Sample records for antibody fragments stabs

  1. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans


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

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

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    Gustav Rodrigo


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

  3. Pressure-balanced stab connection

    Energy Technology Data Exchange (ETDEWEB)

    Best, M.J.A.; Jones, R.H.; Hynes, J.H.; Pond, R.J.; Gordon, C.R.


    This patent describes an apparatus for sealingly connecting a flow bore of a wellhead to a flowline. It consists of: a stab member including a stab body. The stab body has a stab body bore. One end of the stab body bore exiting from the stab body and being in fluid communication with the flow bore and the other end of the stab body bore exiting from a side of the stab body through a stab body side port; a stab receptacle mounted on the wellhead and having a stab receiving bore for receiving the stab body; the stab receptacle having a receptacle flow bore. One end of the receptacle flow bore being in fluid communication with the stab receiving bore and the other end of the receptacle flow bore exiting the stab receptacle and being in fluid communication with the flowline; means for establishing fluid communication between the stab body side port and the one end of the receptacle flow bore when the stab body is received within the stab receiving bore. It includes means disposed on the stab member for orienting the stab member with respect to the Christmas tree for ensuring the alignment of the stab body side port with the one end of the receptable flow bore when the stab body is received within the stab receiving bore; and first and second sealing means disposed on the stab body above and below the stab body side port, respectively, for sealing engagement with the walls of the stab receiving bore above and below the one end of the receptable flow bore for establishing a non-blow-apart, fluid pressure sealed flow passage between the stab body bore of the stab member and the receptacle flow bore of the stab receptacle when the stab body is landed within the stab receiving bore.

  4. Monoclonal antibodies and Fc fragments for treating solid tumors

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    Eisenbeis AM


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

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

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik


    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...

  6. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay. (United States)

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


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

  7. Targeted cancer therapy through antibody fragments-decorated nanomedicines. (United States)

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


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

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

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    Katerina T. Xenaki


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

  9. Recombinant fragment of an antibody tailored for direct radioiodination

    Czech Academy of Sciences Publication Activity Database

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


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

  10. Environmentally Benign Stab Detonators (United States)


    Steve Santor, Mr. Phil Ramsey , and Mr. Octavio Cervantes. Contributors from TACOM-ARDEC include Mr. Gartung Cheng, and Mrs. Neha Mehta...obtain more reproducible stab initiation results led to size sorting of the sheared material. This was performed using a series of Tyler U.S. standard... Tyler Standard 50 Mesh sieve (particle size range is 300 µm to 500 µm) Sample Thickness (um) Bi-layer Period (nm) Coating Min. Stab energy (mJ

  11. Stab resistant body armour


    Horsfall, I


    There is now a widely accepted need for stab resistant body armour for the police in the UK. However, very little research has been done on knife resistant systems and the penetration mechanics of sharp projectiles are poorly understood. This thesis explores the general background to knife attack and defence with a particular emphasis on the penetration mechanics of edged weapons. The energy and velocity that can be achieved in stabbing actions has been determined for a numb...

  12. Fluorescent labeling of antibody fragments using split GFP.

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    Fortunato Ferrara

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

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

    DEFF Research Database (Denmark)

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


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

  14. Primary stabbing headache. (United States)

    Pareja, Juan A; Sjaastad, Ottar


    Primary stabbing headache is characterized by transient, cephalic ultrashort stabs of pain. It is a frequent complaint with a prevalence of 35.2%, a female preponderance, and a mean age of onset of 28 years (Vågå study). Attacks are generally characterized by moderate to severe, jabbing or stabbing pain, lasting from a fraction of a second to 3s. Attack frequency is generally low, with one or a few attacks per day. The paroxysms generally occur spontaneously, during daytime. Most patients exhibit a sporadic pattern, with an erratic, unpredictable alternation between symptomatic and non-symptomatic periods. Paroxysms are almost invariably unilateral. Temporal and fronto-ocular areas are most frequently affected. Attacks tend to move from one area to another, in either the same or the opposite hemicranium. Jabs may be accompanied by a shock-like feeling and even by head movement - "jolts" -or vocalization. On rare occasions, conjunctival hemorrhage and monocular vision loss have been described as associated features. Primary stabbing headache may concur, synchronously or independently, with other primary headaches. In contrast to what is the case in adults, in childhood it is not usually associated with other headaches. Treatment is rarely necessary. Indomethacin, 75-150 mg daily, may seem to be of some avail. Celecoxib, nifedipine, melatonin, and gabapentin have been reported to be effective in isolated cases and small series of patients. The drug studies need corroboration. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Environmentally Benign Stab Detonators

    Energy Technology Data Exchange (ETDEWEB)

    Gash, A


    Many energetic systems can be activated via mechanical means. Percussion primers in small caliber ammunition and stab detonators used in medium caliber ammunition are just two examples. Current medium caliber (20-60mm) munitions are detonated through the use of impact sensitive stab detonators. Stab detonators are very sensitive and must be small, as to meet weight and size limitations. A mix of energetic powders, sensitive to mechanical stimulus, is typically used to ignite such devices. Stab detonators are mechanically activated by forcing a firing pin through the closure disc of the device and into the stab initiating mix. Rapid heating caused by mechanically driven compression and friction of the mixture results in its ignition. The rapid decomposition of these materials generates a pressure/temperature pulse that is sufficient to initiate a transfer charge, which has enough output energy to detonate the main charge. This general type of ignition mix is used in a large variety of primers, igniters, and detonators.[1] Common primer mixes, such as NOL-130, are made up of lead styphnate (basic) 40%, lead azide (dextrinated) 20%, barium nitrate 20%, antimony sulfide 15%, and tetrazene 5%.[1] These materials pose acute and chronic toxicity hazards during mixing of the composition and later in the item life cycle after the item has been field functioned. There is an established need to replace these mixes on toxicity, health, and environmental hazard grounds. This effort attempts to demonstrate that environmentally acceptable energetic solgel coated flash metal multilayer nanocomposites can be used to replace current impact initiated devices (IIDs), which have hazardous and toxic components. Successful completion of this project will result in IIDs that include innocuous compounds, have sufficient output energy for initiation, meet current military specifications, are small, cost competitive, and perform as well as or better than current devices. We expect flash

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

    NARCIS (Netherlands)

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

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

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

    NARCIS (Netherlands)

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


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

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

    NARCIS (Netherlands)

    Dolk, E.


