetic properties of the SD magnetite are typical for magnetotactic bacteria. The SP samples show no remanence. Two samples were prepared from the SD-magnetite to serve as the starting material. The SP-component was then added incrementally to the SD samples. Experimental results from these tests show that the magnetization and coercivity ratios are lower than the theoretical prediction for both sample mixtures. The reasons for this discrepancy will be discussed.

  13. Formation of single domain magnetite by green rust oxidation promoted by microbial anaerobic nitrate-dependent iron oxidation

    Science.gov (United States)

    Miot, Jennyfer; Li, Jinhua; Benzerara, Karim; Sougrati, Moulay Tahar; Ona-Nguema, Georges; Bernard, Sylvain; Jumas, Jean-Claude; Guyot, François

    2014-08-01

    Biomineralization of magnetite is a central geomicrobiological process that might have played a primordial role over Earth’s history, possibly leaving traces of life in the geological record or controlling trace metal(loid)s and organic pollutants mobility in modern environments. Magnetite biomineralization has been attributed to two main microbial pathways to date (namely magnetotactic bacteria and dissimilatory iron-reducing bacteria). Here, we uncover a new route of magnetite biomineralization involving the anaerobic nitrate-reducing iron(II) oxidizing bacterium Acidovorax sp. strain BoFeN1. Using transmission electron microscopy, scanning transmission X-ray microscopy, transmission Mössbauer spectroscopy and rock magnetic analyses, this strain is shown to promote the transformation of hydroxychloride green rust in equilibrium with dissolved Fe(II) to (1) periplasmic lepidocrocite (γ-FeOOH) and (2) extracellular magnetite, thus leading to strong redox heterogeneities at the nanometer scale. On the one hand, lepidocrocite was associated with protein moieties and exhibited an anisotropic texture, with the elongated axis parallel to the cell wall. On the other hand, magnetite crystals exhibited grain sizes and magnetic properties consistent with stable single domain particles. By comparison, abiotic controls led to a very slow (4 months vs. 2 days in BoFeN1 cultures) and incomplete oxidation of hydroxychloride green rust towards magnetite. As this abiotic magnetite exhibited the same size and magnetic properties (stable single domain particles) as magnetite produced in BoFeN1 cultures, only the co-occurrence of textured Fe(III)-oxides and magnetite, associated with the persistence of organic carbon molecules, might constitute valuable biosignatures to be looked for in the geological record. Our results furthermore contribute to a more complex picture of Fe redox cycling in the environment, providing an additional process of Fe(II)-bearing phase

  14. Leptospirosis en camélidos sudamericanos. Estudio de prevalencia serológica en distintas regiones de la Argentina Leptospirosis in south-american camelids. A study on the serological prevalence in different regions of Argentina

    Directory of Open Access Journals (Sweden)

    P. LLORENTE

    2002-01-01

    Full Text Available Se evaluó la seroprevalencia en Camélidos Sudamericanos de Leptospirosis, zoonosis de distribución mundial, producida por espiroquetas patógenas del género Leptospira. Se examinaron 494 animales (llamas, guanacos y vicuñas, clínicamente sanos sin vacunar, de diferentes regiones geográficas de la República Argentina. Se utilizó la técnica, serovar específica, de microaglutinación (MAT. El estudio reveló prevalencias entre 47.3 y 96.2% en llamas, entre 0 y 13% en guanacos y entre 9 y 62.8% en vicuñas. De los serovares que se usaron como antígeno en las determinaciones, los que más frecuentemente reaccionaron con los anticuerpos séricos de los camélidos, fueron copenhageni y castellonisLeptospirosis is a zoonotic infectious disease, affecting wild and domestic animals and human beings, caused by pathogenic spirochetes, spread world wide, belonging to the genus Leptospira. It is transmited by direct contact with infected animal urine or tissues, and indirectly through contaminated water and soil. Leptospirosis has a negative economic impact on porcine and bovine productions. It causes abortions, stillbirths, placental retention, infertility and chronical renal deficiency, causing disturbance of flow milk and quality in dairy cattle. Studies on south-american camelids productive aspects, have increased during the last decades, in order to promote alternative regional economies. There exist three species in Argentina, llama (Lama glama, guanaco (Lama guanicoe and vicuña (Vicugna vicugna. The knowledge of physiological parameters and susceptibility and immune response to infectious agents of these animals, are required to improve their breeding efficiency. Leptospirosis is an infectious disease, which may affect reproduction efficiency. Leptospira antibody prevalence in 494 sera obtained from healthy non vaccinated llamas, vicuñas and guanacos from different geographic zones in Argentina, was evaluated. The serovar specific

  15. Electro-magnetic properties and engineering applications of single-domain high temperature superconductor yttrium barium copper oxide

    Science.gov (United States)

    Qu, Dehui

    industrial applications. In this dissertation, we report levitation force values of single-domain samples of various orientations. The samples are sectioned from one large single-domain material processed using the seeded melt growth (SMG) method. We also discuss the possible physical mechanism responsible for the levitation behavior observed. (Abstract shortened by UMI.)

  16. Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

    Directory of Open Access Journals (Sweden)

    Li Xu

    Full Text Available Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

  17. Exchange biasing of single-domain Ni nanoparticles spontaneously grown in an antiferromagnetic MnO matrix

    Energy Technology Data Exchange (ETDEWEB)

    Shoemaker, Daniel P; Grossman, Madeleine; Seshadri, Ram [Materials Department, Materials Research Laboratory, University of California, Santa Barbara, CA 93106 (United States); Department of Chemistry and Biochemistry, Materials Research Laboratory, University of California, Santa Barbara, CA 93106 (United States)], E-mail: dshoe@mrl.ucsb.edu

    2008-05-14

    Exchange biased composites of ferromagnetic single-domain Ni nanoparticles embedded within and on the surface of large grains of MnO have been prepared by the reduction of Ni{sub x}Mn{sub 3-x}O{sub 4} monoliths in flowing 5% H{sub 2}/N{sub 2}. The Ni precipitates are 20-30 nm in extent, and the majority are completely encased within MnO. The manner in which the Ni nanoparticles are spontaneously formed-starting from a single homogeneous phase-ensures a high degree of dispersion of the Ni and large Ni/MnO interface/volume ratios. The interfaces between ferromagnetic Ni and antiferromagnetic MnO result in magnetic exchange bias below the Neel temperature of MnO. A threefold change in exchange bias fields (from 40 to 120 Oe at 5 K) is observed, depending on the starting Ni content x in the precursor Ni{sub x}Mn{sub 3-x}O{sub 4}, with smaller x values-fewer or smaller Ni nanoparticles-resulting in larger exchange fields. The exchange bias leads to significant hardening of the magnetization, with coercive fields scaling nearly linearly with the exchange field. The strategies outlined here suggest new and simple routes for creating highly interfacial, functional biphasic composites of nanoparticles embedded within or supported on an oxide matrix.

  18. Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

    Institute of Scientific and Technical Information of China (English)

    GUO Shu-yuan; LI Jie; CHEN Zhen; HE Kang-lai

    2014-01-01

    Domain I of the activated Crystal protein from Bacillus thuringiensis has a sevenα-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and puriifed in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspeciifc binding with the brush border membrane vesicles of the Asian corn borer.

  19. Thyroid Antibodies

    Science.gov (United States)

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

  20. ab-plane anisotropy in single-domain Bi 2Sr 2CaCu 2O 8 high-temperature superconductors

    Science.gov (United States)

    Quijada, M. A.; Tanner, D. B.; Kelley, R. J.; Onellion, M.

    1994-12-01

    The anisotropy of the ab-plane optical conductivity and de resistivity of single-domain Bi 2Sr 2CaCu 2O 8 crystals has been measured between 20 and 300 K. There is a modest normal-state anisotropy, optical anisotropy below Tc, and an unexpected anisotropy in the resistive transition.

  1. Magnetic Properties for the Single-domain CoFe2O4 Nano-particles Synthesized by the Hydrothermal Method

    Institute of Scientific and Technical Information of China (English)

    YANG Zhi; ZHANG Yue; SONG Yu; WANG Jiawei; CHEN Yuang; ZHANG Zhe; DUAN Nian; RUAN Xuefeng

    2015-01-01

    The aim of this work was to investigate the size-related magnetism for the single-domain CoFe2O4 nano-particles synthesized using the hydrothermal method. The effects of the reaction temperature and the reaction time on the lattice constants, particle morphologies, and the room-temperature magnetic properties were studied from the X-ray diffraction, the transmission electron microscope, and the vibrating-sample magnetometer. The experimental results show that the samples are composed of CoFe2O4 nano-particles with an average crystallite size (D) smaller than 40 nm, and the magnetic properties of the samples can be manipulated in a wide range:the MS values vary from smaller than 50 emu/g to close to 80 emu/g, and the HC values are between about 200 Oe and 2000 Oe. Additionally, the relationship between HC and 1/D3/2 satisifes linearship, showing the characteristic of single-domain structure. These results indicate that the single-domain CoFe2O4 nano-particles with size controlled between the superparamagnetic critical size and single-domain critical size can be easily prepared using this hydrothermal method.

  2. The formation of right-handed and left-handed chiral nanopores within a single domain during amino acid self-assembly on Au(111).

    Science.gov (United States)

    Yang, Sena; Jeon, Aram; Driver, Russell W; Kim, Yeonwoo; Jeon, Eun Hee; Kim, Sehun; Lee, Hee-Seung; Lee, Hangil

    2016-05-25

    We report the formation of both right- and left-handed chiral nanopores within a single domain during the self-assembly of an amino acid derivative on an inert Au(111) surface using STM. DFT calculations employed to rationalize this unusual result identified that intermolecular interactions between chiral, windmill-shaped tetramers are crucial for self-assembly.

  3. Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

    Science.gov (United States)

    Cancela, Martín; Corvo, Ileana; DA Silva, Edileuza; Teichmann, Aline; Roche, Leda; Díaz, Alvaro; Tort, José Fransisco; Ferreira, Henrique B; Zaha, Arnaldo

    2017-11-01

    Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

  4. Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain.

    Science.gov (United States)

    Nunes-Silva, Sofia; Gangnard, Stéphane; Vidal, Marta; Vuchelen, Anneleen; Dechavanne, Sebastien; Chan, Sherwin; Pardon, Els; Steyaert, Jan; Ramboarina, Stephanie; Chêne, Arnaud; Gamain, Benoît

    2014-12-09

    VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA.

  5. Successful use of camelid (alpaca) antivenom to treat a potentially lethal tiger snake (Notechis scutatus) envenomation in a dog.

    Science.gov (United States)

    Padula, Andrew M; Winkel, Kenneth D

    2016-05-01

    This report describes a confirmed clinical case of tiger snake (Notechis scutatus) envenomation in a domestic dog that was successfully treated with a novel polyvalent camelid (alpaca; Llama pacos) antivenom. Samples collected from the dog were assayed for tiger snake venom (TSV) using a highly sensitive and specific ELISA. The TSV concentration in serum and urine at initial presentation was 365 ng/mL and 11,640 ng/mL respectively. At the time of initial presentation whole blood collected from the dog did not clot and the Prothrombin Time was abnormally increased (>300 s). Serum was also visibly hemolysed. The dog was administered antihistamine, dexamethasone and 4000 Units (sufficient to neutralise 40 mg of TSV) of a novel polyvalent alpaca antivenom diluted in 0.9% NaCl. At 4 h post-antivenom treatment the dog's clinical condition had improved markedly with serum TSV concentrations below the limit of detection (improve by 4 h and had fully normalised by 16 h post-antivenom. Venom concentrations in both serum and urine remained undetectable at 16 h post-antivenom. The dog made a complete recovery, without complications, suggesting that the alpaca-based antivenom is both clinically safe and effective.

  6. Antimitochondrial antibody

    Science.gov (United States)

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

  7. The effects of multi-domain versus single-domain cognitive training in non-demented older people: a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Cheng Yan

    2012-03-01

    Full Text Available Abstract Background Whether healthy older people can benefit from cognitive training (CogTr remains controversial. This study explored the benefits of CogTr in community dwelling, healthy, older adults and compared the effects of single-domain with multi-domain CogTr interventions. Methods A randomized, controlled, 3-month trial of CogTr with double-blind assessments at baseline and immediate, 6-month and 12-month follow-up after training completion was conducted. A total of 270 healthy Chinese older people, 65 to 75 years old, were recruited from the Ganquan-area community in Shanghai. Participants were randomly assigned to three groups: multi-domain CogTr, single-domain CogTr, and a wait-list control group. Twenty-four sessions of CogTr were administrated to the intervention groups over a three-month period. Six months later, three booster training sessions were offered to 60% of the initial training participants. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A, the Color Word Stroop test (CWST, the Visual Reasoning test and the Trail Making test (TMT were used to assess cognitive function. Results Multi-domain CogTr produced statistically significant training effects on RBANS, visual reasoning, and immediate and delayed memory, while single-domain CogTr showed training effects on RBANS, visual reasoning, word interference, and visuospatial/constructional score (all P Conclusions Cognitive training can improve memory, visual reasoning, visuospatial construction, attention and neuropsychological status in community-living older people and can help maintain their functioning over time. Multi-domain CogTr enhanced memory proficiency, while single-domain CogTr augmented visuospatial/constructional and attention abilities. Multi-domain CogTr had more advantages in training effect maintenance. Clinical Trial Registration Chinese Clinical Trial Registry. Registration number: ChiCTR-TRC-09000732.

  8. The diagnostic value of controlled oral word association test-FAS and category fluency in single-domain amnestic mild cognitive impairment.

    Science.gov (United States)

    Malek-Ahmadi, Michael; Small, Brent J; Raj, Ashok

    2011-01-01

    Recent studies have shown that decreases in both letter fluency and category fluency may be present in addition to memory impairment in single-domain amnestic mild cognitive impairment (aMCI). However, the clinical utility of these fluency measures is unclear. The aim of this study was to determine what, if any, diagnostic value letter and category fluency provide in differentiating single-domain aMCI from normal cognition. Data from 66 individuals [33 cognitively normal (CN) and 33 aMCI] between the ages of 66 and 87 years participating in the Florida Alzheimer's Disease Research Center were compared on the Controlled Oral Word Association Test (COWAT)-FAS and Category Fluency test, both in terms of raw and scaled scores. Participants were matched on age, education and sex. Two-tailed independent sample t-tests found statistically significant differences between the CN and aMCI groups for both raw and scaled scores of COWAT-FAS and Category Fluency (p Fluency did not significantly improve diagnostic accuracy when combined with the Hopkins Verbal Learning Test-Revised delayed recall. Although decreased COWAT-FAS and Category Fluency performance may be present in single-domain aMCI, these tests do not improve the ability of the Hopkins Verbal Learning Test-Revised delayed recall to differentiate aMCI from CN individuals. Copyright © 2011 S. Karger AG, Basel.

  9. OSTEOMETRÍA Y GENÉTICA DE LOS CAMÉLIDOS MOCHICA, COSTA NORTE DEL PERÚ / Mochica camelids osteometry and genetics, north coast of Peru

    Directory of Open Access Journals (Sweden)

    Víctor Félix Vásquez Sánchez

    2009-12-01

    Full Text Available El objetivo de este trabajo es evaluar los resultados de la osteometría a dos muestras óseas de camélidos de sitios de la época Mochica (siglos I y VII d.C. en la costa norte de Perú. Los huesos analizados en este estudio son primeras falanges que provienen de dos sitios arqueológicos Mochica (Cerro Chepén y Zona Urbana Moche. Se realizaron análisis discriminantes a una muestra de 44 primeras falanges, tomando en cuenta las cinco variables que son medidas en este hueso. Los resultados obtenidos mostraron la identificación de dos especies de camélidos domésticos en ambos sitios, Lama glama “llama” y Lama pacos “alpaca”. La interpretación de los resultados es discutida mediante la explicación de un modelo de especiación geográfica o vicariante de los camélidos que habitaron la costa peruana en la época Mochica. Este modelo toma en cuenta que actualmente en esta ecología no hay crianza de camélidos, así como factores genéticos y evolutivos de una nueva forma doméstica, incluyendo el efecto de la Regla de Bergmann y los nuevos pastos en la morfología de esta nueva forma de camélido doméstico que vivió en la época Mochica.Palabras claves: osteometría, genética, camélidos, especiación, Mochica  AbstractThe objective of this study was to evaluate the results of osteometric analyses applied to camelid bones samples from Moche sites (I and VII AD located on the north coast of Peru. The bones analyzed in this study correspond to first phalanges from two Moche archaeological sites (Cerro Chepén and Moche Urban Zone. Discriminant analysis was conducted on a sample of 44 first phalanges, taking into account five measurments in each bone. The results showed the identification in both sites of two species of domestic camelids, Lama glama “llama” and Lama pacos “alpaca.” The interpretation of the results is discussed by explaining a model of geographic or vicariant speciation of camelids that inhabited the coast

  10. Analysis of mitochondrial DNA in Bolivian llama, alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids.

    Science.gov (United States)

    Barreta, J; Gutiérrez-Gil, B; Iñiguez, V; Saavedra, V; Chiri, R; Latorre, E; Arranz, J J

    2013-04-01

    The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT-CYB gene and 513 bp of the D-loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT-CYB was more variable than D-loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D-loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations.

  11. Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates

    OpenAIRE

    Kershaw, C.M.; Evans, G; Rodney, R.; Maxwell, W.M.C.

    2016-01-01

    In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1 mg mL–1, 20 min, 37°C) eliminat...

  12. Interspecies embryo transfer in camelids: the birth of the first Bactrian camel calves (Camelus bactrianus) from dromedary camels (Camelus dromedarius).

    Science.gov (United States)

    Niasari-Naslaji, A; Nikjou, D; Skidmore, J A; Moghiseh, A; Mostafaey, M; Razavi, K; Moosavi-Movahedi, A A

    2009-01-01

    Interspecies embryo transfer is a possible approach that can be used to conserve endangered species. It could provide a useful technique to preserve the Iranian and wild Bactrian camels, both of which are threatened with extinction. In the present study, one Bactrian camel was superovulated using decreasing doses of FSH (60, 40, 30, 30, 20, 20 mg, b.i.d.; Folltropin-V; Bioniche, London, ON, Canada) for 6 days, followed by a single injection of FSH (20 mg, i.m.) on Day 7. Daily ovarian ultrasonography was performed until most of the growing follicles had reached a mature size of 13-17 mm, at which time the camel was mated twice, 24 h apart, with a fertile male Bactrian camel. At the time of first mating, female camels were given 20 microg, i.v., buserelin (Receptal; Intervet, Boxmeer, The Netherlands). One day after the donor camel had been mated, the dromedary recipients (n = 8) were injected with 25 mg, i.v., porcine LH (Lutropin-V; Bioniche) to induce ovulation. Embryos were recovered on Day 8.5 after the first mating and transferred non-surgically into recipients on Day 7.5 after LH injection. Pregnancy was diagnosed 25 days after embryo transfer. Healthy Bactrian camel calves (n = 4) were born without any particular complications at the time of parturition (e.g. dystocia and neonatal diseases). The present study is the first report of the birth of Bactrian camel calves from dromedary camels, as well as the first report of interspecies embryo transfer in old world camelids.