    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

  19. Recombinant human antibody fragment against tetanus toxoid produced by phage display (United States)

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


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

  20. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus. (United States)

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

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

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

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    Dorota Smolarek


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

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

    Czech Academy of Sciences Publication Activity Database

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


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

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

    NARCIS (Netherlands)

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


    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

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

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    Hust Michael


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


    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.


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

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

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


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

  7. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. (United States)

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


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

  8. Chemical design of radiolabeled antibody fragments for low renal radioactivity levels. (United States)

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


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

  9. Computational identification of antigen-binding antibody fragments. (United States)

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


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

  10. Environmentally Benign Stab Detonators

    Energy Technology Data Exchange (ETDEWEB)

    Gash, A E


    The coupling of energetic metallic multilayers (a.k.a. flash metal) with energetic sol-gel synthesis and processing is an entirely new approach to forming energetic devices for several DoD and DOE needs. They are also practical and commercially viable manufacturing techniques. Improved occupational safety and health, performance, reliability, reproducibility, and environmentally acceptable processing can be achieved using these methodologies and materials. The development and fielding of this technology will enhance mission readiness and reduce the costs, environmental risks and the necessity of resolving environmental concerns related to maintaining military readiness while simultaneously enhancing safety and health. Without sacrificing current performance, we will formulate new impact initiated device (IID) compositions to replace materials from the current composition that pose significant environmental, health, and safety problems associated with functions such as synthesis, material receipt, storage, handling, processing into the composition, reaction products from testing, and safe disposal. To do this, we will advance the use of nanocomposite preparation via the use of multilayer flash metal and sol-gel technologies and apply it to new small IIDs. This work will also serve to demonstrate that these technologies and resultant materials are relevant and practical to a variety of energetic needs of DoD and DOE. The goal will be to produce an IID whose composition is acceptable by OSHA, EPA, the Clean Air Act, Clean Water Act, Resource Recovery Act, etc. standards, without sacrificing current performance. The development of environmentally benign stab detonators and igniters will result in the removal of hazardous and toxic components associated with their manufacturing, handling, and use. This will lead to improved worker safety during manufacturing as well as reduced exposure of Service personnel during their storage and or use in operations. The

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

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga


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

  12. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries. (United States)

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


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

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

    NARCIS (Netherlands)

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


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

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

    Czech Academy of Sciences Publication Activity Database

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


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

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

    Directory of Open Access Journals (Sweden)

    Barth Stefan


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

    Directory of Open Access Journals (Sweden)

    J. Mala


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

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

    International Nuclear Information System (INIS)

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


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

  3. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies. (United States)

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


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

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

    DEFF Research Database (Denmark)

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


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

  5. Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging (United States)


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

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

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee


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

  10. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su


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

  13. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    "Babaei MH


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

  15. Efficient production of antibody Fab fragment by transient gene expression in insect cells. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Keith R Miller

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

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

    DEFF Research Database (Denmark)

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


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

  18. Multifunctional PSCA antibody fragments for PET and optical prostate cancer imaging (United States)


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

  19. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

  1. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries. (United States)

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


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

  2. Textile sensors for stab and cut detection (United States)

    Graßmann, C.; Obermann, M.; Lempa, E.; Bache, T.; Siegel, P. K.; Freyer, T.; Paschko, S.; Beyer, T.; Kirsche, M.; Schwarz-Pfeiffer, A.


    Manufacturers are aiming for more flexible and lightweight protective clothing to increase wearing comfort. A cardigan with a knitted stab-resistant inlay and an alarm system is presented. The stab-resistant inlay is based on a multilayer ultra-high molecular weight poly ethylene (UHMW-PE) fabric. Stab resistance was evaluated according to the standard of the Association of Test Laboratories for Bullet, Stab or Pike Resistant Materials and Construction Standard (VPAM 2011). Furthermore sensors for the detection of cuts and pressure were integrated. Both sensors can trigger alarms if the wearer is attacked. Normal pressure occurring through leaning on a wall or sitting is filtered out and does not trigger an alarm.

  3. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS. (United States)

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


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

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Amram Torgeman


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

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

    International Nuclear Information System (INIS)

    Bischof-Delaloye, A.; Delaloye, B.


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

  7. Packing motifs as predictors of the propensity of antibody fragments to crystallize (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

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


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

  10. The imaging of stab injuries

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Coert S. de; Africa, Mogoeemang; Gebremariam, Fekade A.; Rensburg, J. Janse van; Otto, Susan F.; Potgieter, Henrik F. (Dept. of Diagnostic Radiology, Faculty of Health Sciences, Univ. of the Free State and Academic Health Complex, Free State Province Dept. of Health, Bloemfontein (South Africa)), e-mail:


    In the trauma unit of the Bloemfontein Academic Complex, the total number of stab wounds seen represents approximately 70.5% of penetrating injuries, which is 6.4% of 5004 trauma cases seen in a period of 1 year. The other cases are gunshot wounds and pedestrian or motor vehicle accidents. Specific guidelines and protocols are followed for penetrating trauma management. All imaging modalities are utilized, with chest radiography the mainstay of thoracic imaging in patients having sustained sharp penetrating chest injuries. Computed tomography (CT) is being used more frequently as the primary imaging modality in the evaluation of hemodynamically stable patients with penetrating injuries. The improved speed of data acquisition and superior image reconstruction of multidetector CT (MDCT) has further driven this change in imaging approach. Although digital subtraction angiography (DSA) has been the reference standard for the diagnosis of traumatic vascular injuries, it is giving way to faster, less invasive, and less personnel-intensive imaging techniques, e.g., MDCT angiography. Given the fact that we work in an academic environment and that we have a dedicated interventional unit, arteriography is still frequently performed and still has its place as the 'gold standard' in the diagnosis of vascular injuries. Penetrating chest injuries suspected of traversing the mediastinum or extending near the posterior mediastinal structures dictate esophageal and tracheal evaluation. Although radiology has a role to play, direct visualization (esophagoscopy, bronchoscopy) remains the most reliable method of excluding injuries to these structures. Transthoracic ultrasound (echocardiography) has become indispensable in helping to evaluate injuries to the heart and the ascending and descending aortas. More recent work has demonstrated that ultrasonography can also be used to detect hemothoraces and pneumothoraces with accuracy

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

    International Nuclear Information System (INIS)

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


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

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

    Directory of Open Access Journals (Sweden)

    Cesar Andres Diaz Arias

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

  13. Penetrating Stab Wound of the Right Ventricle

    Directory of Open Access Journals (Sweden)

    Onursal Buğra


    Full Text Available 18 years old male patient was admitted to our emergency unit with a penetrating stab wound to the right ventricle. A stab wound to the right ventricle was found to be 3 cm in diameter. The bleeding was controlled by insertion of a Foley catheter and inflation of the balloon. The stab wound had transected distal acute marginal side ofthe right coronary artery. A successful repair was performed with the use of a foley catheter and application of the Medtronic Octopus Tissue Stabilization System. The wound was closed with pledgeted mattress sutures. The distal acute marginal side of the right coronary artery was ligated. In this presentation, the surgical intervention method was reported and followed by a discussion of emergency surgical procedures of the heart.

  14. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements. (United States)

    Crivianu-Gaita, Victor; Thompson, Michael


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

  15. Mapping of Monoclonal Antibody Binding Sites on CNBr Fragments of the S- Layer Protein Antigens of Rickettsia Typhi and Rickettsia Prowazekii (United States)


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

  17. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment. (United States)

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


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

  18. 76 FR 69764 - Stab-Resistant Body Armor Standard Workshop (United States)


    ... of Justice Programs Stab-Resistant Body Armor Standard Workshop AGENCY: National Institute of Justice... Standards and Technology (NIST) are jointly hosting a workshop focused on the NIJ Stab-resistant Body Armor... the revised NIJ Stab-resistant Body Armor Standard and to receive input, comments, and recommendations...

  19. Fab fragments of ovine antibody to colchicine enhance its clearance in the rat. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Klatt Stephan


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

  1. Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Ch’ng Wei-Choong


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

  3. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display. (United States)

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


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


    NARCIS (Netherlands)



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

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

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


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

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Tombesi Marina


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

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

    Directory of Open Access Journals (Sweden)

    Tong Li

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

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

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


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

  10. Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments. (United States)

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


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

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


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


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

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

    Directory of Open Access Journals (Sweden)

    Su WANG


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

  13. Isolation of populations of antipeptide antibodies directed against different epitopes of the same fragment. (United States)

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


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

  14. Serum and urine analysis of the aminoterminal procollagen peptide type III by radioimmunoassay with antibody Fab fragments. (United States)

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


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

  15. 17th STAB/DGLR Symposium

    CERN Document Server

    Heller, Gerd; Kreplin, Hans-Peter; Nitsche, Wolfgang; Peltzer, Inken


    This volume contains the contributions to the 17th Symposium of STAB (German Aerospace Aerodynamics Association). STAB includes German scientists and engineers from universities, research establishments and industry doing research and project work in numerical and experimental fluid mechanics and aerodynamics, mainly for aerospace but also for other applications. Many of the contributions collected in this book present results from national and European Community sponsored projects. This volume gives a broad overview of the ongoing work in this field in Germany and spans a wide range of topics: airplane aerodynamics, multidisciplinary optimization and new configurations, hypersonic flows and aerothermodynamics, flow control (drag reduction and laminar flow control), rotorcraft aerodynamics, aeroelasticity and structural dynamics, numerical simulation, experimental simulation and test techniques, aeroacoustics as well as the new fields of biomedical flows, convective flows, aerodynamics and acoustics of high-s...

  16. 18th STAB/DGLR Symposium

    CERN Document Server

    Heller, Gerd; Krämer, Ewald; Kreplin, Hans-Peter; Nitsche, Wolfgang; Rist, Ulrich


    This book presents contributions to the 18th biannual symposium of the German Aerospace Aerodynamics Association (STAB). The individual chapters reflect ongoing research conducted by the STAB members in the field of numerical and experimental fluid mechanics and aerodynamics, mainly for (but not limited to) aerospace applications, and cover both nationally and EC-funded projects. By addressing a number of essential research subjects, together with their related physical and mathematics fundamentals, the book provides readers with a comprehensive overview of the current research work in the field, as well as its main challenges and new directions. Current work on e.g. high aspect-ratio and low aspect-ratio wings, bluff bodies, laminar flow control and transition, active flow control, hypersonic flows, aeroelasticity, aeroacoustics and biofluid mechanics is exhaustively discussed here.  .