  13. Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos).

    Science.gov (United States)

    Liguori, G; Squillacioti, C; Assisi, L; Mirabella, N; Langella, E; Costagliola, A; Vittoria, A

    2017-02-08

    The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1 ) and receptor 2 (OX2 ) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2 -IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2 . Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.

  14. A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

    Science.gov (United States)

    Jiang, Wenzhi; Cossey, Sarah; Rosenberg, Julian N; Oyler, George A; Olson, Bradley J S C; Weeks, Donald P

    2014-09-25

    Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. Camelid antibody

  15. Effect of induced shielding current transmission in longitudinal direction on levitation force of melt grown single-domain YBa2Cu3O7-x cylindrical superconductor

    Institute of Scientific and Technical Information of China (English)

    YANG Wanmin; ZHOU Lian; FENG Yong; ZHANG Pingxiang; R.Nicolsky; R.de Andrade Jr

    2004-01-01

    A novel layer deletion method is used to experimentally investigate the effect of induced shielding current transmission (ISCT) in the longitudinal direction on the levitation force of a single-domain YBa2Cu3O7-x (YBCO) cylindrical superconductor (φ30x7 mm). In the experiment the sample was gradually sliced into two equal sheets, at the middle height along a diameter with 5 mm every step. The experimental results show that the levitation force is closely related with the ISCT in the longitudinal direction. Any layer deletion, even a small piece of layer deletion can reduce the levitation force of the sample. After the whole layer was deleted the levitation force can diminish about 50%. It is also found that the levitation force is directly proportional to the effective factor of surface area, which is equal to the top surface area divided by the total surface area parallel to the top surface of the sample.

  16. 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.

  17. Magnetic characterization of non-ideal single-domain monoclinic pyrrhotite and its demagnetization under hydrostatic pressure up to 2 GPa with implications for impact demagnetization

    Science.gov (United States)

    Bezaeva, Natalia S.; Chareev, Dmitriy A.; Rochette, Pierre; Kars, Myriam; Gattacceca, Jérôme; Feinberg, Joshua M.; Sadykov, Ravil A.; Kuzina, Dilyara M.; Axenov, Sergey N.

    2016-08-01

    Here we present a comprehensive magnetic characterization of synthesized non-ideal single-domain (SD) monoclinic pyrrhotite (Fe7S8). The samples were in the form of a powder and a powder dispersed in epoxy. "Non-ideal" refers to a powder fraction of predominantly SD size with a minor contribution of small pseudo-single-domain grains; such non-ideal SD pyrrhotite was found to be a remanence carrier in several types of meteorites (carbonaceous chondrites, SNC…), which justifies the usage of synthetic compositions as analogous to natural samples. Data were collected from 5 to 633 K and include low-field magnetic susceptibility (χ0), thermomagnetic curves, major hysteresis loops, back-field remanence demagnetization curves, first-order reversal curves (FORCs), alternating field and pressure demagnetization of saturation isothermal remanent magnetization (SIRM), low temperature data (such as zero-field-cooled and field-cooled remanence datasets together with room temperature SIRM cooling-warming cycles) as well as XRD and Mössbauer spectra. The characteristic Besnus transition is observed at ∼33 K. FORC diagrams indicate interacting SD grains. The application of hydrostatic pressure up to 2 GPa using nonmagnetic high-pressure cells resulted in the demagnetization of the sample by 32-38%. Repeated cycling from 1.8 GPa to atmospheric pressure and back resulted in a total remanence decrease of 44% (after 3 cycles). Pressure demagnetization experiments have important implications for meteorite paleomagnetism and suggest that some published paleointensities of meteorites with non-ideal SD monoclinic pyrrhotite as remanence carrier may be lower limits because shock demagnetization was not accounted for.

  18. Defects/strain influenced magnetic properties and inverse of surface spin canting effect in single domain CoFe{sub 2}O{sub 4} nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Simrjit; Khare, Neeraj, E-mail: nkhare@physics.iitd.ernet.in

    2016-02-28

    Graphical abstract: - Highlights: • Synthesized single domain CoFe{sub 2}O{sub 4} nanoparticles with different amount of strain. • Demonstrated a correlation between size, strain and magnetic properties of CoFe{sub 2}O{sub 4}. • Strain induces cationic redistribution at tetrahedral and octahedral sites of CoFe{sub 2}O{sub 4}. • Inverse of spin canting effect due to the redistribution of Fe{sup 3+} ions is demonstrated. - Abstract: Single domain CoFe{sub 2}O{sub 4} nanoparticles with different amount of defects/strain have been synthesized by varying the growth temperature in the hydrothermal method. Nanoparticles grown at lower temperature are of larger size and exhibit more planar defects and oxygen vacancies as compared to nanoparticles grown at higher temperatures which are of smaller sizes and exhibit less planar defects and oxygen vacancies. The nanoparticles with larger amount of defects also possess a higher value of intrinsic strain as compared to nanoparticles with fewer defects. The presence of intrinsic strain in the nanoparticles is found to shift the cationic distribution at the tetrahedral and octahedral sites. The saturation magnetization (M{sub s}) of the nanoparticles is found to depend upon both the intrinsic strain and size of the nanoparticles. The M{sub s} increases with the decrease in the nanoparticles size from 32 nm to 20 nm, and this is correlated to the inverse of spin canting effect due to decrease in the intrinsic strain which leads to shifting of Co{sup 2+} ions from tetrahedral to octahedral sites. However, with further decrease in the size of the nanoparticles (16 nm), the size effect dominates over the strain effect leading to decrease in M{sub s}. The coercivity is found to be higher in the nanoparticles with larger amount of defects/strain and has been attributed to strain induced strong spin canting and pinning due to defect sites. The variation of coercivity with particle size (D) exhibits deviation from D{sup 3

  19. Direct visualization of chemical and thermo-remanent magnetization of pseudo-single-domain magnetite grains and the implications for reliable paleomagentic signal acquisition

    Science.gov (United States)

    Almeida, T.; Muxworthy, A. R.; Kasama, T.; Williams, W.; Kovács, A.; Dunin-Borkowski, R.; Hansen, T. W.

    2015-12-01

    In order to reliably interpret paleomagnetic measurements, the mechanisms of chemical remanent magnetization (CRM) and thermoremanent magnetization (TRM) must be fully understood. Currently, most models of CRM and TRM processes only exist for the smallest, uniformly magnetized grains, termed single domain (SD). However, the magnetic signal from rocks is often dominated by slightly larger grains containing non-uniform magnetization states, termed pseudo-SD (PSD) grains. Magnetite (Fe3O4) is the most magnetic naturally occurring mineral on Earth, carrying the dominant magnetic signature in rocks and providing a critical tool in paleomagnetism. The oxidation of Fe3O4 to other iron oxides, such as maghemite (γ-Fe2O3) and hematite (α-Fe2O3), is of particular interest as it influences the preservation of remanence of the Earth's magnetic field by Fe3O4. Further, TRM in Fe3O4 grains is acquired in the direction of the ambient geomagnetic field as they cool below their Curie temperature (TC) of ~ 580 ˚C. The latest transmission electron microscopy (TEM) techniques like electron holography and environmental TEM (ETEM) allows for the imaging of magnetization in nano-scale minerals during in situ heating under vacuum and controlled atmospheres. In the present study, synthetic Fe3O4 particles in the PSD size range (CRM was visualized using electron holography, in the form of reconstructed magnetic induction maps, where the oxidized grains exhibited a loss of overall remanence and change in remanent direction. The thermomagnetic behavior of Fe3O4 particles in the PSD size range is also investigated using off-axis electron holography. Magnetic induction maps, which are recorded during in situ heating up to above the TC, reveal the PSD nature of several Fe3O4 grains by visualizing their vortex domain states. The vortex states in small Fe3O4 grains (Fig. 1a & b) are shown to rotate or collapse into a single-domain state close to its unblocking temperature (Fig. 1c), rather than

  20. Growth of Highly c-axis Oriented and/or Epitaxial Single-Domain b-axis Oriented La5Ca9Cu24O41 Thin Films by Pulsed Laser Deposition

    Science.gov (United States)

    2016-04-01

    AFRL-AFOSR-UK-TR-2016-0004 Growth of highly c-axis oriented and/or epitaxial single-domain b-axis oriented La5Ca9Cu24O41 thin films by pulsed laser...to 29-Sep-2015 4. TITLE AND SUBTITLE Growth of highly c-axis oriented and/or epitaxial single-domain b-axis oriented La5Ca9Cu24O41 thin films by...to the surface of the substrate, counting success as demonstration of b-axis or c-axis oriented LCCO films along with measurement of bulk thermal

  1. Magnetic single-domain state of the monochiral helimagnet MnSi in the zero-field limit: Magnetic properties study

    Science.gov (United States)

    Narozhnyi, V. N.; Krasnorussky, V. N.

    2015-04-01

    Attention is drawn to the possibility of obtaining the monochiral helimagnet MnSi in the magnetic single-domain state (SDS) in a zero magnetic field limit. It is shown that this metastable zero-field magnetic SDS can be achieved by a gradual decrease of the field down to zero after an initial transformation of MnSi to a spin-polarized state in a high field H . This can be achieved only for H ∥[111 ] . Investigations of MnSi in the magnetic SDS give us the possibility of determining the two components of the low field magnetic susceptibility of this compound, χ⊥(T ) and χ∥(T ) [χ⊥ and χ∥ correspond to H ⊥(111 ) and H ∥(111 ) planes containing magnetic moments in a helically ordered state]. These results are compared with the macroscopic magnetic susceptibility of MnSi that was determined earlier for this compound, but only in a magnetic multidomain state. In addition, our results are compared with the data reported for some nonmonochiral helimagnets. The characteristic features of monochiral helimagnets are elucidated.

  2. Application of mixed colloidal magnetic fluid of single domain Fe3O4 and NiFe2O4 ferrite nanoparticles in audio speaker

    Directory of Open Access Journals (Sweden)

    S. D. Kemkar

    2017-01-01

    Full Text Available Ferrofluids are stable suspensions of colloidal ferrimagnetic particles in suitable non – magnetic carrier liquids. They have attracted a lot of attention from scientists and engineers due to their many interesting properties and applications in various branches of engineering. The present work reports the performance of colloidal fluid of single domain nanoparticles of NiFe2O4 and Fe3O4. The thermal properties and its dynamics on magnetization as well as its effect on thermal conductivity on the colloidal fluid are studied here. Advantages of the increased thermal conductivity and optimization of magnetization of mixed colloidal fluid is used to extract the heat from voice coil. Nanoparticles of 21 nm of Fe3O4 and 12 nm of NiFe2O4 are used for mixed colloidal fluid. The suspension of particles is achieved by coating the nanoparticles with mono-corboxylic group on both the types of particles. The higher size (21 nm of Fe3O4 and 12 nm of NiFe2O4 particles are taken for synthesizing colloidal fluid, to have magnetic property of mixed colloidal liquid at elevated temperature of voice coil of speaker (Higher sized particles gives better magnetization. Oil is used as a carrier. Mixed magnetic colloidal fluid is used as a medium for damping so that noise is reduced at higher temperature of voice coil.

  3. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    Science.gov (United States)

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.

  4. Theoretical analysis and numerical calculation of 3D trapped field distribution of single domain SmBCO bulks by Sm+011 TSIG methods

    Science.gov (United States)

    Yang, Wanmin; Yang, Pengtao; Wang, Yanan; Li, Qiang

    2017-09-01

    The lower critical temperature Tc and critical current density Jc are serious weaknesses of SmBCO bulk superconductors fabricated in air for practical applications, because of the Sm3+/Ba2+ solid solution in Sm1+xBa2-xCu3Oy crystals. In this paper, high quality single domain SmBCO bulk samples S1 (ϕ20 mm) and S2 (ϕ32 mm) have been fabricated in air by a new Sm+011 TSIG method. The trapped field of the samples is 0.8 T and 1.15 T at liquid nitrogen temperature for the samples S1 and S2 respectively, which is the strongest trapped field of the SmBCO samples fabricated in air today. The theoretical formula for 3D trapped field distribution have been derived for a cylindrical model with uniformly distributed critical current density Jc based on the Biot Savart law; the cylindrical sample is divided into a series of concentric rings with the same width and thickness, the trapped field of the samples is the summation of magnetic field produced by all the rings, while the magnetic field generated by each ring was worked out by trapezoidal numerical integration based on the Biot Savart law with the critical current density Jc of the samples. It is found that the calculated field of the samples is well in agreement with the experimental results if the reasonable Jc of the samples is adopted. The theoretical calculation result also indicates that the larger the diameter and the thickness of the samples, the stronger the trapped flux density, but the optimal diameter/thickness ratio should be of a reasonable value around one, and it is not so good to fabricated samples with too larger diameter or thickness for practical applications.

  5. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  6. High levels of antibodies to multiple domains and strains of VAR2CSA correlate with the absence of placental malaria in Cameroonian women living in an area of high Plasmodium falciparum transmission

    DEFF Research Database (Denmark)

    Tutterrow, Yeung L; Avril, Marion; Singh, Kavita;

    2012-01-01

    DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated...

  7. PHASE STABILITY OF SINGLE DOMAIN EPITAXIAL FERROELECTRIC FILMS%单畴外延铁电薄膜的相结构与稳定性

    Institute of Scientific and Technical Information of China (English)

    周志东; 张春祖; 蒋泉

    2012-01-01

    The phase stability of single domain ferroelectric PbTiO3(PTO) epitaxial thin film as a function of the misfit strain and film thickness is studied and discussed systematically using the dynamic Ginzburg-Landau (DGL) equation by taking surface eigenstrain relaxation and depolarization field. Misfit strain-film thickness phase diagrams for various temperatures and misfit strain-temperature phase diagrams for various film thicknesses are obtained,respectively. The numerical results show that the phase diagrams in high strain zone are markedly different from previous thermodynamic predictions due to misfit dislocations. Especially, we present that there are c-phase and rotational r-phase in a broader misfit tensile strain zone. The results also express that effects of surface and depolarization field obviously shift the paraelectrie phase to lower temperature and shrink the ferroelectric phase with decreasing thickness of the films.%论义采用动态金茨堡-朗道(DGL)方程研究了薄膜厚度与错配应变对(001)取向单畴外延PbTiO3(PTO)铁电薄膜相结构与稳定性的影响,结合平面内松弛应变(等效应变)、表面效应与退极化场等机电耦合边界条件,通过数值求解DGL方程获得外延单畴铁电薄膜错配应变厚度相图和错配应变温度相图.数值分析结果显示,由于生成的界面位错松弛了薄膜内错配应变,在理论高应变区相图与传统分析结果有较大差别,文中发现在更广的理论错配拉应变区出现稳定的四方相(c相)结构和单斜相(r相)结构.结果也显示,随着薄膜厚度的减小,表面效应与退极化效应会把顺电相扩展到更低温度区域,从而压缩稳定的铁电相存在的温度区域.

  8. Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig-binding proteins (IBPs with four kinds of Ig molecules

    Directory of Open Access Journals (Sweden)

    Jia Jian-An

    2008-08-01

    Full Text Available Abstract Background Protein A, protein G and protein L are three well-defined immunoglobulin (Ig-binding proteins (IBPs, which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting. Results We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D and PA(A respectively, B2 domain of protein G (PG and B3 domain of protein L (PL for affinity selection with human IgG (hIgG, human IgM (hIgM, human IgA (hIgA and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A-PG and PA(A-PL were obtained in hIgG (hIgG1-Fc and hIgM (hIgA post-selection populations respectively. In addition, the linking peptides among all PA(A-PG and PA(A-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A-PG and PA(A-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively. Conclusion In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A-PG and PA(A-PL, were obtained, and demonstrate the novel Ig binding properties.

  9. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  10. Single domain SmCo5@Co exchange-coupled magnets prepared from core/shell Sm[Co(CN)6]·4H2O@GO particles: a novel chemical approach.

    Science.gov (United States)

    Yang, Ce; Jia, Lihui; Wang, Shouguo; Gao, Chen; Shi, Dawei; Hou, Yanglong; Gao, Song

    2013-12-20

    SmCo5 based magnets with smaller size and larger maximum energy product have been long desired in various fields such as renewable energy technology, electronic industry and aerospace science. However, conventional relatively rough synthetic strategies will lead to either diminished magnetic properties or irregular morphology, which hindered their wide applications. In this article, we present a facile chemical approach to prepare 200 nm single domain SmCo5@Co core/shell magnets with coercivity of 20.7 kOe and saturation magnetization of 82 emu/g. We found that the incorporation of GO sheets is responsible for the generation of the unique structure. The single domain SmCo5 core contributes to the large coercivity of the magnets and the exchange-coupled Co shell enhances the magnetization. This method can be further utilized in the synthesis other Sm-Co based exchange-coupled magnets.

  11. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    Science.gov (United States)

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  12. Presence, distribution and steroidogenic effect of the peptides orexin A and receptor 1 for orexins in the testis of the South American camelid alpaca (Vicugna pacos).

    Science.gov (United States)

    Liguori, Giovanna; Assisi, Loredana; Squillacioti, Caterina; Paino, Salvatore; Mirabella, Nicola; Vittoria, Alfredo

    2012-10-01

    The orexins A (oxA) and B are peptides discovered in the rat hypothalamus and successively found in some peripheral organs of the mammalian body. They binds two protein G-coupled receptors defined receptor 1 (ox1r) and 2 for orexins, the first of which is highly specific for oxA while the second binds both the peptides with equal affinity. This work aimed to detect the presence of oxA and ox1r in the testis of the South American camelid alpaca (Vicugna pacos) and investigate the role played by them on Leydig cell steroidogenesis. The species alpaca acquired, in the last years, increasing zootechnical interest for the quality of the wool produced and its breeding spread from the country of origin to USA, Australia and Europe. Immunohistochemistry allowed us to detect oxA in Leydig and Sertoli cells, spermatogonia, resting spermatocytes, round and oval spermatids. Ox1r-immunoreactivity was found in Leydig cells and round, oval and elongated spermatids. The expression of the two peptides in tissue extracts was established by using Western blotting technique. Such results demonstrated that in the alpaca testis exists in a cellular complex able to produce and/or internalize oxA. Finally, the effect of oxA on steroidogenesis was investigated by means of in vitro cultured thin testis slices which were added with oxA or/and Müllerian Inhibiting Substance (MIS), a steroidolitic agent basally produced by the Sertoli cell. OxA evoked increase of testosterone production while MIS a decrease. The consecutive addition of oxA and MIS, or vice versa, highlighted an antagonistic interplay between the two substances which has been thought to be the main molecular event at the basis of the oxA-stimulated steroidogenesis mechanism.

  13. Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance.

    Science.gov (United States)

    Anderson, George P; Liu, Jinny L; Zabetakis, Dan; Legler, Patricia M; Goldman, Ellen R

    2017-03-01

    Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein. A subset of the monoclonal antibodies was immobilized to characterize antigen capture in solution. It can be advantageous to utilize antibodies that recognize distinct epitopes when choosing reagents for detection and diagnostic assays. We determined the uniqueness of the epitope recognized by the anti-VP40 antibodies using a checkerboard format that exploits the 6×6 array of interactions monitored by the Bio-Rad ProteOn XPR36 SPR instrument. The results demonstrate the utility of surface plasmon resonance to characterize monoclonal and recombinant antibodies. Additionally, the analysis presented here enabled the identification of pairs of anti-VP40 antibodies which could potentially be utilized in sandwich type immunoassays for the detection of Ebola virus.

  14. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    Science.gov (United States)

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  15. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele

    2015-01-01

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions. PMID:26216991

  16. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins.

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A; Vendruscolo, Michele

    2015-08-11

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

  17. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  18. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    Science.gov (United States)

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  19. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  20. Molecular Targeting of CEACAM6 Using Antibody Probes of Different Sizes

    Science.gov (United States)

    Niu, Gang; Murad, Yanal M.; Gao, Haokao; Hu, Shuo; Guo, Ning; Jacobson, Orit; Nguyen, Thanh-Dung; Zhang, Jianbing; Chen, Xiaoyuan

    2012-01-01

    Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is overexpressed in a number of human malignancies, especially in pancreatic cancer. It has been demonstrated that CEACAM6 is a potential target for monoclonal antibody (mAb) therapy with a safe therapeutic index. Here, we labeled three anti-CEACAM6 antibodies of different sizes, including a single-domain antibody 2A3 (16 kDa), a heavy chain antibody 2A3-mFc (80 kDa) and a full length antibody 9A6 (150 kDa), with 64Cu to image CEACAM6 expression in a xenografted pancreatic tumor model. For positron emission tomography (PET) imaging, the tumor mice were intravenously injected with 64Cu-DOTA-antibodies and static scans were obtained at 5 min, 0.5, 1, 2, 4, 8 and 24 h post-injection (p.i.). All three antibodies showed strong CEACAM6 binding. Ex vivo immunostaining on tumor sections at 24 h after Ab injection demonstrated specific tumor targeting of both 2A3-mFc and 9A6. 64Cu-DOTA-2A3 showed fast BxPC3 tumor uptake and rapid whole-body clearance. At 24 h p.i., the tumor uptakes were 98.2 ± 6.12 %ID/g for 64Cu-DOTA-2A3-mFc and 57.8 ± 3.73 %ID/g for 64Cu-DOTA-9A6, respectively. Compared with the full length antibody 9A6, the heavy chain antibody 2A3-mFc showed higher tumor uptake, lower liver uptake and shorter circulation half-life. All the data supported that the heavy chain antibody 2A3-mFc is superior to the single domain antibody and the full-length antibody with regard to tumor detection and pharmacokinetics, which has great potential to be developed for CEACAM6-targeted pancreatic cancer imaging and therapy. PMID:22568933

  1. ESTÁNDARES MÉTRICOS Y VARIABILIDAD EN FALANGES PROXIMALES DE CAMÉLIDOS SUDAMÉRICANOS. SU IMPORTANCIA COMO CONJUNTO COMPARATIVO PARA INTERPRETACIONES EN ARQUEOLOGÍA / Metric standards and variability in South American camelids proximal phalanges

    Directory of Open Access Journals (Sweden)

    Andrés D. Izeta

    2009-12-01

    obtained from South American camelids appendicular skeleton bones have been used in recent years as a benchmark for interpreting different aspects of archaeological studies. The shape and size of bones have been linked to various factors which may be related to environmental restrictions and human management. This is of great importance for interpreting the history of human-camelid relationships through time, since the many remains obtained from archaeological deposits permit reconstructing situations that occurred in the past, which can be interpreted as the result of paleoeconomic activity or environmental changes. Therefore, this paper aims to present metric data obtained from contemporary individuals of South American camelids, which have been considered relevant for use as comparative material. An evaluation of this data is given a critical look in order to rethink the use of local and nonlocal standards for “taxonomic” assignment of archaeological samples. In order to assess the variability in shape and size of the first phalanx, the application of Principal Component Analysis and Cluster Analysis (UPGMA is presented. This takes into account differences in size, shape and geographical origin in order to observe the contribution of each variable in the formation of this sample. As a result, it is concluded that the use of both techniques, as well as data from the geographical provenience of the modern samples, allows a finer-grained species determination.   Keywords: South American camelids, osteometry, postcranial skeleton, proximal phalanx, variability, comparative standards.  

  2. The art of blocking ADP-ribosyltransferases (ARTs): nanobodies as experimental and therapeutic tools to block mammalian and toxin ARTs.

    Science.gov (United States)

    Menzel, Stephan; Rissiek, Björn; Haag, Friedrich; Goldbaum, Fernando A; Koch-Nolte, Friedrich

    2013-08-01

    In 1901, the first Nobel Prize in Physiology or Medicine was awarded to Emil von Behring for his ground-breaking discovery of serum therapy: serum from horses vaccinated with toxin-containing culture medium of Corynebacterium diphtheriae contained life-saving 'antitoxins'. The molecular nature of the ADP-ribosylating toxin and the neutralizing antibodies were unraveled only 50 years later. Today, von Behring's antibody therapy is being refined with a new generation of recombinant antibodies and antibody fragments. Nanobodies, which are single-domain antibodies derived from the peculiar heavy-chain antibodies of llamas and other camelids, are emerging as a promising new class of highly specific enzyme inhibitors. In this review, we illustrate the potential of nanobodies as tools to block extracellular and intracellular ADP-ribosyltransferases (ARTs), using the toxin-related membrane-bound mammalian ecto-enzyme ARTC2 and the actin-ADP-ribosylating Salmonella virulence plasmid factor B toxin of Salmonella enterica as examples.

  3. 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.

    2016-01-01

    Abstract 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-1BaL 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. PMID:26950606

  4. INDUCTION OF SUPEROVULATION IN CAMELIDS

    Directory of Open Access Journals (Sweden)

    Ratto MH

    2015-12-01

    Full Text Available El desarrollo de las técnicas de reproducción asistida en Camélidos Sudamericanos ha sido establecido con bastante lentitud al compararlas con el desarrollo que han tenido en otras especies como son los rumiantes. Sin embargo técnicas como superovulación, transferencia de embriones y fecundación in vitro han sido abordadas en los últimos años con resultados bastante alentadores y que podrán en un futuro hacer posible de uso en forma comercial. Similar a los rumiantes las respuestas a los protocolos de superovulación continúan siendo muy variables a pesar de tener un control de la actividad ovárica al comienzo de los mismos. La recuperación de blastocistos eclosionados mediante lavado uterino continúa siendo una limitante para poder manipular al embrión posterior a su colección con otras metodologías como congelación o vitrificación. Un gran paso se ha realizado en lo que respecta a los métodos de colección de semen y su uso para la inseminación artificial o para fecundación in vitro. Así también, se ha logrado de obtener ovocitos de animales vivos utilizando punción folicular guiada por ecografía transvaginal. Quizás el impedimento más complejo para lograr un avance significativo del método de fecundación in vitro has sido la escasez en la obtención de ovocitos de matadero, una fuente muy importante de material que pudiera ser utilizado para investigación de la misma forma que lo ha sido para los sistemas in vitro en rumiantes. La intención de esta revisión es dar a conocer los principales protocolos de superovulación que han sido desarrollados hasta la fecha en llamas y alpacas.

  5. Induction of superovulation in Camelids

    Directory of Open Access Journals (Sweden)

    Ratto MH

    2015-08-01

    Full Text Available El desarrollo de las técnicas de reproducción asistida en Camélidos Sudamericanos ha sido establecido con bastante lentitud al compararlas con el desarrollo que han tenido en otras especies como son los rumiantes. Sin embargo técnicas como superovulación, transferencia de embriones y fecundación in vitro han sido abordadas en los últimos años con resultados bastante alentadores y que podrán en un futuro hacer posible de uso en forma comercial. Similar a los rumiantes las respuestas a los protocolos de superovulación continúan siendo muy variables a pesar de tener un control de la actividad ovárica al comienzo de los mismos. La recuperación de blastocistos eclosionados mediante lavado uterino continúa siendo una limitante para poder manipular al embrión posterior a su colección con otras metodologías como congelación o vitrificación. Un gran paso se ha realizado en lo que respecta a los métodos de colección de semen y su uso para la inseminación artificial o para fecundación in vitro. Así también, se ha logrado de obtener ovocitos de animales vivos utilizando punción folicular guiada por ecografía transvaginal. Quizás el impedimento más complejo para lograr un avance significativo del método de fecundación in vitro has sido la escasez en la obtención de ovocitos de matadero, una fuente muy importante de material que pudiera ser utilizado para investigación de la misma forma que lo ha sido para los sistemas in vitro en rumiantes. La intención de esta revisión es dar a conocer los principales protocolos de superovulación que han sido desarrollados hasta la fecha en llamas y alpacas.

  6. Antinuclear antibody panel

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003535.htm Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  7. Lyme disease antibody

    Science.gov (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  8. Acetylcholine receptor antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

  9. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  10. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer....

  11. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  12. Circular single domains in hemispherical Permalloy nanoclusters

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, Clodoaldo I. L de, E-mail: dearaujo@ufv.br; Fonseca, Jakson M. [Departamento de Física, Universidade Federal de Viçosa, 36570-900 Viçosa-MG (Brazil); Sinnecker, João P. [Centro Brasileiro de Pesquisas Físicas, 22290-180 Rio de Janeiro-RJ (Brazil); Delatorre, Rafael G.; Garcia, Nicolas; Pasa, André A. [Departamento de Física, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC (Brazil)

    2014-11-14

    We have studied ferromagnetic Permalloy clusters obtained by electrodeposition on n-type silicon. Magnetization measurements reveal hysteresis loops almost independent on temperature and very similar in shape to those obtained in nanodisks with diameter bigger than 150 nm. The spin configuration for the ground state, obtained by micromagnetic simulation, shows topological vortices with random chirality and polarization. This behavior in the small diameter clusters (∼80 nm) is attributed to the Dzyaloshinskii-Moriya interaction that arises in its hemispherical geometries. This magnetization behavior can be utilized to explain the magnetoresistance measured with magnetic field in plane and out of sample plane.

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

    Directory of Open Access Journals (Sweden)

    Ole Aalund Mandrup

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

  14. Expression of recombinant antibodies.

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

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

  15. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  16. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  17. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

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

    2015-01-01

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

  18. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  19. Bi2O3掺杂对TSMTG法单畴YBCO超导块材性能的影响%The Effect of Bi2 O3 Additions on the Properties of Single Domain YBCO Bulks by TSMTG process

    Institute of Scientific and Technical Information of China (English)

    高平; 杨万民

    2014-01-01

    本文采用顶部籽晶熔融织构法(TSM TG)成功地将Bi2 O3掺杂到YBCO超导块材中,研究了Bi2 O3掺杂含量对单畴 YBCO 超导块材生长形貌和磁悬浮力的影响.结果表明,在YBa2 Cu3 O7-δ(Y123):Y2 BaCuO5(Y211)=1:0.4不变的情况下,掺杂的Bi2 O3粉体在样品内部均生成了Y2 Ba4 CuBiOx纳米粒子.当Bi2 O3添加量 x≤1.5w t%时,样品均可长成完整的单畴YBCO超导块材,且样品的磁悬浮力随着Bi2 O3掺杂量的增加而增大;当 x>1.5 w t%时,YBCO超导块材的单畴区域随着Bi2 O3掺杂量的增加而逐渐减小,且随机成核现象严重,磁悬浮力降低;当 x=1.5 wt%时,样品的磁悬浮力最大.该结果对缩短样品制备的周期及进一步提高超导块材性能具有十分重要的意义.%The YBCO bulk superconductors with different Bi2 O3 content have been successfully fab‐ricated by the top‐seeded melt texture growth process (TSMTG) ,and the effect of Bi2O3 doping content on the magnetic levitation force is investigated based on the precursors of YBa2 Cu3 O7‐δ:Y2 BaCuO5 =1 :0.4 .It is found that the Y2 Ba4 CuBiOx nano‐particles can be formed with Bi2 O3 doped .Completely single domain YBCO bulks have been synthesized w hen Bi2 O3 doping content is less than 1 .5 w t% ,T he mag‐netic levitation force of the samples increase with the rising Bi2 O3 amount ;When Bi2 O3 addition is more than 1 .5 w t% ,the single domain areas are small and random nucleations are more and more serious ,so the magnetic levitation force is decreasing ;The largest levitation force of the YBCO superconducting bulks can be obtained when Bi2O3 addition equal to 1 .5 wt% .The results are very important for further shortening the period of sample preparation and improving the properties of YBCO bulk superconduc‐tors .

  20. TRAPPED MAGNETIC FIELDS MEASUREMENT SYSTEM OF HTS SINGLE DOMAIN BULK BASED ON LABVIEW%基于Labview的高温超导单畴块材俘获磁场测试系统

    Institute of Scientific and Technical Information of China (English)

    连博文; 徐克西; 左鹏翔; 曹越; 胡顺波

    2012-01-01

    A trapped magnetic fields measurement system of high temperature superconductor single domain bulk has been designed based on virtual instrument. Distribution of the whole magnetic fields can be obtained accurately and timely. The software platform is built by Labview, which can achieve the precise communication and control between computer and measurement equipments, and then the collected data will be processed and expressed. The underlying hardware includes stepper motor drive, 3D control frame, Gauss meter, digital multimeter, low temperature Hall probe, superconducting magnets, cryogenic dewar flask, vacuum pumps and cryoeooler. The whole meas- urement system is accurate, efficient, convenient, and highly automated. This system provides a reliable experimental techniques program for the research of applications and theoretical analysis of high temperature superconductors.%本文利用虚拟仪器技术设计出一套高温超导单畴块材俘获磁场测试系统,能够准确及时地反馈出超导块材整体磁通冻结场的分布.软件平台由Labview搭建,实现计算机与测量设备的精确通信与控制,并对采集到的数据进行处理和表达.底层硬件包括步进电机驱动器,可控支架,高斯测量仪,数字多用表,低温霍尔探头,超导强磁体,低温杜瓦瓶,真空泵及制冷机.整套测量系统准确、高效、便捷、自动化成度高,为进一步高温超导体应用特性的研究和机理理论分析提供了可靠的实验技术方案.

  1. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  2. Antiphospholipid antibody syndrome.

    Science.gov (United States)

    Kutteh, William H; Hinote, Candace D

    2014-03-01

    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

  3. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  4. Antithyroid microsomal antibody

    Science.gov (United States)

    ... to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also used to find ... positive test may be due to: Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also ...

  5. Serum herpes simplex antibodies

    Science.gov (United States)

    ... 2. HSV-1 most often causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test ... whether a person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  6. Heterogeneity of monoclonal antibodies.

    Science.gov (United States)

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

    2008-07-01

    Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

  7. Induction of strain-transcendent antibodies to placental-type isolates with VAR2CSA DBL3 or DBL5 recombinant proteins

    Directory of Open Access Journals (Sweden)

    Hathaway Marianne J

    2011-02-01

    Full Text Available Abstract Background Pregnancy associated malaria is a severe clinical syndrome associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, which adheres to chondroitin sulphate A (CSA. VAR2CSA is a large and polymorphic protein that has six Duffy binding-like (DBL domains. There is still limited understanding as to how effective individual VAR2CSA domains are at generating inhibitory antibodies or the number of domain variants needed for universal vaccine coverage. Methods To investigate the immunogenic properties of single domain VAR2CSA recombinant proteins, rats or rabbits were immunized with five of the six VAR2CSA domains produced in Pichia pastoris. Immune plasma was analysed against a geographically diverse panel of CSA-binding lab lines to assess antibody breadth and inhibitory activity. Results Of the five domains, DBL3, and to a lesser extent DBL5, induced antibodies that cross-reacted on five diverse CSA-binding parasite lines by flow cytometry. By comparison, anti-DBL6 antibodies were highly strain-specific and anti-DBL1 and anti-DBL4 antibodies were poorly reactive by flow cytometry. From this series of recombinant proteins, adhesion-blocking activity was restricted to a single rat immunized against a DBL4 recombinant protein. Conclusions Single domain VAR2CSA recombinant proteins produced in P. pastoris had limited efficacy in eliciting adhesion blocking antibody responses, but VAR2CSA DBL3 and DBL5 domains contain strain-transcendent epitopes that can be targeted by vaccination and may have application for vaccine development.

  8. LA PRESENCIA DE CAMÉLIDOS EN EL AUSTRO ECUATORIANO: EVIDENCIA ARQUEOLÓGICA DE TEXTILERÍA EN LA MESETA DE PACHAMAMA (The Presence of Camelids in Southern Ecuador: Archaeological Evidence of Textile Industry on the Pachamama Plateau

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Novillo Verdugo

    2016-09-01

    Full Text Available La meseta de Pachamama (Azuay, Ecuador constituye un espacio de gran interés arqueológico pues los restos culturales en él registrados denotan actividades específicas de culturas prehispánicas asentadas en la región. De esta manera, el presente artículo resulta una primera aproximación a la caracterización de la actividad textilera en el área a partir de elementos como fusayolas, mandíbulas de camélidos, corrales, con el fin de discutir y aportar nuevos conocimientos históricos en el austro ecuatoriano. Se matiza el área de estudio como un escenario propicio para el desarrollo de flora y fauna, pues el recurso hídrico en la zona es permanente y se presenta en toda la extensión de la meseta. Esta característica es perceptible por los diferentes momentos en que fue ocupada la meseta de Pachamama, donde se construyeron sistemas de terracería y caminos que conectan diferentes áreas constituyendo una red vial. ENGLISH: The Pachamama Plateau (Azuay, Ecuador is an area of great archaeological interest, as it contains evidence regarding the specific activities of pre-Hispanic cultures in the region. This article is an initial approach to the characterization of textile-making activity in the area based on artifacts such as spindle whorls, camelid mandibles, and corrals, in order to provide new historical knowledge about southern Ecuador. The study area is a favorable environment for the development of flora and fauna, as it contains permanent water resources across the extent of plateau. This environmental scenario was similar during periods of past occupations of the Pachamama Plateau, where terraces and roads were built to connect different areas, constituting a road network.