  17. Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    Directory of Open Access Journals (Sweden)



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

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

    Directory of Open Access Journals (Sweden)

    Laura Frigotto


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

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

    International Nuclear Information System (INIS)

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


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

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

    DEFF Research Database (Denmark)

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


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

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


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


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

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

    International Nuclear Information System (INIS)

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


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

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

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


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

  6. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells. (United States)

    Toleikis, Lars; Frenzel, André


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

  7. Herpes simplex virus following stab phlebectomy. (United States)

    Hicks, Caitlin W; Lum, Ying Wei; Heller, Jennifer A


    Herpes simplex virus infection following surgery is an unusual postoperative phenomenon. Many mechanisms have been suggested, with the most likely explanation related to latent virus reactivation due to a proinflammatory response in the setting of local trauma. Here, we present a case of herpes simplex virus reactivation in an immunocompetent female following a conventional right lower extremity stab phlebectomy. Salient clinical and physical examination findings are described, and management strategies for herpes simplex virus reactivation are outlined. This is the first known case report of herpes simplex virus reactivation following lower extremity phlebectomy.

  8. Unusual Presentation of Meningitis following Stab Neck | Motsitsi ...

    African Journals Online (AJOL)

    Background: A case report of stab neck presenting at Kalafong Hospital, Pretoria, South Africa with atypical meningitis. The objective was to illustrate the challenge of diagnosing this unusual and late presentation of meningitis. Case Report: A 48 year-old male patient presented to us two days after a stab neck. He was ...

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

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.


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

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

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

    Mach, J.P.; Buchegger, F.


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

  12. The effect of varying the peptide linker length in a single chain variable fragment antibody against wogonin glucuronide. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Edyta Petters


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

  14. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1. (United States)

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


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

  15. A Role for Small Antibody Fragments to Bind and Neutralize HIV | Center for Cancer Research (United States)

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

  16. Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity. (United States)

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


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

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

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


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

  18. [Use of antibodies or their fragments for the treatment of tumors]. (United States)

    Douillard, J Y; Chatal, J F


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

  19. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

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

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

    International Nuclear Information System (INIS)

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


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

  3. Renal brush border enzyme-cleavable linkages for low renal radioactivity levels of radiolabeled antibody fragments. (United States)

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


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

  4. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro


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

  7. Conservative treatment of abdominal stab wounds with omental protrusion

    DEFF Research Database (Denmark)

    Duus, B R; Damm, P; Jensen, F U


    Two cases of abdominal stab wounds with omental protrusion treated conservatively are presented. Omental protrusion in a patient without signs of shock or peritoneal irritation is not an absolute indication for explorative laparotomy.......Two cases of abdominal stab wounds with omental protrusion treated conservatively are presented. Omental protrusion in a patient without signs of shock or peritoneal irritation is not an absolute indication for explorative laparotomy....

  8. Characteristics and rehabilitation for patients with spinal cord stab injury (United States)

    Wang, Fangyong; Zhang, Junwei; Tang, Hehu; Li, Xiang; Jiang, Shudong; Lv, Zhen; Liu, Shujia; Chen, Shizheng; Liu, Jiesheng; Hong, Yi


    [Purpose] The objective of the study was to compare the incidence, diagnosis, treatment, and prognosis of patients with spinal cord stab injury to those with the more common spinal cord contusion injury. [Subjects] Of patients hospitalized in China Rehabilitation Research Center from 1994 to 2014, 40 of those having a spinal cord stab injury and 50 with spinal cord contusion were selected. [Methods] The data of all patients were analyzed retrospectively. The cases were evaluated by collecting admission and discharge ASIA (American Spinal Injury Association) and ADL (activity of daily living) scores. [Results] After a comprehensive rehabilitation program, ASIA and ADL scores of patients having both spinal cord stab injury and spinal cord contusion significantly increase. However, the increases were noted to be higher in patients having a spinal cord stab injury than those having spinal cord contusion. [Conclusion] Comprehensive rehabilitation is effective both for patients having spinal cord stab injury and those with spinal cord contusion injury. However, the prognosis of patients having spinal cord stab injury is better than that of patients with spinal cord contusion. PMID:26834329

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

    NARCIS (Netherlands)

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


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

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

    NARCIS (Netherlands)

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


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

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

    NARCIS (Netherlands)

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


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

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

    International Nuclear Information System (INIS)

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


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

  13. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display. (United States)

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


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

  14. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference? (United States)

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


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

  15. Tuning the Hydrolytic Stability of Next Generation Maleimide Cross-Linkers Enables Access to Albumin-Antibody Fragment Conjugates and tri-scFvs. (United States)

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


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

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

    International Nuclear Information System (INIS)

    Liu Guangda


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

  17. Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase. (United States)

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


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

  18. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Guus Koch


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

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

    Directory of Open Access Journals (Sweden)

    Jason H Stafford

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

  1. Selective Nonoperative Management of Abdominal Stab Wounds. (United States)

    Murry, Jason S; Hoang, David M; Ashragian, Sogol; Liou, Doug Z; Barmparas, Galinos; Chung, Rex; Alban, Rodrigo F; Margulies, Daniel R; Ley, Eric J


    Stab wounds (SW) to the abdomen traditionally require urgent exploration when associated with shock, evisceration, or peritonitis. Hemodynamically stable patients without evisceration may benefit from serial exams even with peritonitis. We compared patients taken directly to the operating room with abdominal SWs (ED-OR) to those admitted for serial exams (ADMIT). We retrospectively reviewed hemodynamically stable patients presenting with any abdominal SW between January 2000 and December 2012. Exclusions included evidence of evisceration, systolic blood pressure ≤110 mm Hg, or blood transfusion. NON-THER was defined as abdominal exploration without identification of intra-abdominal injury requiring repair. Of 142 patients included, 104 were ED-OR and 38 were ADMIT. When ED-OR was compared with ADMIT, abdominal Abbreviated Injury Score was higher (2.4 vs 2.1; P = 0.01) and hospital length of stay was longer (4.8 vs 3.3 days; P = 0.04). Incidence of NON-THER was higher in ED-OR cohort (71% vs 13%; P ≤ 0.001). In a regression model, ED-OR was a predictor of NON-THER (adjusted odds ratio 16.6; P patient from ED-OR expired after complications from NON-THER. There were no deaths in the ADMIT group. For those patients with abdominal SWs who present with systolic blood pressure ≥110 mm Hg, no blood product transfusion in the emergency department and lacking evisceration, admission for serial abdominal exams may be preferred regardless of abdominal exam.

  2. Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology. (United States)

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


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

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

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


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

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

    International Nuclear Information System (INIS)

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


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

  5. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

  7. Primary stabbing headache in adults and pediatrics: a review. (United States)

    Hagler, Suzanne; Ballaban-Gil, Karen; Robbins, Matthew S


    Primary stabbing headache (PSH) is an under-recognized primary headache disorder, which often goes undiagnosed. It is mainly characterized by its ultrashort stabbing quality and can be easily overlooked both by patients and providers as it is often not severe enough to interfere significantly with daily life. However, PSH may be severe and require therapy, and it is important for providers to recognize this headache type, both in adult and pediatric populations, as well as to be able to distinguish it from secondary headache disorders. PSH also may be more common than previously thought.

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

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


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

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

    International Nuclear Information System (INIS)

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


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

  10. The correlation between stabbing-related upper extremity wounds and survival of stabbing victims with abdominal and thoracic injuries. (United States)

    Rozenfeld, Michael; Peleg, Kobi; Givon, Adi; Kessel, Boris


    When treating patients with stab injuries of the torso, clinicians often lack timely information about the degree and nature of internal organ damage. An externally observable sign significantly associated with characteristics of torso injuries may therefore be useful for practitioners. One such potential sign is the presence of wounds to the hands, sometimes sustained during victims' attempt to defend themselves during the violent altercation. Thus, the primary aim of this study was to evaluate the association between presence of upper extremity wounds and the severity of the thoracic and intra-abdominal injuries due to stabbing. This study was carried out retrospectively using data on 8714 patients with stabbing-related injuries from 19 trauma centers that participated in the Israeli National Trauma Registry (INTR) between January 1st1997 and December 31st 2013. Patients with wounds of upper extremities in addition to torso injuries (UE group) were compared to other patients with torso injuries (TO group) in terms of demographics, injury characteristics and clinical outcome. The compared groups were found to be homogeneous in terms of age and systolic blood pressure; the number of sustained torso injuries was also identical. The UE group comprised a slightly greater percentage of females, however both groups were predominantly male. Patients with upper extremity injuries had a lower proportion of internal organ damage (36% vs. 38.5%) and lower mortality (0.9% vs. 2%). The higher mortality of patients without upper extremity wounds remained significantly different even when adjusted by other epidemiological parameters (OR 2.46, 95% CI 1.33-5.08).The number of sustained upper extremity injuries was positively associated with deeper penetration of the torso by the stabbing instrument. Patients with stabbing-related upper extremity wounds had a significant survival advantage over patients without such injuries. However, a greater number of sustained upper extremity

  11. Scientific Letter: Stabbing nails into the neck: an unusual self ...

    African Journals Online (AJOL)

    Scientific Letter: Stabbing nails into the neck: an unusual self-damaging behavior mandating neurosurgery. A Aghabiklooei, R Aghabiklooei, N Zamani. Abstract. Scientific Letter - No Abstract Available. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  12. An optimal dynamic interval stabbing-max data structure?

    DEFF Research Database (Denmark)

    Agarwal, Pankaj Kumar; Arge, Lars; Yi, Ke


    In this paper we consider the dynamic stabbing-max problem, that is, the problem of dynamically maintaining a set S of n axis-parallel hyper-rectangles in Rd, where each rectangle s ∈ S has a weight w(s) ∈ R, so that the rectangle with the maximum weight containing a query point can be determined...

  13. Non-operative management of abdominal stab wounds -an analysis ...

    African Journals Online (AJOL)

    Patients with abdominal stab wounds presenting with peritonitis, haemodynamic instability, organ evisceration and high spinal cord injury underwent emergency laparotomy. No local wound exploration, diagnostic peritoneal lavage or ultrasound was used. Haematuria in patients without an indication for emergency surgery ...

  14. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. (United States)

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


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

  15. Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product. (United States)

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


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

  16. SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells. (United States)

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


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

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

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    Grasso Felicia


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

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

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


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

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

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    John M Louis

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

  20. Abdominal and lower back pain in pediatric idiopathic stabbing headache. (United States)

    Kakisaka, Yosuke; Ohara, Tomoichiro; Hino-Fukuyo, Naomi; Uematsu, Mitsugu; Kure, Shigeo


    Idiopathic stabbing headache (ISH) is a primary headache syndrome characterized by transient, sharp, stabbing pains located in the first division of the trigeminal nerve. Reports of pediatric ISH are rare, and extracephalic pain in pediatric ISH is extremely rare. Here we report the case of a 7-year-old male patient suffering from frequent, short, stabbing headache, which was occasionally associated with abdominal and lower back pain. Various investigations were normal. He was diagnosed with ISH, and valproic acid was administered to relieve his headache and accompanying symptoms. Our case demonstrates that abdominal and lower back pain may occur in pediatric ISH. This case may provide new evidence linking ISH and migraine by showing that extracephalic symptoms accompanying ISH are similar to those of migraine. We hypothesize that the mechanism underlying the headache and abdominal and lower back pain associated with ISH may be similar to that of a migraine headache. Accumulating additional cases by asking specific questions regarding the presence of the unusual symptoms presented in our case may help to establish a detailed clinical profile of these unfamiliar and peculiar symptoms in the pediatric ISH population.