  9. Heparin-Induced Thrombocytopenia Antibody Test

    Science.gov (United States)

    ... Global Sites Search Help? Heparin-induced Thrombocytopenia PF4 Antibody Share this page: Was this page helpful? Also known as: Heparin-PF4 Antibody; HIT Antibody; HIT PF4 Antibody; Heparin Induced Antibody; ...

  10. Direct Observations of Correlation between Si-2p Components and Surface States on Si(110)-16 × 2 Single-Domain Surface Using Si-L23VV Auger-Electron and Si-2p Photoelectron Coincidence Measurements

    Science.gov (United States)

    Kakiuchi, Takuhiro; Yoshizaki, Yuya; Kubota, Hiroyuki; Sato, Yuki; Nagaoka, Shin-ichi; Mase, Kazuhiko

    2017-05-01

    A Si(110)-16 × 2 single-domain (SD) surface is investigated in a site-selective way using Si L23VV Auger-electron Si-2p photoelectron coincidence spectroscopy (Si-L23VV-Si-2p APECS) and Si-2p photoelectron Si-L23VV Auger-electron coincidence spectroscopy (Si-2p-Si-L23VV PEACS). The Si(110)-16 × 2 SD consists of five Si-2p surface components (SC1-SC5) and has four semiconducting surface states (S1-S4). The Si-L2VV-Si-2p1/2 APECS spectrum of the Si(110)-16 × 2 SD measured in coincidence with Si-2p1/2 photoelectrons of SC3, SC4, and SC5 shows two small shoulders in the higher Auger electron kinetic energy (AeKE) region. These shoulders suggest Auger processes involving the surface states S1 and S3. The spectral weights of SC3, SC4, and SC5 Si-2p components are greatly enhanced in the Si-2p-Si-L23VV PEACS spectrum measured at Auger electrons with an AeKE of +5.0 eV relative to the Si L23VV peak. On the other hand, the spectral weights of SC1 and SC2 Si-2p components in the Si-2p-Si-L23VV PEACS spectrum show a maximum peak at a relative AeKE of +3 eV. These results directly support the correlations between the five surface components (SC1-SC5) and four surface states (S1-S4) in the adatom-buckling model for the Si(110)-16 × 2 SD proposed by Sakamoto et al. [https://doi.org/10.1103/PhysRevB.79.045304" xlink:type="simple">Phys. Rev. B 79, 045304 (2009)].

  11. Study on the pulsed-field magnetization of single domain YBCO bulk%单畴 YBCO 超导块材的脉冲充磁磁化规律

    Institute of Scientific and Technical Information of China (English)

    李佳伟; 杨万民

    2015-01-01

    为了提高氧化钇钡铜(YBCO)超导体的捕获磁场,成功制备了单畴 YBCO 超导块材,研究了不同强度脉冲磁场和充磁次数对 YBCO 超导体捕获磁场的影响。结果表明:对直径20 mm 的YBCO 超导体,存在一个最佳的脉冲磁场强度,使得捕获磁场最强;当采用最佳脉冲磁场充磁时,增加充磁次数可以显著增加 YBCO 超导体的捕获磁场。进一步采用 Bean 模型解释了该磁化规律。本文的研究结果有助于提高 YBCO 超导体的磁化效果和理解 YBCO 超导体的磁化机制。%In order to improve the trapped field of YBCO bulk,a single domain YBCO bulk was successfully fabricated,and the effects of the intensity and the magnetization time of pulsed mag-netic fields on the trapped field have been investigated in detail.The result shows that the intensi-ty of the trapped field in YBCO superconductor with the diameter of 20 mm may reach its maxi-mum when the magnetic field with an optimal intensity(4.8 T)is applied.The trapped field in-creases significantly when the sample is magnetized for several times by the optimal magnetic field.Furthermore,the magnetization process is explained by Bean model.The results in this work helps to improve the magnetization efficiency of YBCO superconductors and understand the mag-netization mechanism.

  12. [New antibodies in cancer treatment].

    Science.gov (United States)

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  13. Engineering antibodies for cancer therapy.

    Science.gov (United States)

    Boder, Eric T; Jiang, Wei

    2011-01-01

    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  14. A complete set of material properties of single domain 0.26Pb(In(12)Nb(12))O(3)-0.46Pb(Mg(13)Nb(23))O(3)-0.28PbTiO(3) single crystals.

    Science.gov (United States)

    Liu, Xiaozhou; Zhang, Shujun; Luo, Jun; Shrout, Thomas R; Cao, Wenwu

    2010-01-04

    Pb(In(12)Nb(12))O(3)-Pb(Mg(13)Nb(23))O(3)-PbTiO(3) (PIN-PMN-PT) single crystals have been developed recently, which can increase the operating temperature by at least 20 degrees C compared to PMN-PT crystals. We have measured a complete set of material properties of single domain PIN-PMN-PT crystal, which is urgently needed in theoretical studies and electromechanical device designs using this crystal. Because the rotated values of d33*=1122 pCN and k33*=89% along [001](c) calculated using the single domain data obtained here are in good agreement with the [001](c) poled multidomain PIN-PMN-PT crystals, one may conclude that the physical origin of the ultrahigh piezoelectric properties mainly come from orientation effect.

  15. 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...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  16. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...... 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...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...

  17. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  18. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1979-03-15

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  19. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  20. Human germline antibody gene segments encode polyspecific antibodies.

    Science.gov (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  1. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn Thorup;

    2012-01-01

    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  2. Antibody Blood Tests

    Science.gov (United States)

    ... What do I do if I have a negative blood test (or panel) but I’m still having symptoms? While it is rare, it is possible for patients to have a negative antibody test results and still have celiac disease. ...

  3. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  4. RBC Antibody Screen

    Science.gov (United States)

    ... test also may be used to help diagnose autoimmune-related hemolytic anemia in conjunction with a DAT. This condition may be caused when a person produces antibodies against his or her own RBC antigens. This can happen with some autoimmune disorders , such as lupus , with diseases such as ...

  5. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  6. Anti-smooth muscle antibody

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the ...

  7. What Is Antiphospholipid Antibody Syndrome?

    Science.gov (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  8. Red Blood Cell Antibody Identification

    Science.gov (United States)

    ... ID, RBC; RBC Ab ID Formal name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC ... I should know? How is it used? Red blood cell (RBC) antibody identification is used as a follow- ...

  9. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  10. Antibody Engineering and Therapeutics Conference

    OpenAIRE

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A.; Burton, Dennis R.; Adams, Gregory P.; Weiner, Louis M.; Scott, Jamie K.; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M.

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Bi...

  11. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    Science.gov (United States)

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

  12. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  13. IDENTIFICACIÓN INTERESPECÍFICA DE CAMÉLIDOS EN EL VALLE DE AMBATO (CATAMARCA, ARGENTINA. UNA APROXIMACIÓN A LA PROBLEMÁTICA DESDE DISTINTAS LÍNEAS DE ANÁLISIS/Interspecific identification of camelids in Ambato valley (Catamarca, Argentina...

    Directory of Open Access Journals (Sweden)

    Mariana Dantas

    2012-11-01

    Full Text Available La identificación interespecífica de camélidos y la distinción entre morfotipos de llamas en los materiales óseos recuperados en sitios arqueológicos constituyen dos aspectos de suma importancia dentro de los estudios zooarqueológicos del noroeste argentino y de la región andina en general. En este trabajo se pretende contribuir a esta problemática, mediante el entrecruzamiento de distintas líneas de análisis aplicadas sobre los restos de camélidos procedentes del Valle de Ambato, Catamarca, entre los siglos VI y X d.C. De este modo, la integración de estudios osteométricos y de isótopos estables posibilitó establecer la existencia en este valle de camélidos domésticos en función de su tamaño relativo y una dieta selectiva concentrada en plantas C4. Dentro de aquellos también fue posible distinguir dos grupos que podrían responder a distintas variedades de tamaños: un grupo de animales de mayor porte, o llamas grandes, y otro de menor porte, o llamas pequeñas, cuyo tamaño se superpone con el rango de tamaños de los guanacos norandinos. En definitiva, se puede afirmar, que la combinación de ambos análisis permitió alcanzar un grado de diferenciación que de otro modo no hubiera sido posible.   Abstract   Interspecific identification of camelids and distinction between llamas’ morphotypes in bone materials from archaeological sites are two major aspects in zooarchaeological studies of northwestern Argentina and the Andean region in general. This paper aims at contributing to the issue along different analytical approaches applied to camelids remains from Ambato Valley, Catamarca, between the 6th and 10th centuries AD. Thus, the integration of osteometric and stable isotope studies allowed establishing/confirming the presence of domestic camelids in this valley, based on their relative size and a selective diet mostly derived from C4 plants. It was also possible to distinguish two probably differently-sized groups

  14. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

  15. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi

    2014-01-01

    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic......More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...

  16. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  17. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  18. How antibodies use complement to regulate antibody responses.

    Science.gov (United States)

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  19. Antibody response in Heterodontus.

    Science.gov (United States)

    Litman, G W; Erickson, B W; Lederman, L; Mäkelä, O

    1982-05-28

    Appropriately selected phylogenetic models are capable of providing insight into genetic mechanisms which may have become obscured during the passage of evolutionary time. In higher vertebrates a complex multigenic family encodes immunoglobulin-variable regions. The mechanisms involved in the expansion of the gene family and the stable maintenance of large numbers of individual genes presently are not understood. By defining the nature of antibody diversity in lower vertebrate species, it may be possible to approach such issues at a more fundamental level. Analyses of the immunoglobulins in Heterodontus francisci (horned shark), a representative phylogenetically primitive elasmobranch, indicate that this species may represent a useful developmental model.

  20. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  1. VIRAL ANTIBODIES IN PRESCHOOL CHILDREN

    Directory of Open Access Journals (Sweden)

    S. Saidi

    1974-08-01

    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  2. Cancer imaging with radiolabeled antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Goldenberg, D.M. (Center for Molecular Medicine and Immunology, Newark, NJ (US))

    1990-01-01

    This book presents a perspective of the use of antibodies to target diagnostic isotopes to tumors. Antibodies with reasonable specificity can be developed against almost any substance. If selective targeting to cancer cells can be achieved, the prospects for a selective therapy are equally intriguing. But the development of cancer detection, or imaging, with radiolabeled antibodies has depended upon advances in a number of different areas, including cancer immunology and immunochemistry for identifying suitable antigen targets and antibodies to these targets, tumor biology for model systems, radiochemistry for he attachment of radionuclides to antibodies, molecular biology for reengineering the antibodies for safer and more effective use in humans, and nuclear medicine for providing the best imaging protocols and instrumentation to detect minute amounts of elevated radioactivity against a background of considerable noise. Accordingly, this book has been organized to address the advances that are being made in many of these areas.

  3. Human anti-mouse antibodies.

    Science.gov (United States)

    Klee, G G

    2000-06-01

    Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

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

    NARCIS (Netherlands)

    van Schouwenburg, P.A.

    2012-01-01

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

  5. The effects of different particle raze Y2Ba4CuBiOy nanoparticles doped on the properties of single domain YBCO bulk superconductors by TSIG process%不同粒径纳米Y2Ba4CuBiOy相掺杂对TSIG法单畴YBCO超导块材性能的影响

    Institute of Scientific and Technical Information of China (English)

    王妙; 杨万民; 张晓菊; 唐艳妮; 王高峰

    2012-01-01

    Single domain YBCO bulk superconductors are fabricated by the top-seeded infiltration and growth process(TSIG). The effects of different particle size Y2Ba4CuBiOy nanoparticles doped on the growth morphology, microstrncture and levitation force of single domain YBCO bulk superconductors are investigated. The mean diameters of the initial Y2Ba4CuBiOy nanoparticles respectively are 283 nm, 170.4 nm and 82.5 nm, and the amount of Y2Ba4CuBiOyadded to Y2BaCuO5 is 2 wt%. The results show that the single domain YBCO bulk superconductors can be fabricated, and the surface of the sample has significant cross pattern and four single domain sectors become smooth and flat. The second phase Y2Ba4CuBiOy nanoparticles are well-distributed into the single domain YBCO bulk samples, and the particles sizes of Y2Ba4CuBiOy nanoparticles in the YBCO bulk sample are respectively 270 nm, 150 nm and 50 nm, smaller than that the initial powder. The magnetic levitation forces of the samples increase gradually with the decrease of initial powders of Y2Ba4CuBiOy nanoparticles, which are respectively 10 N, 17 N and 22 N. The results are very important for further studying the method of flux pinning of nanoparticles and improving the properties of YBCO bulk superconductors.%本文采用顶部籽晶熔渗方法(TSIG),研究了不同粒径纳米Y2Ba4CuBiOy粒子对单畴YBCO超导块材的生长形貌、微观结构及其磁悬浮力的影响.实验所用纳米Y2Ba4CuBiOy粉体的平均粒度分别为283.0nm,170.4nm以及82.5nm,每种粉体在YBCO超导块材中的含量均为2wt%.研究结果表明:在掺杂量为2wt%的情况下,Y2Ba4CuBiOy粉体的粒度并不影响样品的宏观形貌,均可制备出单畴YBCO块材;并且成功地将纳米Y2Ba4CuBiOy粒子引入单畴YBCO块材中,且使其均匀分布,但样品中的Y2Ba4CuBiOy粒子均小于其初始粉体的粒度,分别减小到270nm,150nm和50nm;随着Y2Ba4CuBiOy粉体初始粒度

  6. Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures.

    Science.gov (United States)

    Simmons, David P; Streltsov, Victor A; Dolezal, Olan; Hudson, Peter J; Coley, Andrew M; Foley, Michael; Proll, David F; Nuttall, Stewart D

    2008-04-01

    Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of

  7. Reproduction in female South American domestic camelids.

    Science.gov (United States)

    Sumar, J B

    1999-01-01

    Alpacas and llamas are induced ovulators. They show marked reproductive seasonality in the Andean region, but under Northern Hemisphere conditions of feeding and management, they are non-seasonal breeders. Puberty is attained when they reach 50% of adult body weight. When they are not exposed to a male, females show successive waves of follicular maturation and atresia. Growth, maintenance and regression of a follicle each require an average of 4 and 6 days in alpacas and llamas, respectively. After sterile mating, progesterone concentrations in blood were increased from day 5, reached maximum concentrations on day 7-8, and declined rapidly at 9-10 days after mating. A fertile mating results in formation of a corpus luteum that remains functional throughout gestation. The duration of gestation is 340-346 days. Almost all fetuses were found to occupy the left uterine horn, even though ovulation occurs from both ovaries with equal frequency. Several methods of pregnancy diagnosis have been described. Mating is recommended within 15-20 days after parturition to obtain good fertility rates and one offspring per year. The factors that contribute to high rates of embryonic mortality are unknown. Reproductive technologies, such as AI, superovulation, embryo transfer and IVF, have not been used very extensively in these species but can be successfully applied.

  8. Reproductive physiology of the male camelid.

    Science.gov (United States)

    Bravo, P W; Johnson, L W

    1994-07-01

    The physiology of reproduction with emphasis on endocrinology of llamas and alpacas is addressed. Information regarding male anatomy, puberty, testicular function, semen description, and sexual behavior is also included.

  9. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

    Science.gov (United States)

    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D

    2004-08-24

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

  10. New engineered antibodies against prions

    Science.gov (United States)

    Škrlj, Nives; Dolinar, Marko

    2014-01-01

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

  11. Pathogenic role of antiphospholipid antibodies

    NARCIS (Netherlands)

    Salmon, J. E.; de Groot, P. G.

    2008-01-01

    The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that AP

  12. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  13. Antibodies and Plasmodium falciparum merozoites

    NARCIS (Netherlands)

    Ramasamy, R; Ramasamy, M; Yasawardena, S

    There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium

  14. Educational paper: Primary antibody deficiencies

    NARCIS (Netherlands)

    G.J.A. Driessen (Gertjan); M. van der Burg (Mirjam)

    2011-01-01

    textabstractPrimary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA a

  15. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  16. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive...... and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...

  17. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells

    Science.gov (United States)

    1987-01-01

    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  18. Simultaneous expression of displayed and secreted antibodies for antibody screen.

    Directory of Open Access Journals (Sweden)

    Yuanping Zhou

    Full Text Available The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS analysis, as well as in conditioned medium in a secreted version for function analysis.

  19. Construction, expression and identification of anti-human AFP VH single domain antibody fusion protein gene%抗AFP重链可变区单域抗体融合蛋白的构建、表达及初步鉴定

    Institute of Scientific and Technical Information of China (English)

    方丽; 王振达; 张平; 赵宗蓉; 章崇杰

    2007-01-01

    目的 构建抗甲胎蛋白(AFP)重链可变区(VH)单域抗体融合蛋白基因在大肠杆菌中的表达,并初步鉴定表达产物的活性.方法 从已构建的抗AFP单链抗体(ScFv)的载体中,经PCR扩增出VH 单域抗体基因,再克隆到融合蛋白表达载体pET32a(+)中进行表达;用SDS-PAGE及Western-blot鉴定表达产物;并通过竞争抑制ELISA分析TrxA-VH融合蛋白的结合活性;细胞免疫组化染色分析其内化及结合情况.结果 VH 单域抗体基因全长339 bp,将其克隆到pET32a(+)中,转化大肠杆菌可获得高效表达,Western-blot证实在相应分子质量处,有TrxA-VH融合蛋白的显色条带.经初步纯化和复性后,获得抗AFP的VH单域抗体融合蛋白.竞争抑制ELISA及细胞免疫组化证明,表达产物具有与AFP特异结合的活性.结论 成功构建了抗AFP的VH单域抗体融合蛋白,为临床的应用研究奠定了基础.

  20. Antibodies to a full-length VAR2CSA immunogen are broadly strain-transcendent but do not cross-inhibit different placental-type parasite isolates.

    Directory of Open Access Journals (Sweden)

    Marion Avril

    Full Text Available The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL domains to that of a three-domain construct (DBL4-6 in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species.

  1. Fragmentation of monoclonal antibodies

    Science.gov (United States)

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  2. Antibodies to watch in 2016

    OpenAIRE

    Reichert, Janice M

    2015-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, ar...

  3. Evolution of antiphospholipid antibody syndrome.

    Science.gov (United States)

    Baviskar, Rutuja R; Amonkar, Gayathri P; Chaudhary, Vinod A; Balasubramanian, Meenakshi; Mohite, Shailesh C; Puranik, Gururaj V

    2012-12-01

    Antiphospholipid antibody syndrome is a very important cause of cerebral infarction, myocardial infarction, and repeated pregnancy losses in women. We present an extremely rare case of a 44-year-old man with antiphospholipid syndrome who collapsed and died suddenly. At autopsy, he was found to have both cerebral and myocardial infarction. In all young patients with cerebral infarction, myocardial infarction, pulmonary embolism, recurrent miscarriages, and unexplained low platelet count, one must consider the strong possibility of antiphospholipid antibody syndrome.

  4. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  5. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  6. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    Science.gov (United States)

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  7. Production and Purification of Polyclonal Antibodies.

    Science.gov (United States)

    Nakazawa, Masami; Mukumoto, Mari; Miyatake, Kazutaka

    2016-01-01

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

  8. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    Science.gov (United States)

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

  9. Antibodies to watch in 2016.

    Science.gov (United States)

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015.

  10. Epigenetics of the antibody response.

    Science.gov (United States)

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-09-01

    Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

  11. Antibodies to watch in 2013

    Science.gov (United States)

    Reichert, Janice M

    2013-01-01

    The transitions of antibody therapeutics to late-stage clinical development, regulatory review and the market are proceeding at a rapid pace in 2013. Since late 2012, two monoclonal antibody (mAb) therapeutics (itolizumab, trastuzumab emtansine) received their first approvals, first marketing applications for three mAbs (vedolizumab, ramucirumab, obinutuzumab) were submitted to regulatory agencies, and five mAbs (brodalumab, MABp1, moxetumomab pasudotox, tildrakizumab, rilotumumab) entered their first Phase 3 studies. The current total of commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies is 30. Recently announced study results for farletuzumab, naptumomab estafenatox, and tabalumab indicate that clinical endpoints were not met in some Phase 3 studies of these product candidates. PMID:23727858

  12. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  13. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  14. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  15. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  16. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  17. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2017-03-28

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  18. Genetic Engineering of Single-Domain Magnetic Bacteria.

    Science.gov (United States)

    1992-09-25

    iron-uptake gene (fVuA), a ferrichrome-mediated iron-uptake gene (fhuB), the consensus FUR binding site, the tonB gene, and the btuB gene. In these...experiments, we were able to identify fragments that hybridized with the tonB -specific probes. However, upon cloning and sequencing, no significant...homology was detected between the cloned fragments and the tonB sequence of E. coli. We obtained similar results using the polymerase chain reactior (PCR

  19. Coaxial magnetic brakes using single-domain YBCO

    Energy Technology Data Exchange (ETDEWEB)

    Putman, P.T. [Texas Center for Superconductivity and Department of Mechanical Engineering, University of Houston, Houston, TX 77204 (United States)], E-mail: pputman@uh.edu; Salama, K. [Texas Center for Superconductivity and Department of Mechanical Engineering, University of Houston, Houston, TX 77204 (United States)

    2008-06-15

    In coaxial magnetic brakes, the changing field produced by movement of a solenoidal magnet induces a current in the wall of a conductive tube. The interaction of the field and current leads to a repulsive force that slows the motion of the magnet. For brake applications that require high force density, melt-textured YBCO is a clear choice of material for the magnet because it can carry high currents at a given field and temperature, and is inherently capable of operating in persistent current mode. We present calculations of the performance of this type of brake as a function of magnet current density for catch tubes composed of aluminum and titanium. These results are validated with low speed (20 m/s) tests. Calculations indicate that melt-textured magnets can decelerate projectiles with a mass of 1 kg from 2000 m/s to rest in distances on the order of 10 m. This suggests that this type of brake is suitable for use in hypervelocity experiments, which sometimes requires nondestructive deceleration of projectiles for diagnostic purposes.

  20. Antiphospholipid antibody syndrome and autoimmune diseases.

    Science.gov (United States)

    Ostrowski, Rochella A; Robinson, John A

    2008-02-01

    The arbitrary division between antiphospholipid antibody syndrome and secondary antiphospholipid antibody syndrome has not proven useful. Antiphospholipid antibodies in the absence of antiphospholipid antibody syndrome often occur as epiphenomena in many autoimmune diseases. They are very common in systemic lupus erythematosus. Antiphospholipid antibody syndrome is a significant comorbidity in lupus but is uncommon in Sjögren's syndrome, rheumatoid arthritis, scleroderma, and systemic vasculitis. Evidence is growing that antiphospholipid antibodies may have a pathogenic role in pulmonary hypertension and accelerated atherosclerosis of autoimmune diseases.

  1. Monoclonal antibodies for the identification and purification of vNAR domains and IgNAR immunoglobulins from the horn shark Heterodontus francisci.

    Science.gov (United States)

    Juarez, Karla; Dubberke, Gudrun; Lugo, Pavel; Koch-Nolte, Friedrich; Buck, Friedrich; Haag, Friedrich; Licea, Alexei

    2011-08-01

    In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.

  2. Mathematical and experimental analyses of antibody transport in hollow-fiber-based specific antibody filters.

    Science.gov (United States)

    Hout, Mariah S; Federspiel, William J

    2003-01-01

    We are developing hollow fiber-based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within the SAF and to use this information to guide the choice of design and operational parameters that maximize the SAF-based antibody removal rate. In this study, we formulated a simple mathematical model of SAF-based antibody removal and performed in vitro antibody removal experiments to test key predictions of the model. Our model revealed three antibody transport regimes, defined by the magnitude of the Damköhler number Da (characteristic antibody-binding rate/characteristic antibody diffusion rate): reaction-limited (Da /= 10). For a given SAF geometry, blood flow rate, and antibody diffusivity, the highest antibody removal rate was predicted for diffusion-limited antibody transport. Additionally, for diffusion-limited antibody transport the predicted antibody removal rate was independent of the antibody-binding rate and hence was the same for any antibody-antigen system and for any patient within one antibody-antigen system. Using SAF prototypes containing immobilized bovine serum albumin (BSA), we measured anti-BSA removal rates consistent with transport in the intermediate regime (Da approximately 3). We concluded that initial SAF development work should focus on achieving diffusion-limited antibody transport by maximizing the SAF antibody-binding capacity (hence maximizing the characteristic antibody-binding rate). If diffusion-limited antibody transport is achieved, the antibody removal rate may be raised further by increasing the number and length of the SAF fibers and by increasing the blood flow rate through the SAF.

  3. Monoclonal antibodies to Treponema Pallidum.

    NARCIS (Netherlands)

    H.J.M. van de Donk; J.D.A. van Embden; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractThree successive fusions of mouse myeloma cells and spleen lymphocytes of a mouse immunized with Treponema Pallidum resulted in one hybridoma producing anti T. pallidum antibodies for each fusion. The mice were immunized with live pallidum cells respectively 1, 3 and 5 months before fusi

  4. Antibody Isotype Switching in Vertebrates.

    Science.gov (United States)

    Senger, Kate; Hackney, Jason; Payandeh, Jian; Zarrin, Ali A

    2015-01-01

    The humoral or antibody-mediated immune response in vertebrates has evolved to respond to diverse antigenic challenges in various anatomical locations. Diversification of the immunoglobulin heavy chain (IgH) constant region via isotype switching allows for remarkable plasticity in the immune response, including versatile tissue distribution, Fc receptor binding, and complement fixation. This enables antibody molecules to exert various biological functions while maintaining antigen-binding specificity. Different immunoglobulin (Ig) classes include IgM, IgD, IgG, IgE, and IgA, which exist as surface-bound and secreted forms. High-affinity autoantibodies are associated with various autoimmune diseases such as lupus and arthritis, while defects in components of isotype switching are associated with infections. A major route of infection used by a large number of pathogens is invasion of mucosal surfaces within the respiratory, digestive, or urinary tract. Most infections of this nature are initially limited by effector mechanisms such as secretory IgA antibodies. Mucosal surfaces have been proposed as a major site for the genesis of adaptive immune responses, not just in fighting infections but also in tolerating commensals and constant dietary antigens. We will discuss the evolution of isotype switching in various species and provide an overview of the function of various isotypes with a focus on IgA, which is universally important in gut homeostasis as well as pathogen clearance. Finally, we will discuss the utility of antibodies as therapeutic modalities.

  5. Pharmacokinetics interactions of monoclonal antibodies.

    Science.gov (United States)

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  6. Development of Antibody Against Sulfamethazine

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  7. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

    Science.gov (United States)

    2016-02-01

    ECBC-TR-1339 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY...CHARACTERIZATION: CHARACTERIZATION OF AN MS2 HUMAN IGG ANTIBODY PRODUCED BY ANAPTYSBIO, INC. DARPA ATP Standardized Test Bed for Antibody...Characterization: Characterization of an MS2 human IgG antibody produced by AnaptysBio DARPA ATP Standardized Test Bed for Antibody

  8. Antibody Engineering for Pursuing a Healthier Future

    Science.gov (United States)

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

    2017-01-01

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

  9. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

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

  10. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2013-01-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  11. Platelet antigens and antibodies. Literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2014-07-01

    Full Text Available Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.

  12. [Neuroimmunological diseases associated with VGKC complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-05-01

    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  13. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  14. Chemical engineering of cell penetrating antibodies.

    Science.gov (United States)

    Zhao, Y; Lou, D; Burkett, J; Kohler, H

    2001-08-01

    Antibodies, being exquisitely specific tools in biology, are routinely used to detect and identify intra-cellular structures. However, current intra-cellular application of antibodies requires that the membrane be rendered leaky, resulting in the death of cells. Here, we present a novel method to allow antibodies to penetrate the cellular membrane of living cells without affecting cell viability. A peptide (MTS, membrane transport sequence) that facilitates transport across membranes has been site-specifically attached to antibodies. MTS-antibodies enter the living cells in culture and can be detected by immunofluorescence and ELISA after extraction. Cellular structures are visualized in living cells using a specific MTS-antibody. Antibodies with membrane penetrating properties can become an important tool for the study of intra-cellular processes in living cells. Furthermore, such membrane penetrating antibodies can be used to selectively stimulate or suppress functions of the cellular machinery.

  15. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  16. Recombinant bispecific antibodies for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Roland E KONTERMANN

    2005-01-01

    Bispecific antibodies can serve as mediators to retarget effector mechanisms to disease-associated sites. Studies over the past two decades have revealed the potentials but also the limitations of conventional bispecific antibodies. The development of recombinant antibody formats has opened up the possibility of generating bispecific molecules with improved properties. This review summarizes recent developments in the field of recombinant bispecific antibodies and discusses further requirements for clinical development.

  17. Engineered single chain antibody fragments for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Huhalov, A.; Chester, K. A. [Cancer Research UK Imaging and Targeting Group Royal Free, London (United Kingdom). Department of Oncology; University College Medical School Royal Free Campus, London (United Kingdom)

    2004-12-01

    An ideal molecule to deliver radioimmunotherapy (RIT) would be target specific and have prolonged residence time at high concentrations in the tumour with rapid clearance from normal tissues. It would also be non-immunogenic. These features can be rationally introduced into recombinant antibody-based proteins using antibody engineering techniques. This reviews focuses on the use of antibody engineering in the design and development of RIT molecules which have single chain Fv (scFv) antibody fragments as building blocks.

  18. Anti-DNA antibodies in SLE

    Energy Technology Data Exchange (ETDEWEB)

    Voss, E.W.

    1988-01-01

    This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.

  19. Antibodies to staphylococcal enterotoxin in laboratory personnel.

    OpenAIRE

    Jozefczyk, Z; Robbins, R N; Spitz, J M; Bergdoll, M S

    1980-01-01

    Eighty-five percent of laboratory personnel working with staphylococcal enterotoxin had antibodies to enterotoxin in their sera, whereas only 23% of the control group had antibodies specific for enterotoxin. Two persons who carried enterotoxin B-producing staphylococci in their noses, throats, or both, had antibodies to enterotoxin B in their sera.

  20. Nanoparticles for the delivery of therapeutic antibodies

    DEFF Research Database (Denmark)

    Sousa, Flávia; Castro, Pedro; Fonte, Pedro;

    2016-01-01

    INTRODUCTION: Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy...

  1. Heavy chain-only IgG2b llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of coreceptor- and CD4-binding sites.

    Science.gov (United States)

    Acharya, Priyamvada; Luongo, Timothy S; Georgiev, Ivelin S; Matz, Julie; Schmidt, Stephen D; Louder, Mark K; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R; Kwong, Peter D

    2013-09-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

  2. 抗前列腺特异抗原重链可变区单域抗体基因的构建及表达%Construction and expression of anti-prostate specific antigen VH sin gle domain antibody gene

    Institute of Scientific and Technical Information of China (English)

    袁建林; 王智; 武国军; 郝晓柯

    2001-01-01

    目的 扩增出抗前列腺特异抗原(PSA)单克隆抗体(MAb)的重链可变区(V H)单域抗体基因,并在大肠杆菌中表达. 方法 从分泌抗PSA mAb的杂交瘤细胞系E 4B7中提取总RNA,经RT-PCR扩增VH单域抗体基因,将其克隆到融合蛋白表达载体pGEX-4T-1中进行表达. 结果 VH单域抗体基因全长363 bp,含起始码和终止码.将其克隆 到pGEX-4T-1内,转化大肠杆菌DH5α,获得高效表达,表达量占全菌总蛋白质的38%,以包涵体形式存在.经初步纯化和复性后,用谷胱甘肽(GST)亲和色谱纯化,再经凝血酶水解获得抗PSA VH单域抗体. 竞争结合抑制实验证明,该表达产物具有前列腺癌细胞亲和活性. 结论  构建成功抗PSA的VH单域抗体,为进一步临床应用奠定基础.%AIM To amplify the VH single domain gene of anti-p rostate specific antigen (PSA) monoclonal antibody(mAb), and express it in E. coli. METHODS Total RNA was extracted from the hybridoma cell line E4B7, which se- creted McAb against PSA, and subjected to reverse transcri ption. The VH single domain antibody gene was amplified by PCR, clo ned into the prokaryotic fusion protein expression vector pGEX-4T-1 and expressed in E. c oli. RESULTS The VH single domain antibody gene consisted of 363 bp, which was cloned into an exp ression vector pGEX-4T-1 and transformed into E. coli DH5α. Clones harboring plasmid pGE X-VH produced a 40 kDa of fusion protein in E. coli at a level of 38% of the total cellular pro tein. After primary purification and renaturation, the fusion protein was further purified by GST-a ffinity chromatography. The fusion protein was digested with thrombin and the VH single domain antibod y was separated, the binding activity of anti-PSA VH single domain antibody was confirmed by competi tive binding inhibition assay in vitro. CONCLUSION Anti-PSA single dom ain antibody has been successfully constructed for the use in clinical studies.

  3. Antisperm antibodies and human reproduction.

    Science.gov (United States)

    Check, J H

    2010-01-01

    To present strategies in diagnosing and treating infertility related to antisperm antibodies. Antisperm antibodies (ASA) were detected on sperm using the direct immunobead (IBD) test. Treatments included intrauterine insemination (IUI) with pretreatment with chymotrypsin/galactose vs. in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). Intrauterine insemination with protein digestive enzyme treatment was much more effective than IUI without enzymatic therapy. However IVF with ICSI provided three times the pregnancy rate for males with sperm coated with ASA than IUI with chymotrypsin treated sperm. It is advisable to include measurement for ASA on the initial semen analysis. However, another option is to perform it initially only with an abnormal post-coital test. The decision for IUI with chymotrypsin pretreatment of the sperm vs. IVF with ICSI may depend on insurance and financial issues.

  4. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

  5. Phenotypic screening: the future of antibody discovery.

    Science.gov (United States)

    Gonzalez-Munoz, Andrea L; Minter, Ralph R; Rust, Steven J

    2016-01-01

    Most antibody therapeutics have been isolated from high throughput target-based screening. However, as the number of validated targets diminishes and the target space becomes increasingly competitive, alternative strategies, such as phenotypic screening, are gaining momentum. Here, we review successful phenotypic screens, including those used to isolate antibodies against cancer and infectious agents. We also consider exciting advances in the expression and phenotypic screening of antibody repertoires in single cell autocrine systems. As technologies continue to develop, we believe that antibody phenotypic screening will increase further in popularity and has the potential to provide the next generation of therapeutic antibodies.

  6. Bovine milk antibodies for health.

    Science.gov (United States)

    Korhonen, H; Marnila, P; Gill, H S

    2000-11-01

    The immunoglobulins of bovine colostrum provide the major antimicrobial protection against microbial infections and confer a passive immunity to the newborn calf until its own immune system matures. The concentration in colostrum of specific antibodies against pathogens can be raised by immunising cows with these pathogens or their antigens. Immune milk products are preparations made of such hyperimmune colostrum or antibodies enriched from it. These preparations can be used to give effective specific protection against different enteric diseases in calves and suckling pigs. Colostral immunoglobulin supplements designed for farm animals are commercially available in many countries. Also, some immune milk products containing specific antibodies against certain pathogens have been launched on the market. A number of clinical studies are currently in progress to evaluate the efficacy of immune milks in the prevention and treatment of various human infections, including those caused by antibiotic resistant bacteria. Bovine colostrum-based immune milk products have proven effective in prophylaxis against various infectious diseases in humans. Good results have been obtained with products targeted against rotavirus, Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutans, Cryptosporidium parvum and Helicobacter pylori. Some successful attempts have been made to use immune milk in balancing gastrointestinal microbial flora. Immune milk products are promising examples of health-promoting functional foods, or nutraceuticals. This review summarises the recent progress in the development of these products and evaluates their potential as dietary supplements and in clinical nutrition.

  7. Broadly Neutralizing Antibodies for HIV Eradication.

    Science.gov (United States)

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  8. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic...