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

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    Urszula Jarocka


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

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

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


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

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

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    Torsten Rülker

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

  4. Terrorist Stabbings-Distinctive Characteristics and How to Prepare for Them. (United States)

    Merin, Ofer; Sonkin, Roman; Yitzhak, Avraham; Frenkel, Hagai; Leiba, Adi; Schwarz, Alon D; Jaffe, Eli


    The number of terror attack incidents is on the increase worldwide. The knife is one of the weapons most commonly used among terrorists. Appropriate preparation in trauma units for coping with the increasing numbers of terrorist-inflicted stabbings is different from the preparation suitable for civilian stabbings. Therapeutic and logistic guidelines need to be adjusted to accommodate those differences. Characterize the unique injuries related to terrorist stabbing, and suggest preparedness actions. Retrospective data on all terrorist-inflicted stabbing incidents between September 2015 and May 2016 were retrieved from the database of the national Israeli emergency medical services and from the Israeli Defense Forces Medical Corps records. There were a total of 414 civilian victims (34 fatalities) of terror incidents. Of these, 161 involved stabbings during 106 separate incidents. There was more than 1 stab wound per patient in approximately 60% of cases, and more than 1 victim in approximately 40% of cases. Unlike civilian stabbings, terrorist stabbings were characterized by more commonly occurring to the upper part of the body, being executed by large knives with high force, and involving multiple and more severe injuries. There is a clear distinction between the characteristics of wounds resulting from civilian stabbings and those incurred by acts of terror. Terrorists intend to injure as many random victims as possible, and trauma units need to be prepared to cope with the simultaneous admission of multiple patients with penetrating and often life-threatening knife wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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


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


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    Shabarni Gaffar


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

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

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


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

  8. Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients. (United States)

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


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

  9. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. (United States)

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


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

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

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


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

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

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    Els Beirnaert


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

  12. Forensic evidence in apparel fabrics due to stab events. (United States)

    Kemp, S E; Carr, D J; Kieser, J; Niven, B E; Taylor, M C


    Stab injuries and fatalities have been reported to be the most common crimes of violence in several countries, particularly in those where access to firearms is restricted [J.M. Taupin, F.-P. Adolf, J. Robertson, Examination of damage to textiles, in: J. Robertson, M. Grieve (Eds.), Forensic Examination of Fibres, CRC Press, United States of America, 1999, pp. 65-87; A.C. Hunt, R.J. Cowling, Murder by stabbing, Forensic Sci. Int. 52 (1991) 107-112; D.A. Rouse, Patterns of stab wounds: a six year study, Med. Sci. Law 34 (1994) 67-71]. Analysis of damaged apparel may provide important information about the cause of death and the events leading up to and after the victim's final moments [M.T. Pailthorpe, N.A.G. Johnson, The private forensic scientist and the criminal justice system, in: D. Biles, J. Vernon (Eds.), Private Sector and Community Involvement in the Criminal Justice System: Conference Proceedings, vol. 23, Australian Institute of Criminology, Wellington, 1994, 231-240]. A high proportion of stab wounds occur in the chest and as this area is generally clothed many sharp force cases involve damage to fabrics [J.M. Taupin, F.-P. Adolf, J. Robertson, Examination of damage to textiles, in: J. Robertson, M. Grieve (Eds.), Forensic Examination of Fibres, CRC Press, United States of America, 1999, pp. 65-87; A.C. Hunt, R.J. Cowling, Murder by stabbing, Forensic Sci. Int. 52 (1991) 107-112; D.A. Rouse, Patterns of stab wounds: a six year study, Med. Sci. Law 34 (1994) 67-71]. The structural stabilisation and degradation of fabric due to laundering significantly alters fabric properties [S.E. Gore, R.M. Laing, C.A. Wilson, D.J. Carr, B.E. Niven, Standardizing a pre-treatment cleaning procedure and effects of application on apparel fabrics, Text. Res. J. 76 (2006) 455-464], yet the effect of such on severance morphology does not appear to have been investigated. In this work the effect of blade type (hunting knife, kitchen knife, screwdriver) on new and laundered

  13. The application of PA/CF in stab resistance body armor (United States)

    Yuan, M. Q.; Liu, Y.; Gong, Z.; Qian, X. M.


    Stab resistance body armor (SRBA) is an essential defensive equipment to protect human body against injuries from stabbing. The conventional SRBAs shared low wearing frequency since they are heavy and poor in flexibility. This paper designed a structured stab-resistance plate using the model of crocodile armor and manufactured using 3D printing technology-laser sintering (LS). CF(Carbon fiber) was applied to enhance the stab resistance properties of SRBA. The effects of the material and structure were analysed through the stab resistance property tests based on the national standard GA68-2008. It is found that the stab resistance property of flat plates sintered by PA powder and PA/CF are both weaker than that of the structured plate. The penetrating depth of PA/CF structured plate is significantly 2-mm-less than the pure PA structured plate. The SEM observations confirmed the conclusion that addition of the CF largely improved the plate stab resistance property. Moreover, using PA/CF structured plate to produce the stab resistance body armor would result in a weight reduction by about 30-40% as compared to the existing SRBA that was made up of metal plates, which could largely reduce the wearer physical burden and improve the wearing frequency.

  14. 76 FR 35024 - National Institute of Justice Stab-Resistant Body Armor Standard Workshop (United States)


    ... of Justice Programs National Institute of Justice Stab-Resistant Body Armor Standard Workshop AGENCY... Technology (NIST) are jointly hosting workshop focused on the NIJ Stab-Resistant Body Armor Standard. It is... planning to attend are responsible for their own travel arrangements. Registration information may be found...