  9. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories.Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases,inflammatory diseases,cancer,and other immune-associated diseases.This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis,an inflammatory disease of the heart,could be a novel approach in the future.In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis,we,through a significant amount of literature research both domestic and abroad,developed a systematic elaboration of monoclonal antibodies,pathogenesis of myocarditis,and application of monoclonal antibodies in myocarditis.This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future.Under conventional therapy,myocarditis is typically associated with congestive heart failure as a progressive outcome,indicating the need for alternative therapeutic strategies to improve long-term results.Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis,we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above.However,several issues remain.The technology on howto make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues.If we are to further stimulate

  10. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies.

    Science.gov (United States)

    Gown, A M; Vogel, A M

    1982-11-01

    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

  11. The effect of oxide Bi2O3 doping on the levitation force of single domain YBCO bulk superconductors%Bi2O3氧化物掺杂对单畴YBCO超导块材磁悬浮力的影响

    Institute of Scientific and Technical Information of China (English)

    王妙; 杨万民; 马俊; 唐艳妮; 张晓菊; 王高峰

    2012-01-01

    In this paper, single domain YBCO superconductors, with compositions of Bi2O3:Y5BaCuO5=x:(1 -x), (x=0.1; 0.3; 0,5; 0.7; 0.9; 2, units: wt%), have been successfully fabricated by the top-seeding infiltration and growth process (TS1G). The effect of the different doping amounts of Bi2O3 particles on the growth morphology, the magnetic levitation force properties and microstruclure of single domain YBCO bulk superconductors has been investigated. The results show that the nanoscale Y2Ba4CuBiOx(YBi2411) particles which were formatted by oxide Bi2Oi doping can work as effective flux pinning centers to improve the properties of YBCO bulk samples. It is found that the levitation force is increasing from 7 N to 25 N with the increasing of Bi2O3 addition from 0.1wt% to 0.7wt%, and decreasing from 25 N to 6 N with the increasing of Bi2O3 addition from 0.7wt% to 2wt%. The results are very important for further study in flux pinning of oxide Bi2O3 doping and improvement in the properties of YBCO bulk superconductors.%采用顶部籽晶熔渗工艺(TSIG)制备出了配比为Bi2O3∶Y2BaCuO5=x∶(1-x)的系列单畴YBCO超导块材(其中x=0.1,0.3,0.5,0.7,0.9,2,单位为wt%),并且研究了不同比例的氧化物Bi2O3掺杂对样品的生长形貌、磁悬浮力以及其微观结构的影响.实验结果表明了,Bi2O3粒子的掺杂在样品中生成Y2Ba4CuBiOx(YBi2411)纳米粒子从而可以有效地提高样品的磁悬浮性能.当Bi2O3粒子掺杂量x从0.1wt%(质量分数,下同)增加到0.7wt%时,样品的磁悬浮力从7N增加到25N;当其掺杂量从0.7wt%增加到2wt%时,样品的磁悬浮力从25N降低到6N.该实验结果对于我们进一步研究氧化物掺杂对磁通钉扎作用的影响以及提高YBCO超导块材的性能有着重要的影响.

  12. Effect of assembled bar magnet configuration on levitation force of single domain GdBCO bulk superconductor%条状永磁体的组合形式及间距对单畴GdBCO超导体磁悬浮力的影响

    Institute of Scientific and Technical Information of China (English)

    马俊; 杨万民

    2011-01-01

    By measuring the levitation forces between a single domain GdBCO bulk superconductor and assembled bar magnets (ABM) in different configurations at liquid nitrogen temperature, the effects of ABM configurations on the levitation force of single domain GdBCO bulk superconductor are investigated. The maximum levitation force is obtained at the same vertical gap distance Z = 5 mm between the superconductor and the ABM for configurations with different lateral gap distance(D)between the magnets of the ABM. It is found that 1) for the ABM consisting of 3 bar magnet, the levitation force of the GdBCO bulk decreases from 22.8N to 9.7N with the D value increasing from 0 to 30 mm, when the magnetic pole N of the middle magnet is pointed upwards and the directions of magnetic pole N of two side magnets are pointed to the middle magnet in horizontal direction; the levitation force of the GdBCO bulk increases first from 9.2N to 13.9N and then decreases tol0.4 N with D value increasing from 0 to 30ram, if the magnetic pole N of the middle magnet is pointed upwards and the directions of magnetic pole N of two side magnets are pointed downwards;2)for the ABM consisting of 2 bar magnets, the levitation force of the GdBCO bulk decreases from l 1.2N to 1.2N with D value increasing from 0 to 30mm, when the directions of magnetic pole N of two side magnets are pointed upwards ; the levitation force of the GdBCO bulk increases first from 6. ON to 6.8N and then decreases to 2.9N with D value increasing from 0 to 30mm, if the directions of magnetic pole N of two magnets are anti-parallel in horizontal direction; 3) for the ABM consisting of only 1 bar magnets, D = 0, and the levitation force of the GdBCO bulk is about 9.5N. The results indicate that the magnet configuration and its detailed parameters of ABM are very important for improving the levitation force of a superconductor and helpful for designing and application based on the superconducting magnetic levitation

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

    Science.gov (United States)

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

    2011-11-01

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

  14. Surface activity of a monoclonal antibody.

    Science.gov (United States)

    Mahler, Hanns-Christian; Senner, Frank; Maeder, Karsten; Mueller, Robert

    2009-12-01

    The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation.

  15. Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

    Science.gov (United States)

    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. PMID:23658521

  16. 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.

  17. 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.

  18. Controlled delivery of antibodies from injectable hydrogels.

    Science.gov (United States)

    Fletcher, Nathan A; Babcock, Lyndsey R; Murray, Ellen A; Krebs, Melissa D

    2016-02-01

    Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

  19. Antibody response to measles immunization in India*

    OpenAIRE

    Job, J. S.; John, T J; Joseph, A.

    1984-01-01

    Antibody response to measles vaccine was measured in 238 subjects aged 6-15 months. Seroconversion rates ranged from 74% at 6 months of age to 100% at 13-15 months; the differences in age-specific rates were not statistically significant. The postimmunization antibody titres increased with increasing age of the vaccinee. Seroconversion rates and antibody titres in 49 subjects with grades I and II malnutrition were not significantly different from those in the 189 normal subjects.

  20. In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.

    Science.gov (United States)

    Kopsidas, George; Roberts, Anthony S; Coia, Gregory; Streltsov, Victor A; Nuttall, Stewart D

    2006-11-15

    We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.

  1. Antiphospholipid Antibodies and Systemic Scleroderma

    Directory of Open Access Journals (Sweden)

    Awa Oumar Touré

    2013-03-01

    Full Text Available Objective: Antiphospholipid antibodies (APLs could be associated with an increased risk of vascular pathologies in systemic scleroderma. The aim of our study was to search for APLs in patients affected by systemic scleroderma and to evaluate their involvement in the clinical manifestations of this disease. Materials and Methods: We conducted a cross-sectional descriptive study, from January 2009 until August 2010, with patients received at the Department of Dermatology (Dakar, Senegal. Blood samples were taken at the hematology laboratory and were analyzed for the presence of APLs. Results: Forty patients were recruited. Various types of either isolated or associated APLs were found in 23 patients, i.e. 57.5% of the study population. The most frequently encountered antibody was IgG anti-β2 GPI (37.5% of the patients, followed by anticardiolipins (17.5% and lupus anticoagulants (5%. No statistically significant association of positive antiphospholipid-related tests to any of the scleroderma complications could be demonstrated. Conclusion: A high proportion of patients showing association of systemic scleroderma and APLs suggests the presence of a morbid correlation between these 2 pathologies. It would be useful to follow a cohort of patients affected by systemic scleroderma in order to monitor vascular complications following confirmation of the presence of antiphospholipid syndrome.

  2. Exceptional Antibodies Produced by Successive Immunizations.

    Directory of Open Access Journals (Sweden)

    Patricia J Gearhart

    2015-12-01

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

  3. 6th Annual European Antibody Congress 2010

    Science.gov (United States)

    2011-01-01

    The 6th European Antibody Congress (EAC), organized by Terrapinn Ltd., was held in Geneva, Switzerland, which was also the location of the 4th and 5th EAC.1,2 As was the case in 2008 and 2009, the EAC was again the largest antibody congress held in Europe, drawing nearly 250 delegates in 2010. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations and representatives from the US Food and Drug Administration (FDA). The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations of golimumab (Simponi®) and canakinumab (Ilaris®) by Johnson & Johnson and Novartis, respectively, updates on antibodies in late clinical development (obinutuzumab/GA101, farletuzumab/MORAb-003 and itolizumab/T1 h, by Glycart/Roche, Morphotek and Biocon, respectively) and success rates for this fast-expanding class of therapeutics (Tufts Center for the Study of Drug Development). Case studies covering clinical progress of girentuximab (Wilex), evaluation of panobacumab (Kenta Biotech), characterization of therapeutic antibody candidates by protein microarrays (Protagen), antibody-drug conjugates (sanofi-aventis, ImmunoGen, Seattle Genetics, Wyeth/Pfizer), radio-immunoconjugates (Bayer Schering Pharma, Université de Nantes) and new scaffolds (Ablynx, AdAlta, Domantis/GlaxoSmithKline, Fresenius, Molecular Partners, Pieris, Scil Proteins, Pfizer, University of Zurich) were presented. Major antibody structural improvements were showcased, including the latest selection engineering of the best isotypes (Abbott, Pfizer, Pierre Fabre), hinge domain (Pierre Fabre), dual antibodies (Abbott), IgG-like bispecific antibodies (Biogen Idec), antibody epitope mapping case studies (Eli Lilly), insights in FcγRII receptor (University of Cambridge), as well as novel tools for antibody fragmentation (Genovis). Improvements

  4. Isoimmunization with anti-U antibody.

    Science.gov (United States)

    Turner, R J; Holder, W T; McCord, D L

    1984-03-01

    Isoimmunization with anti-U antibody is a rare but significant cause of hemolytic disease in black newborns. In this case report, an lgG antibody stimulated by fetomaternal transfusion produced a positive direct Coombs' test on cord blood but not neonatal hyperbilirubinemia. A review of the literature suggests the pathophysiology is similar to Rh isoimmunization. The anti-U antibody may develop as a result of pregnancy or blood transfusion in the 1.2 percent of American blacks who are at risk for developing the antibody. The principles of treatment employed in Rh isoimmunization can be successfully used in isoimmunization due to anti-U.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

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

  7. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  8. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  9. Antibodies to watch in 2017.

    Science.gov (United States)

    Reichert, Janice M

    Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6-9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other 'antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these studies may

  10. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  11. Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

  12. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  13. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  14. Anti-influenza M2e antibody

    Science.gov (United States)

    Bradbury, Andrew M.

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  15. Receptor antibodies as novel therapeutics for diabetes

    DEFF Research Database (Denmark)

    Ussar, Siegfried; Vienberg, Sara Gry; Kahn, C Ronald

    2011-01-01

    Antibodies to receptors can block or mimic hormone action. Taking advantage of receptor isoforms, co-receptors, and other receptor modulating proteins, antibodies and other designer ligands can enhance tissue specificity and provide new approaches to the therapy of diabetes and other diseases....

  16. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  17. Anti-influenza M2e antibody

    Science.gov (United States)

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  18. Photonic crystal fiber based antibody detection

    OpenAIRE

    Duval, A.; Lhoutellier, M; Jensen, J. B.; Hoiby, P E; Missier, V; Pedersen, L. H.; Hansen, Theis Peter; Bjarklev, Anders Overgaard; Bang, Ole

    2004-01-01

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy and the use of a transversal illumination setup.

  19. Antibody-drug conjugates: Intellectual property considerations.

    Science.gov (United States)

    Storz, Ulrich

    2015-01-01

    Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.

  20. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  1. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    Science.gov (United States)

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  2. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B

    2004-01-01

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy and ...

  3. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    1989-01-01

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  4. Methods for Selecting Phage Display Antibody Libraries.

    Science.gov (United States)

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

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

    Science.gov (United States)

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

    2016-04-01

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

  6. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors...... such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  7. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  8. Monoclonal antibodies in chronic lymphocytic leukemia.

    Science.gov (United States)

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  9. Antibody-based resistance to plant pathogens.

    Science.gov (United States)

    Schillberg, S; Zimmermann, S; Zhang, M Y; Fischer, R

    2001-01-01

    Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

  10. Utilización de los test de Fluorescencia Polarizada (FP y Elisa de Competencia (C-Elisa en el diagnóstico de brucelosis de camélidos The use of polarized fluorescence assay (PF and competitive ELISA test (C-Elisa for the diagnosis of Brucellosis in South American camelids

    Directory of Open Access Journals (Sweden)

    X Rojas

    2004-01-01

    Full Text Available En Chile, como en muchos países de Latinoamérica, la brucelosis es una zoonosis presente, representando no sólo un factor de riesgo para las explotaciones pecuarias sino también para quienes laboran y hacen usufructo de ella, especialmente por el consumo de leche cruda y sus derivados. Como la Brucella afecta a diferentes especies de animales, el objetivo de este trabajo fue utilizar las técnicas de unión primaria, como son Fluorescencia Polarizada (FP y Elisa de Competencia (C-Elisa, para la detección de anticuerpos anti-Brucella en suero de camélidos y, comparar sus respuestas con las pruebas de Rosa de Bengala (RB, Aglutinación Lenta en Tubo (SAT y Fijación de Complemento (FC en atención a que estas especies representan un potencial pecuario de cierta trascendencia. Para ello se analizaron 336 sueros, de los cuales 315 pertenecían a llamas y 21 a alpacas, provenientes de un predio de la Novena Región clínica y serológicamente libre de Brucella. Los resultados obtenidos señalan que el umbral de positividad para FP, calculado según la mediana del último percentil, fue de 98.7 milipolarizaciones (mP y, el corte para C-Elisa con el mismo método fue de 32% de Inhibición (% I. Todas las muestras analizadas dieron resultados negativos a RB y SAT. Los resultados con FC no pudieron ser considerados, por cuanto un alto porcentaje de las muestras fueron anticomplementarias. Con las técnicas de C-Elisa y FP, dos muestras reaccionaron positivamente en cada uno de los test con valores en el nivel de corte o levemente superiores. Se discute la significación de ellas.Brucellosis is a zoonotic disease representing a risk contact with, or consuming, their raw milk and raw dairy several animal species, including camelids, the aim of this Polarization assay (PF and Competitive ELISA (C-ELISA, along with conventional serological tests such as the Rose Complement Fixation test (CF. A total of 336 serum samples herd of the IXth Region (Chile

  11. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  12. Antiphospholipid Antibodies in Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Ioannis Parodis

    Full Text Available Lupus nephritis (LN is a major manifestation of systemic lupus erythematosus (SLE. It remains unclear whether antiphospholipid antibodies (aPL alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204 or without (n = 294 LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous, before and after induction treatment (short-term outcomes. Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR and the Chronic Kidney Disease (CKD stage, after a median follow-up of 11.3 years (range: 3.3-18.8. Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all, but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are

  13. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries.

  14. 抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定%Preparation and characterization of recombinant VHH antibody against H5N1 hemagglutinin from avian influenza virus

    Institute of Scientific and Technical Information of China (English)

    夏立亮; 吴标; 程亚庭; 包文静; 赵国屏; 王颖

    2012-01-01

    目的:获得具有中和活性、高特异性和稳定性的抗H5N1禽流感病毒血凝素蛋白(HA)的羊驼重链单域(VHH)抗体.方法:利用pET-22b表达载体诱导表达抗H5N1禽流感病毒HA VHH抗体蛋白,以包涵体形式表达的VHH抗体蛋白采用最优复性方法进行复性后,获得高纯度的VHH抗体,分别采用ELISA法鉴定VHH抗体的亲和力和热稳定性,采用血凝抑制实验鉴定抗体的特异性和体外中和活性.结果:经复性的抗H5N1禽流感HA VHH抗体对H5N1禽流感病毒HA具有良好的特异性.通过对三种不同复性方法比较,利用柱上复性的VHH23抗体具有较好的热稳定性,亲和力为9.1×10-7 mol/L,同时对H5N1禽流感病毒HA具有良好的体外中和活性.结论:实验结果表明通过原核表达获得具有较好中和活性、特异性及稳定性的抗H5N1禽流感病毒VHH抗体,为进一步开展抗体的体内病毒中和试验奠定良好基础.%Objective; To prepare and characterize camelid VHH antibody against hemagglutinin from H5N1 avian influenza virus. Methods: Recombinant expression vector pET-22b-VHH23 was constructed and VHH antibody against hemagglutinin from H5N1 a-vian influenza virus was expressed in E. Coli BL2l( DE3 ) strain by IPTG induction. As VHH antibody protein was expressed in inclusion body,there different refolding methods were compared for the refolding of VHH antibody in which the optimal methods were adopted for preparation of VHH antibody. The activity and thermal stability of VHH antibodies were tested by ELISA while hemagglutination-inhibition assay was perfomed to determine the specificity and neutralizing activity. Results; By using on-column refolding procedure, VHH antibody has been obtained with highest yield and good quality. This antibody specifically recognized hemagglutinin derived from H5N1 avian influenza virus as well as in vitro inhibition against hemagglutinin-mediated red blood cell agglutination. The affinity constant of VHH

  15. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  16. Next generation of antibody therapy for cancer

    Institute of Scientific and Technical Information of China (English)

    Zhenping Zhu; Li Yan

    2011-01-01

    Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective altematives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-basod regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.

  17. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

  18. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene

    2012-02-01

    Full Text Available Biologics such as monoclonal antibodies (mAb and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.

  19. Antibodies as predictors of complex autoimmune diseases.

    Science.gov (United States)

    Vojdani, A

    2008-01-01

    Emerging evidence has suggested environmental factors such as infections and xenobiotics and some dietary proteins and peptides in the pathogenesis of many autoimmune diseases. Considering the fact that autoantibodies can often be detected prior to the onset of a disease, in this study an enzyme immunoassay was used for measurement of antibodies against different highly purified antigens or synthetic peptides originating not only from human tissue, but also from cross-reactive epitopes of infectious agents, dietary proteins and xenobiotics. The measurement of antibodies against a panel of antigens allows for identification of patterns or antibody signatures, rather than just one or two markers of autoimmunity, thus establishing the premise for increased sensitivity and specificity of prediction, as well as positive predictive values. This panel of different autoantibodies was applied to 420 patients with different autoimmune diseases, including pernicious anemia, celiac disease, thyroiditis, lupus, rheumatoid arthritis, osteoarthritis, Addison's disease, type 1 diabetes, cardiovascular disease and autoimmunity, which are presented in this article. In all cases, the levels of these antibodies were significantly elevated in patients versus controls. Antibody patterns related to neuroautoimmune disorders, cancer, and patients with somatic hypermutation will be shown in a subsequent article. We believe that this novel 96 antigen-specific autoantibody or predictive antibody screen should be studied for its incorporation into routine medical examinations. Clinicians should be aware that the detection of antibodies should not automatically mean that a patient will definitely become ill, but would rather give a percentage of risk for autoimmune disease over subsequent months or years.

  20. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  1. Combinatorial antibody libraries: new advances, new immunological insights.

    Science.gov (United States)

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  2. Distinct Therapeutic Mechanisms of Tau Antibodies

    Science.gov (United States)

    Funk, Kristen E.; Mirbaha, Hilda; Jiang, Hong; Holtzman, David M.; Diamond, Marc I.

    2015-01-01

    Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo. PMID:26126828

  3. Advances in recombinant antibody manufacturing.

    Science.gov (United States)

    Kunert, Renate; Reinhart, David

    2016-04-01

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

  4. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  5. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    Science.gov (United States)

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    Science.gov (United States)

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  7. Uses of monoclonial antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  8. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  9. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  10. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  11. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  12. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  13. Serum Antibodies to Glycans in Peripheral Neuropathies.

    Science.gov (United States)

    Sonnino, Sandro; Chiricozzi, Elena; Ciampa, Maria Grazia; Mauri, Laura; Prinetti, Alessandro; Toffano, Gino; Aureli, Massimo

    2017-03-01

    In peripheral neuropathies, such as sensorimotor neuropathies, motor neuron diseases, or the Guillain-Barré syndrome, serum antibodies recognizing saccharide units, portion of oligosaccharides, or oligosaccharide chains, have been found. These antibodies are called anti-glycosphingolipid (GSL) or anti-ganglioside antibodies. However, the information on the aglycone carrying the hydrophilic oligosaccharide remains elusive. The absolute and unique association of GSL to the onset, development and symptomatology of the peripheral neuropathies could be misleading. Here, we report some thoughts on the matter.

  14. Preparation, Characterization, and Application of Antiharpinxoo Antibody

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  15. 中子辐照对GdBa2Cu3O7-δ单畴超导性能的影响%Effects of Neutron Irradiation on Superconducting Properties of GdBa2Cu3O7-δ Single Domain Superconductor

    Institute of Scientific and Technical Information of China (English)

    张红; 李广; 周桃飞; 刘勇; 李晓光; 陈羽

    2007-01-01

    研究了中子辐照对GdBa2Cu3O7-δ单畴超导性能的影响,使用的辐照剂量为1015 n/cm2.通过高分辨电镜观察发现中子辐照产生球形的缺陷和小的点缺陷,这些球形缺陷的尺寸在4~7 nm,与高温超导相干长度相当,并且在退火过程中稳定存在,而小的点缺陷在退火过程中消失.磁测量结果显示中子辐照使样品的超导电性严重退化,临界电流密度下降并且鱼尾峰效应几乎消失.然而对辐照的样品退火处理后,其超导电性明显改善,临界电流密度显著提高并超过了未经辐照的样品,鱼尾峰也向高场移动.这些结果表明中子辐照和退火处理使GdBa2Cu3O7-δ单畴样品中引入了有效的磁通钉扎中心(即球型的缺陷)从而导致其临界电流密度大幅提高.%The effects of neutron irradiation with a fluence of 1015 n/cm2 on the superconducting properties of GdBa2Cu3O7-δ single domain sample were studied. The point and cascade defects produced by the neutron irradiation were observed by high-resolution transmission electron microscopy. The cascade defects were found to have the sizes of about 4-7 nm which is comparable to the coherence lengths of high temperature superconductors. The point defects disappear while the cascades still exist through thermal annealing.The temperature dependence of magnetization for the magnetic field parallel to the crystallographic c axis shows that the neutron irradiation leads to a dramatic degradation of superconductivity for the as-irradiated sample, a decrease of critical current density (Jc), and the weakening of the fishtail effect in the Jc versus magnetic induction B curve. However, for the as-irradiated sample annealed in the flowing oxygen atmosphere, it shows that Jc under high magnetic fields is greatly enhanced, the fishtail shifts towards higher magnetic fields, and its superconductivity is partially recovered as well due to the remaining effective pinning centers of the cascades

  16. Synthesis of bifunctional antibodies for immunoassays.

    Science.gov (United States)

    DeSilva, B S; Wilson, G S

    2000-09-01

    The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.

  17. Solid phase synthesis of bifunctional antibodies.

    Science.gov (United States)

    DeSilva, B S; Wilson, G S

    1995-12-15

    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

  18. Patient-Derived Antibody Targets Tumor Cells

    Science.gov (United States)

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  19. Correlation between antisperm antibody and Semen parameters

    Directory of Open Access Journals (Sweden)

    farhad Shahsavar

    2004-08-01

    Conclusion: Antisperm antibodies can disrupt normal sperm function by damaging sperm motility. Therefore, it can be suggest that patients with sperm motility than 50% should become Candidate of ASA assay.

  20. Nanobodies - the new concept in antibody engineering

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-17

    Jun 17, 2009 ... form the basis of a new generation of therapeutic antibodies which were named Nanobodies ..... cular characteristics and structural stabilities, but also to ... Hmila I, Ben Abdallah RBA, Saerensb D, Benlasfard Z, Conrathb K, El.

  1. evaluation of an antigen-antibody

    African Journals Online (AJOL)

    GB

    BACKGROUND: Development of “combination” assays detecting in parallel, within a ... METHODS: We compared the Monolisa® HCV Antigen-Antibody Ultra (Bio-Rad Laboratories Limited, ... mediated response in these patients, a rapid viral.

  2. Sneddon's syndrotne with anticardiolipin antibodies complications ...

    African Journals Online (AJOL)

    systemic complications, which included stroke, habitual abortions ... nisone she suffered an acute exacerbation of her previ- .... Antiphospholipid antibodies: a brief. reVIew. Ann Neuro11989; 26: 386-389. 13. Laskin CA ... Ischemic stroke asso-.

  3. Enhanced Phagocytosis and Antibody Production by Tinospora ...

    African Journals Online (AJOL)

    SERVER

    2008-01-18

    Jan 18, 2008 ... antibody production through in vitro and in vivo studies. MATERIALS AND METHODS. Collection of plant .... and were fed orally to each animal for 14 days. After 14 days of .... Rubiadin: A new antioxidant from Rubia cardifolia.

  4. Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  5. Therapeutic monoclonal antibody for Sporotrichosis

    Directory of Open Access Journals (Sweden)

    Sandro eAlmeida

    2012-11-01

    Full Text Available Sporotrichosis is a chronic subcutaneous mycosis that affects either humans or animals and occurs worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits a considerable genetic variability, where recently, was suggesting that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to the cutaneous forms but, recently, occurrences of more severe clinical forms of this mycosis were described, especially among immunocompromized individuals. The immunological mechanisms involved in prevention and control of sporotrichosis are still not very well understood. Some works suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus have not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induce a specific humoral response in infected animals, mainly against the 70-kDa molecules, indicating a possible participation of specific antibodies to this molecule in infection control. In an other work of the our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell deficient mice were used. Drugs of choice in the treatment of sporothrichosis require long periods and frequently relapses are observed, mainly in immunocompromized patients. The strong protection induced by mAb against a 70-kDa glycoprotein makes it a strong candidate for a

  6. Mepanipyrim haptens and antibodies with nanomolar affinity

    OpenAIRE

    Esteve Turrillas, Francesc Albert; Mercader Badia, Josep Vicent; Agulló, Consuelo; Abad Somovilla, Antonio; Abad Fuentes, Antonio

    2013-01-01

    Mepanipyrim is an anilinopyrimidine fungicide used worldwide for crop protection. With the aim of developing useful immunoreagents for mepanipyrim immunoanalysis, two new functionalized derivatives were prepared and antibodies were generated. Affinity and specificity were assessed by direct and indirect competitive ELISA using homologous and heterologous conjugates. Although all antibodies were selective for the target analyte, the immunizing hapten structure was revealed as a determinant for...

  7. Neutralizing antibodies to Haemophilus ducreyi cytotoxin.

    OpenAIRE

    Lagergård, T; Purvén, M

    1993-01-01

    Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single o...

  8. Primary Antiphospholipid Antibody Syndrome: A Case Report.

    Science.gov (United States)

    Kadeli, Deepak K; Hanjagi, Siddaraya Y

    2015-10-01

    Primary Antiphospholipid antibody syndrome is a rare disease associated with thromboembolic events which may affect either the arterial or the venous vasculature. It presents with an increased risk of thrombosis in pregnant woman leading to repeated fetal losses. We present here a case of primary antiphospholipid antibody syndrome in young women who had previous event of gangrene of toes leading to their amputation and repeated fetal losses.

  9. Structure and specificity of lamprey monoclonal antibodies

    OpenAIRE

    Herrin, Brantley R.; Alder, Matthew N; Roux, Kenneth H.; Sina, Christina; Ehrhardt, Götz R. A.; Boydston, Jeremy A.; Turnbough, Charles L.; Cooper, Max D.

    2008-01-01

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores...

  10. Autoimmune encephalitis: Clinical diagnosis versus antibody confirmation

    Directory of Open Access Journals (Sweden)

    Asha Caroline Cyril

    2015-01-01

    Full Text Available Context: Autoimmune encephalitis is a heterogeneous disorder which is being diagnosed with increasing frequency. The diagnosis of these disorders is based on the detection of autoantibodies and characteristic clinical profiles. Aims: We aimed to study the antibody profile in encephalitis patients with suspected autoimmune etiology presenting to a tertiary care center. Settings and Design: The subjects were selected by screening all patients with clinical profile suggesting autoimmune encephalitis admitted in the neuromedical intensive care unit (ICU of a tertiary care center in South India. Materials and Methods: Patients who fulfilled modified Zuliani et al.′s, criteria for autoimmune encephalitis were identified during the period December 2009-June 2013. Blood samples from these subjects were screened for six neuronal antibodies. Statistical analysis used: Chi-square test was applied to compare the antibody positive and negative patients. Results: Out of 1,227 patients screened, 39 subjects (14 males: 25 females were identified with a mean age of 15.95 years and 19 cases were assessed in the acute and 20 in the convalescent phase of the illness. Seizure (87.8 % was the most common presenting symptom; status epilepticus occurred in 23 (60.5% patients during the course of the illness. Fourteen (35.9% patients were N-methyl-D-aspartate receptor (NMDAR antibody-positive and all were negative for the other antibodies tested. Conclusions: One-third of patients presenting with acute noninfective encephalitis would be positive for NMDAR antibodies with the remaining two-thirds with clinically suspected autoimmune encephalitis being antibody-negative. There are few markers in the clinical and investigative profiles to distinguish antibody-positive and -negative patients.

  11. Discovery of functional antibodies targeting ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I; Gardener, Matthew J; Williams, Wendy A

    2015-04-01

    Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

  12. Standardization of anti-DNA antibody assays.

    Science.gov (United States)

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  13. Antibody-mediated resistance against plant pathogens.

    Science.gov (United States)

    Safarnejad, Mohammad Reza; Jouzani, Gholamreza Salehi; Tabatabaei, Meisam; Tabatabaie, Meisam; Twyman, Richard M; Schillberg, Stefan

    2011-01-01

    Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants.

  14. Modern affinity reagents: Recombinant antibodies and aptamers.

    Science.gov (United States)

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  16. IMPORTANCE OF RESEARCH HLA ANTIBODIES CLASS I AND II, AND MICA ANTIBODIES IN KIDNEY TRANSPLANTATION

    Directory of Open Access Journals (Sweden)

    M. Sh. Khubutia

    2011-01-01

    Full Text Available The purpose of this study was to investigate the occurrence of HLA and MICA antibodies in patients from the waiting list for kidney transplantation and their influence on the course of post-transplant period. Determination of HLA antibodies class I and II, and MICA antibodies was performed on a platform of Luminex (xMAP-tech- nology using sets LABScreen ONE LAMBDA (U.S.. A total of 156 patients from the waiting list for kidney transplantation. Revealed the presence of HLA and MICA antibodies in the serum of 31.4% of patients. Regraf- ted patients increased the content of antibodies to the antigens of HLA system was noted in 88.2% of cases, 47% met the combination of antibodies to the I, II classes and MICA. In patients awaiting first kidney transplantation, HLA and MICA antibodies were determined in 23.7% of cases. The presence of pretransplant HLA and MICA antibodies had a significant influence on the course of post-transplant period. Patients with the presence of HLA and MICA in 50% of cases delayed graft function. Sessions of plasmapheresis can reduce the concentration of HLA and MICA antibodies on average by 61.1%. 

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

    Science.gov (United States)

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

    2014-01-25

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

  18. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  19. Antiphospholipid Antibody Syndrome Presenting with Unilateral Adrenal Hemmorhage.

    Science.gov (United States)

    Ullah, Kifayat; Butt, Ghias; Neopane, Sippy; Arshi, Shahana

    2016-06-01

    The antiphospholipid antibody syndrome presents with vascular thrombosis which involve both arterial and venous systems. The clinical presentation of antiphospholipid antibody syndrome includes obstetric complications leading to recurrent abortions, presence of circulating antibodies against phospholipids, and multi-organ thromboembolisms. We report a case of a patient who presented with unilateral adrenal hemorrhage and subsequently found to have antiphospholipid antibody syndrome and lupus nephritis.

  20. Coming-of-Age of Antibodies in Cancer Therapeutics.

    Science.gov (United States)

    Ayyar, B Vijayalakshmi; Arora, Sushrut; O'Kennedy, Richard

    2016-12-01

    Antibody-based therapies have garnered considerable success in recent years. This is due to the availability of strategies to successfully engineer antibodies into humanized forms, better understanding of the biological processes involved in cancer development, the availability of novel recombinant antibody formats, better antibody selection platforms, and improved antibody conjugation methodologies. Such achievements have led to an explosion in the generation of antibodies and antibody-associated constructs for the treatment of cancer and other diseases. In this review, we critically assess recent trends in the development and applications of bispecific antibodies (bsAbs), antibody-drug conjugates (ADCs), and immune checkpoint inhibitors (ICIs) as cancer therapeutics. We also highlight recent US FDA approvals and clinical trials of antibody-based cancer therapies.

  1. Detección de anticuerpos para BVDV y BoHV-1 en llamas de la región de Tandil - Provincia de Buenos Aires Detection of antibodies to BVDV and BoHV-1 in llamas from Tandil - Province of Buenos Aires

    Directory of Open Access Journals (Sweden)

    P.E. Morán

    2010-12-01

    Full Text Available La llama (Lama glama es la especie predominante de camélidos sudamericanos de la República Argentina, con una población aproximada de 200.000 animales. El aumento de la actividad productiva ha incrementado el transporte y la distribución de animales hacia diferentes regiones del país. Como consecuencia, estas especies interactúan con el ganado doméstico posibilitando la diseminación de agentes infecciosos propios y comunes entre especies. Considerando que los camélidos sudamericanos son susceptibles a la infección con pestivirus y herpesvirus, se planteó realizar un relevamiento serológico de anticuerpos neutralizantes para el virus de la diarrea viral bovina (BVDV y el herpesvirus bovino 1 (BoHV-1. Se analizaron 49 animales de dos rebaños de llamas de la región de Tandil mediante la técnica de seroneutralización sobre cultivos celulares de la línea Madin Darby Bovine Kidney. El 22% (11/49 de los animales expresaron seropositividad a BoHV-1, mientras que el 2% (1/49 fue positivo a BVDV. Estos resultados sugieren circulación viral en la población analizada. Es necesario el monitoreo continuo en las poblaciones de estos rumiantes para identificar factores de riesgo involucrados en la epidemiología de estos agentes virales, considerando los potenciales efectos sobre los programas de control y erradicación.Llama (Lama glama is the predominant species of South American camelids in Argentine Republic, with an estimated population of 200.000 animals. The increasing of productive activity raised the animals transport and distribution to different regions of the country. As a result, the direct contact of these species with cattle increase opportunities for spreading own and common infectious agents among species. Considering that American camelids are susceptible to pestivirus and herpesvirus infections, it was proposed to carry out a serological searching of neutralizing antibodies to bovine viral diarrhea (BVDV and bovine

  2. Relevance of anti-myelin antibodies in Multiple Sclerosis

    OpenAIRE

    2005-01-01

    Antibodies directed against myelin antigens have been described in multiple sclerosis (MS). Although anti-myelin antibodies have been implicated in central nervous system (CNS) demyelination, it is unclear to what extent anti-myelin antibodies contribute to MS pathogenesis. In this dissertation, the role of antibodies in MS and in the animal model experimental allergic encephalomyelitis (EAE) is addressed in eight chapters: Chapter 1: A review on antibodies, complement and Fc receptors in MS ...

  3. Cross-reactive broadly neutralizing antibodies: timing is everything

    OpenAIRE

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  4. Cross-reactive broadly neutralizing antibodies: timing is everything.