  15. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro (United States)

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


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

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

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


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

  17. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface. (United States)

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


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

  18. Analytical assessment of woven fabrics under vertical stabbing - The role of protective clothing. (United States)

    Hejazi, Sayyed Mahdi; Kadivar, Nastaran; Sajjadi, Ali


    Knives are being used more commonly in street fights and muggings. Therefore, this work presents an analytical model for woven fabrics under vertical stabbing loads. The model is based on energy method and the fabric is assumed to be unidirectional comprised of N layers. Thus, the ultimate stab resistance of fabric was determined based on structural parameters of fabric and geometrical characteristics of blade. Moreover, protective clothing is nowadays considered as a strategic branch in technical textile industry. The main idea of the present work is improving the stab resistance of woven textiles by using metal coating method. In the final, a series of vertical stabbing tests were conducted on cotton, polyester and polyamide fabrics. Consequently, it was found that the model predicts with a good accuracy the ultimate stab resistance of the sample fabrics. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. An autopsy case of suicide by hanging with multiple stab wounds of the neck and chest. (United States)

    Maeda, H; Imura, M; Higuchi, T; Noguchi, K


    We report, from the aspect of 'acting' capability, an autopsy case of suicide by hanging in which multiple deep stab wounds were observed in the neck and chest. A 49-year-old man was found dead in a state typical of hanging. His car was found about 100m away with a blood-stained climber's knife on the seat. Multiple incised and stab wounds were in the neck, with a lesion of the right inner jugular vein. Three penetrating stab wounds of the chest punctured the lungs, with pooling of about 500ml of blood in the left thorax and about 100ml in the right. Two other shallow stab wounds were in the chest. All the above wounds were explainable as self-inflicted. From the autopsy findings and the circumstantial evidence, it was concluded that he walked approximately 100m after stabbing his neck and chest and finally committed suicide by hanging.

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

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


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

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

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


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

  2. Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia. (United States)

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


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

  3. Variability of simulants used in recreating stab events. (United States)

    Carr, D J; Wainwright, A


    Forensic investigators commonly use simulants/backing materials to mount fabrics and/or garments on when recreating damage due to stab events. Such work may be conducted in support of an investigation to connect a particular knife to a stabbing event by comparing the severance morphology obtained in the laboratory to that observed in the incident. There does not appear to have been a comparison of the effect of simulant type on the morphology of severances in fabrics and simulants, nor on the variability of simulants. This work investigates three simulants (pork, gelatine, expanded polystyrene), two knife blades (carving, bread), and how severances in the simulants and an apparel fabric typically used to manufacture T-shirts (single jersey) were affected by (i) simulant type and (ii) blade type. Severances were formed using a laboratory impact apparatus to ensure a consistent impact velocity and hence impact energy independently of the other variables. The impact velocity was chosen so that the force measured was similar to that measured in human performance trials. Force-time and energy-time curves were analysed and severance morphology (y, z directions) investigated. Simulant type and knife type significantly affected the critical forensic measurements of severance length (y direction) in the fabric and 'skin' (Tuftane). The use of EPS resulted in the lowest variability in data, further the severances recorded in both the fabric and Tuftane more accurately reflected the dimensions of the impacting knives. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. Management of stab wounds to the anterior abdominal wall

    Directory of Open Access Journals (Sweden)

    João Baptista Rezende-Neto

    Full Text Available The meeting of the Publication "Evidence Based Telemedicine - Trauma and Emergency Surgery" (TBE-CiTE, through literature review, selected three recent articles on the treatment of victims stab wounds to the abdominal wall. The first study looked at the role of computed tomography (CT in the treatment of patients with stab wounds to the abdominal wall. The second examined the use of laparoscopy over serial physical examinations to evaluate patients in need of laparotomy. The third did a review of surgical exploration of the abdominal wound, use of diagnostic peritoneal lavage and CT for the early identification of significant lesions and the best time for intervention. There was consensus to laparotomy in the presence of hemodynamic instability or signs of peritonitis, or evisceration. The wound should be explored under local anesthesia and if there is no injury to the aponeurosis the patient can be discharged. In the presence of penetration into the abdominal cavity, serial abdominal examinations are safe without CT. Laparoscopy is well indicated when there is doubt about any intracavitary lesion, in centers experienced in this method.

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

    International Nuclear Information System (INIS)

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


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

  6. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments. (United States)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  8. Data on crystal organization in the structure of the Fab fragment from the NIST reference antibody, RM 8671. (United States)

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


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

  9. Occurrence of anti-Leptospira spp. antibodies in Rhipidomys spp. from a forest fragment of the Brazilian Cerrado. (United States)

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


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

  10. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody. (United States)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

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


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

  14. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior* (United States)

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


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

  15. An Optimal Dynamic Data Structure for Stabbing-Semigroup Queries

    DEFF Research Database (Denmark)

    Agarwal, Pankaj K.; Arge, Lars; Kaplan, Haim


    {R}$, the stabbing-semigroup query asks for computing $\\sum_{s \\in S(q)} \\omega(s)$. We propose a linear-size dynamic data structure, under the pointer-machine model, that answers queries in worst-case $O(\\log n)$ time and supports both insertions and deletions of intervals in amortized $O(\\log n)$ time....... It is the first data structure that attains the optimal $O(\\log n)$ bound for all three operations. Furthermore, our structure can easily be adapted to external memory, where we obtain a linear-size structure that answers queries and supports updates in $O(\\log_B n)$ I/Os, where B is the disk block size....... For the restricted case of a nested family of intervals (either every pair of intervals is disjoint or one contains the other), we present a simpler solution based on dynamic trees...