    Science.gov (United States)

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  5. 辅助永磁体的引入方式对单畴GdBCO超导块材磁场分布及其磁悬浮力的影响%The effects of magnetization methods with additional permanent magnet on the magnetic field distribution and levitation force of single domain GdBCO bulk superconductor

    Institute of Scientific and Technical Information of China (English)

    马俊; 杨万民; 李佳伟; 王妙; 陈森林

    2012-01-01

    通过对永磁体辅助下单畴GdBCO超导体和方形永磁体在液氮温度、零场冷、轴对称情况下磁悬浮力的测量,研究了三种不同组态中辅助永磁体的引入方式对单畴GdBCO超导块材磁场分布及其磁悬浮力的影响.实验结果表明,如果处在超导体上方的测量用方形永磁体N极向下,则在轴对称情况下,当方形辅助永磁体N极向上与超导体下表面贴在一起时,超导体的最大磁悬浮力从没有引入辅助永磁体磁化的14.3 N增加到31.8 N,提高到222%;当方形辅助永磁体放置在超导体上表面、N极垂直向上且场冷后去掉辅助永磁体时,超导体的最大磁悬浮力从没有引入辅助永磁体磁化的14.3 N增加到21.6N,增加到151%;当方形辅助永磁体放置在超导体上表面、N极垂直向下且场冷后去掉方形辅助永磁体时,超导体的最大磁悬浮力从没有引入辅助永磁体磁化的14.3 N减小到8.6 N,减小为无辅助永磁体时的60%.这些结果说明,只有通过科学合理地设计超导体和永磁体的组合方式,才能获得较高的磁场强度,有效地提高超导体的磁悬浮力特性,该结果对促进超导体的应用具有重要的指导意义.%The effects of magnetization methods with additional permanent magnet on the magnetic field distribution and the levitation force of single domain GdBCO bulk superconductor are investigated with a cubic permanent magnet in their coaxial configuration in zero field cooled state at liquid nitrogen temperature in three different ways. It is found that when the N pole of the cubic permanent magnet, for the levitation force measurement, is placed above the GdBCO bulk superconductor and in the downward direction, the maximal levitation force can be improved to 31.8 N, and that when the N pole of the additional cubic permanent magnet points to upward and sticks to the bottom of the GdBCO bulk, the maximal levitation force is increased up to about 222% of the

  6. A study on associations between antiprothrombin antibodies, antiplasminogen antibodies and thrombosis

    NARCIS (Netherlands)

    Simmelink, MJA; De Groot, PG; Derksen, RHWM

    2003-01-01

    Anti-prothrombin antibodies area frequent cause of lupus anticoagulant (LAC), a thrombotic risk factor. Prothrombin shares structural homology with plasminogen, a kringle protein with an important role in fibrinolysis. Cross-reactivity between antiprothrombin antibodies and plasminogen has been desc

  7. Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

    Science.gov (United States)

    Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J

    2013-05-31

    Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

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

    NARCIS (Netherlands)

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

    1999-01-01

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

  9. Presence of non-maternal antibodies in newborns of mothers with antibody deficiencies.

    NARCIS (Netherlands)

    M. Hahn-Zoric; B. Carlsson; J. Bjö rkander; A.D.M.E. Osterhaus (Albert); L. Mellander; L.A. Hanson

    1992-01-01

    textabstractTo explain the mechanism for induction and production of specific antibodies found in the newborn already at birth, without previous known exposure to the antigen, we chose a model that presumably excluded the possibility of specific antibodies being transferred from the mother to the fe

  10. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    Energy Technology Data Exchange (ETDEWEB)

    Won, JaeSeon; Nam, PilWon; Lee, YongChan [College of Life Sciences and Graduate School of Biotechnology, Korea University, 5-ga Anam-dong, Sungbuk-gu, Seoul 136-701 (Korea, Republic of); Choe, MuHyeon, E-mail: choemh@korea.ac.kr [College of Life Sciences and Graduate School of Biotechnology, Korea University, 5-ga Anam-dong, Sungbuk-gu, Seoul 136-701 (Korea, Republic of)

    2009-04-24

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  11. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    Science.gov (United States)

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  12. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

  13. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    Science.gov (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  14. Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

    Science.gov (United States)

    Okeley, Nicole M; Toki, Brian E; Zhang, Xinqun; Jeffrey, Scott C; Burke, Patrick J; Alley, Stephen C; Senter, Peter D

    2013-10-16

    The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

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

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

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

  16. Monoclonal antibody disulfide reduction during manufacturing

    Science.gov (United States)

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  17. Striational antibodies in a paraneoplastic context.

    Science.gov (United States)

    McKeon, Andrew; Lennon, Vanda A; LaChance, Daniel H; Klein, Christopher J; Pittock, Sean J

    2013-04-01

    The clinical significance of striational antibodies (StrAbs) detected in the course of paraneoplastic antibody testing is unknown. We compared all 203 striational antibody (StrAb)-seropositive patients identified (2004-2005) during evaluation for paraneoplastic antibodies with age- and sex-matched seronegative controls. Thymoma and myasthenia gravis (MG) were significantly more common among cases (Pdetected after StrAb detection were adenocarcinoma in 5 patients and sarcoma in 3 patients. All patients who had a cancer identified after StrAb testing had a titer of ≥ 1:7680 or a coexisting muscle AChR-binding antibody. Autoimmune disorders more commonly observed among cases (with any StrAb value) included: hypothyroidism; rheumatoid arthritis; and pernicious anemia (all P<0.05). StrAbs may serve as a diagnostic clue for an autoimmune diagnosis. There is a low likelihood of oncological significance in patients with StrAb titers <1:7680 without coexisting paraneoplastic Abs. Copyright © 2013 Wiley Periodicals, Inc.

  18. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.

  19. Non-antibody protein-based biosensors

    Science.gov (United States)

    2016-01-01

    Biosensors that depend on a physical or chemical measurement can be adversely affected by non-specific interactions. For example, a biosensor designed to measure specifically the levels of a rare analyte can give false positive results if there is even a small amount of interaction with a highly abundant but irrelevant molecule. To overcome this limitation, the biosensor community has frequently turned to antibody molecules as recognition elements because they are renowned for their exquisite specificity. Unfortunately antibodies can often fail when immobilised on inorganic surfaces, and alternative biological recognition elements are needed. This article reviews the available non-antibody-binding proteins that have been successfully used in electrical and micro-mechanical biosensor platforms. PMID:27365032

  20. Sensitive neutralization test for rubella antibody.

    Science.gov (United States)

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  1. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  2. Limbic encephalitis associated with elevated antithyroid antibodies.

    Science.gov (United States)

    Hacohen, Yael; Joseph, Sonia; Kneen, Rachel; Eunson, Paul; Lin, Jean-Pierre; Vincent, Angela; Lim, Ming

    2014-06-01

    Immune-mediated limbic encephalitis affects both adults and children. Patients typically present with seizures, memory problems, and imaging changes in the medial temporal lobes. Both paraneoplastic and nonparaneoplastic forms have been described in which the antibody to the voltage-gated potassium channel-complex associated protein, leucine-rich glioma-inactivated 1, is most commonly reported. Elevated antithyroid antibodies have also been reported in a range of neurological syndromes with encephalopathy, such as limbic encephalitis, often collectively termed Hashimoto encephalopathy, a condition whereby corticosteroids responsiveness with a complete recovery is commonly observed. Here we describe 3 children presenting with limbic encephalitis with elevated thyroid antibodies that did not respond to corticosteroids alone and required more aggressive immunotherapy, mirroring the slower treatment response that is more frequently seen in other immune-mediated forms of limbic encephalitis.

  3. Origin and pathogenesis of antiphospholipid antibodies

    Directory of Open Access Journals (Sweden)

    C.M. Celli

    1998-06-01

    Full Text Available Antiphospholipid antibodies (aPL are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus, infectious (syphilis, AIDS and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias. Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.

  4. Adsorption of monoclonal antibodies to glass microparticles.

    Science.gov (United States)

    Hoehne, Matthew; Samuel, Fauna; Dong, Aichun; Wurth, Christine; Mahler, Hanns-Christian; Carpenter, John F; Randolph, Theodore W

    2011-01-01

    Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.

  5. Calciphylaxis in catastrophic antiphospholipid antibody syndrome.

    Science.gov (United States)

    Shah, Surbhi; Larson, Andrew; Datta, Yvonne

    2015-06-01

    Antiphospholipid antibody syndrome (APS) is a multisystem disorder characterized by vascular thrombosis and presence of circulating autoantibodies. The presence of APS can predispose to macrovascular as well as microvascular thrombotic events. Renal involvement is a common occurrence especially in the background of systemic lupus erythematosus. Skin appears to be another frequent target organ and a significant proportion of patients may present with skin lesions at the time of diagnosis. We present the case of a patient who presented with skin necrosis secondary to antiphospholipid antibody syndrome despite being on therapeutic anticoagulation and then developed dystrophic calcification secondary to her renal insufficiency. This complex skin condition eventually leads to her demise, as she was not a candidate for surgical management of these lesions. Why is this important? This case brings to our attention the need to consider calciphylaxis as a cause of ecchymotic-appearing skin lesions in dialysis patients on warfarin in patients with antiphospholipid antibody syndrome.

  6. Imaging spectrum of primary antiphospholipid antibody syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Kwon Ha; Won, Jong Jin [Wonkwang University Hospital, Iksan (Korea, Republic of); Ha, Hyun Kwon; Kim, Jung Hoon; Kim, Jeong Gon; Ki, Won Woo; Kim, Pyo Nyun; Lee, Moon Gyu; Auh, Yong Ho [Asan Medical Center, Seoul (Korea, Republic of)

    1998-04-01

    Antiphospholipid antibody syndrome is recognized as one of the most important causes of hypercoagulability. It can be clinically diagnosed if patients have experienced unexplained recurrent venous or arterial thrombosis, recurrent fetal loss, or thrombocytopenia in the presence of circulating autoantibodies to phospholipids, such as anticardiolipin antibody or lupus anticoagulant. Approximately half of all patients with this syndrome do not have associated systemic disease, and their condition is described as primary antiphospholipid antibody syndrome (PAPS). In the remainder, the syndrome is accompanied by systemic lupus erythematosus or other connective tissue diseases, and is known as secondary antiphospholipid syndrome (1). The purpose of this paper is to illustrate the systemic manifestation of PAPS, focusing on the radiological findings of CT, MR and angiography in clinically proven patients. (author). 8 refs., 10 figs.

  7. Utility of feline coronavirus antibody tests.

    Science.gov (United States)

    Addie, Diane D; le Poder, Sophie; Burr, Paul; Decaro, Nicola; Graham, Elizabeth; Hofmann-Lehmann, Regina; Jarrett, Oswald; McDonald, Michael; Meli, Marina L

    2015-02-01

    Eight different tests for antibodies to feline coronavirus (FCoV) were evaluated for attributes that are important in situations in veterinary practice. We compared four indirect immunofluorescent antibody tests (IFAT), one enzyme-linked immunosorbent assay (ELISA) (FCoV Immunocomb; Biogal) and three rapid immunochromatographic (RIM) tests against a panel of samples designated by consensus as positive or negative. Specificity was 100% for all but the two IFATs based on transmissible gastroenteritis virus (TGEV), at 83.3% and 97.5%. The IFAT and ELISA tests were best for obtaining an antibody titre and for working in the presence of virus. The RIM tests were the best for obtaining a result quickly (10-15 mins); of these, the Speed F-Corona was the most sensitive, at 92.4%, followed by FASTest feline infectious peritonitis (FIP; 84.6%) and Anigen Rapid FCoV antibody test (64.1%). Sensitivity was 100% for the ELISA, one FCoV IFAT and one TGEV IFAT; and 98.2% for a second TGEV IFA and 96.1% for a second FCoV IFAT. All tests worked with effusions, even when only blood products were stipulated in the instruction manual. The ELISA and Anigen RIM tests were best for small quantities of sample. The most appropriate FCoV antibody test to use depends on the reason for testing: in excluding a diagnosis of FIP, sensitivity, specificity, small sample quantity, rapidity and ability to work in the presence of virus all matter. For FCoV screening, speed and sensitivity are important, and for FCoV elimination antibody titre is essential. © ISFM and AAFP 2014.

  8. Nutritional requirements and assessing nutritional status in camelids.

    Science.gov (United States)

    Van Saun, Robert J

    2009-07-01

    It has been nearly 30 years since the first imported llamas and alpacas have been commercially raised in the United States. Nutritional requirements for these animals have not been well understood and most feeding practices were based on extrapolated and experiential information. Only recently has a National Research Council committee reviewed the available published information relative to nutrient requirements of llamas and alpacas. This article summarizes current nutrient requirement recommendations and provides some practical feeding recommendations and methods to assess nutritional status.

  9. Anti-DNA antibodies--quintessential biomarkers of SLE.

    Science.gov (United States)

    Pisetsky, David S

    2016-02-01

    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  10. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  11. Behaviour of non-donor specific antibodies during rapid re-synthesis of donor specific HLA antibodies after antibody incompatible renal transplantation.

    Directory of Open Access Journals (Sweden)

    Nithya S Krishnan

    Full Text Available HLA directed antibodies play an important role in acute and chronic allograft rejection. During viral infection of a patient with HLA antibodies, the HLA antibody levels may rise even though there is no new immunization with antigen. However it is not known whether the converse occurs, and whether changes on non-donor specific antibodies are associated with any outcomes following HLA antibody incompatible renal transplantation.55 patients, 31 women and 24 men, who underwent HLAi renal transplant in our center from September 2005 to September 2010 were included in the studies. We analysed the data using two different approaches, based on; i DSA levels and ii rejection episode post transplant. HLA antibody levels were measured during the early post transplant period and corresponding CMV, VZV and Anti-HBs IgG antibody levels and blood group IgG, IgM and IgA antibodies were quantified.Despite a significant DSA antibody rise no significant non-donor specific HLA antibody, viral or blood group antibody rise was found. In rejection episode analyses, multiple logistic regression modelling showed that change in the DSA was significantly associated with rejection (p = 0.002, even when adjusted for other antibody levels. No other antibody levels were predictive of rejection. Increase in DSA from pre treatment to a post transplant peak of 1000 was equivalent to an increased chance of rejection with an odds ratio of 1.47 (1.08, 2.00.In spite of increases or decreases in the DSA levels, there were no changes in the viral or the blood group antibodies in these patients. Thus the DSA rise is specific in contrast to the viral, blood group or third party antibodies post transplantation. Increases in the DSA post transplant in comparison to pre-treatment are strongly associated with occurrence of rejection.

  12. Investigation of Antiphosphatidyl-Serine Antibody and Antiphosphatidyl-Inositol Antibody in Ischemic Stroke Patients

    Directory of Open Access Journals (Sweden)

    Hirohisa Okuma

    2010-01-01

    Full Text Available Antiphospholipid syndrome is characterized by arterial or venous thrombosis and the presence of antiphospholipid antibodies (aPL. We measured β2-GPI aCL, IgGaCL, LA, antiphosphatidyl-serine antibody (PS, and antiphosphatidyl-inositol antibody (PI in each patient at one month after the onset of stroke. In addition, carotid artery echography was performed in patients positive for PI or PS. Among the 250 patients, 13.6% (34/250 were positive for either PI or PS, and 6.8% (17/250 were positive for both. Carotid artery echography performed on these 34 patients showed that the frequencies of increased intimal-medial thickness (IMT of 1.1 mm or more, plaque, and carotid artery stenosis of 50% or more were all significantly higher in patients positive for antinuclear antibody than those negative for the antibody (P<.05. PI and PS are associated with antinuclear antibody and precipitation of atherosclerosis. Ischemic stroke patients with SLE frequently showed a variety of antiphospholipid-protein antibodies.

  13. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    Science.gov (United States)

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  14. Monoclonal antibody technologies and rapid detection assays

    Science.gov (United States)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  15. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana

    1994-01-01

    bronchopulmonary P. aeruginosa infection (CF + P) but in none of the CF patients with no or intermittent P. aeruginosa infection. Anti-beta-lactamase antibodies were present in serum from CF + P patients after six antipseudomonal courses (median) and correlated with infection with a beta-lactam-resistant strain...

  16. IgA Antibodies in Rett Syndrome

    Science.gov (United States)

    Reichelt, K. L.; Skjeldal, O.

    2006-01-01

    The level of IgA antibodies to gluten and gliadin proteins found in grains and to casein found in milk, as well as the level of IgG to gluten and gliadin, have been examined in 23 girls with Rett syndrome and 53 controls. Highly statistically significant increases were found for the Rett population compared to the controls. The reason for this…

  17. Coronavirus antibodies in African bat species.

    Science.gov (United States)

    Müller, Marcel A; Paweska, Janusz T; Leman, Patricia A; Drosten, Christian; Grywna, Klaus; Kemp, Alan; Braack, Leo; Sonnenberg, Karen; Niedrig, Matthias; Swanepoel, Robert

    2007-09-01

    Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV.

  18. Antineutrophil cytoplasmic antibodies in juvenile chronic arthritis

    NARCIS (Netherlands)

    Mulder, L; Horst, G; Limburg, P; deGraeffMeeder, ER; Kuis, W; Kallenberg, C

    1997-01-01

    Objective, To evaluate the diagnostic significance of antineutrophil cytoplasmic antibodies (ANCA) by assessing the prevalence of ANCA in juvenile chronic arthritis (JCA) (n = 93) of either oligoarticular, polyarticular, or systemic onset. To investigate the prevalence of ANCA in other diseases of c

  19. Antiphospholipid Antibody Syndrome Presenting with Hemichorea

    Directory of Open Access Journals (Sweden)

    Yezenash Ayalew

    2012-01-01

    Full Text Available A 25-year-old Bangladeshi lady presented to neurology with a three-month history of involuntary movements of her right arm, associated with loss of power. There was progression to the right leg, and she subsequently developed episodes of slurred speech and blurred vision. At the time of presentation, she was 12 weeks pregnant and the symptoms were reported to have started at conception. Past medical history was unremarkable apart from one first trimester miscarriage and there was no significant family history suggestive of a hereditary neurological condition. MRI of the head revealed no abnormalities but serology showed positive antinuclear antibodies (ANAs at a titre of 1/400. Further investigations revealed strongly positive anticardiolipin antibodies (>120 and positive lupus anticoagulant antibodies. The patient had a second miscarriage at 19 weeks gestation strengthening the possibility that the chorea was related to antiphospholipid antibody syndrome and she was started on a reducing dose of Prednisolone 40 mg daily and aspirin 300 mg daily. Six months later, she had complete resolution of neurological symptoms. There are several reports of chorea as a feature of antiphospholipid syndrome, but no clear consensus on underlying pathophysiology.

  20. Anti-glomerular basement membrane antibodies.

    Science.gov (United States)

    Silvariño, Ricardo; Noboa, Oscar; Cervera, Ricard

    2014-11-01

    Basement membranes form an anatomic barrier that contains connective tissue. They are composed of type IV collagen, laminin and proteoglycans. Anti-basement membrane antibodies bind to the non-collagen site of the α3 chain of type IV collagen. A group of renal diseases, pulmonary diseases and perhaps others affecting different organs have long been associated with the presence of antibodies directed against glomerular basement membrane (GBM), alveolar basement membrane and tubular basement membrane. Goodpasture disease has a frequency of 0.5 to 1 case by million/year, and is responsible for up to 20% of crescentic glomerulonephritis in renal biopsy. It has been associated with genetic and immune abnormalities and there are usually environmental triggers preceding clinical onset. Renal disease can occur isolated or in association with pulmonary hemorrhage. In general, renal disease has a rapid progression that determines severe compromise, with rare spontaneous resolution. The diagnosis of Goodpasture disease requires the presence of the anti-GBM antibody, either in circulation or in renal tissue. The prognosis of non-treated patients is poor. The standard of care is plasma exchange combined with prednisone and cyclophosphamide. Anti-GBM antibody levels must be monitored frequently until their disappearance, and then every 6 months to confirm sustained remission in the absence of clinical signs of recurrence. Prognosis of the disease is strongly associated with its initial presentation. Survival rates are related to the degree of renal compromise at onset of the disease. Recurrence of the disease post-transplantation is low.