  16. Holmes Tremor Secondary to a Stabbing Lesion in the Midbrain

    Directory of Open Access Journals (Sweden)

    Rubens Gisbert Cury


    Full Text Available Background: The development of Holmes tremor (HT after a direct lesion of the midbrain has rarely been reported in the literature, although several etiologies have been linked with HT, such as stroke, brainstem tumors, multiple sclerosis, head trauma, or infections.Phenomenology Shown: A 31-year-old male, having been stabbed in the right eye, presented with a rest and action tremor in the left upper limb associated with left hemiparesis with corresponding post-contrast volumetric magnetic resonance imaging T1 with sagittal oblique reformation showing the knife trajectory reaching the right midbrain.Educational Value: Despite the rarity of the etiology of HT in the present case, clinicians working with persons with brain injuries should be aware of this type of situation.

  17. The spectrum of injuries in buttock stab wounds. (United States)

    Campion, T; Cross, S


    Buttock stab wounds are a surprisingly common and increasing source of presentations to emergency departments. These injuries can have a significant impact on quality of life, and there are a number of often subtle, but significant, injuries that the radiologist must be alert to when interpreting computed tomography examinations in these patients. In this review, we will examine briefly the sociological reasons for the increase in these injuries, discuss appropriate imaging techniques, and provide imaging examples of the clinically important injuries that may be encountered in this region. These injuries include rectal or colonic perforation; genito-urinary trauma, for example urethral injury; injury to the sciatic nerve; and a spectrum of vascular trauma including transection and pseudoaneurysm or arteriovenous fistula formation. Copyright © 2017 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

  18. The persistent diagnostic challenge of thoracoabdominal stab wounds. (United States)

    Berg, Regan J; Karamanos, Efstathios; Inaba, Kenji; Okoye, Obi; Teixeira, Pedro G; Demetriades, Demetrios


    Penetrating thoracoabdominal trauma, with potential injury to two anatomic cavities, significantly challenges surgical management, yet this injury pattern has not been reviewed across a large patient series. The trauma registry of a major level 1 center was queried for all adult patients admitted with thoracoabdominal stab wounds between January 1996 and December 2011. The study identified 617 patients; 11% arrived hypotensive (systolic blood pressure undergoing thoracotomy. Among this group, only 1 (2.4%) of 41 had a major thoracic vessel or aortic injury without cardiac trauma. Diaphragmatic injury (DI) occurred in 224 (38%) of 595, with 72 (32.1%) of these 224 demonstrating no computed tomographic evidence of DI. Either hollow viscus injury or DI occurred in 50%. Only 36.8% of liver, 58% of spleen, and 29.8% of kidney injuries required surgical repair. The need for dual-cavitary intervention was associated with a precipitous increase in patient mortality. Patients with thoracoabdominal stab wounds present considerable clinical challenges due to high surgical need, high occult DI incidence, persistently high rates of negative laparotomy, and significant mortality with dual-cavitary intervention. Many patients with solid-organ injuries do not require intervention. High incidence of hollow viscus injury and DI ultimately limits nonoperative management. Laparoscopy is necessary to exclude occult DI. In unstable patients, determination of which anatomic cavity to explore primarily requires exclusion of cardiac injury. In those with equivocal clinical or ultrasonographic evidence of cardiac trauma, laparotomy, with transdiaphragmatic pericardial window, if a causative abdominal injury is not immediately apparent, seems the most effective strategy. Epidemiologic study, level III.

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

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


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

  20. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas. (United States)

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


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

  1. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen (United States)

    Kuroki, Motomu; Kuroki, Masahide


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

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

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


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

  3. Stab Resistance of Shear Thickening Fluid (STF)-Kevlar Composites for Body Armor Applications

    National Research Council Canada - National Science Library

    Egres Jr., R. G; Decker, M. J; Halbach, C. J; Lee, Y. S; Kirkwood, J. E; Kirwood, K. M; Wagner, N. J; Wetzel, E. D


    The stab resistance of shear thickening fluid (STF)-Kevlar and STF-Nylon fabric composites are investigated and found to exhibit significant improvements over neat fabric targets of equivalent areal density...



    Raikwar; Odiya; Ashish; Sachin


    AIMS AND OBJECTIVES: The aim of this study was to , 1. E valuate the incidence of stab injuries admitting in our institute, t o know various parameter and its association with injuries such as age group, sex ratio, clinical presentation . 2. V arious surgical interventi on and conservative treatment according the nature of stab injuries and through examination. 3. M orbidity and mortality of these patients , settings and design . ...

  5. Perforation of the ileum after a stab wound of the gluteal region: a case report


    van Oldenrijk, Jakob; Ünlü, Çaĝdaş; van Wagensveld, Bart A


    We present a case of a patient with a seemingly insignificant single gluteal stab wound which led to a solitary perforation of the ileum and delayed peritonitis. This case report illustrates that, despite the absence of any signs of bowel perforation on presentation, a patient may deteriorate gradually in the subsequent hours. This demonstrates the role of clinical observation in high risk gluteal stab wound patients.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Laia Montoliu-Gaya

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

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


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


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

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

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen


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

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

    Directory of Open Access Journals (Sweden)

    Philippe Thullier

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

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Shibaguchi H


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

  15. Candida albicans spondylodiscitis following an abdominal stab wound: forensic considerations. (United States)

    Savall, Frederic; Dedouit, Fabrice; Telmon, Norbert; Rougé, Daniel


    Candida albicans spondylodiscitis is a fungal infection of the spine which is still unusual in spite of the increasing frequency of predisposing factors. A 22-year-old man received an abdominal stab wound during a physical assault. Initial medical care included surgery, prolonged use of indwelling vascular catheters with administration of broad-spectrum antibiotics, and hospitalization in intensive care. Two months after the event, the victim experienced back pain in the right lumbar region and septic spondylodiscitis secondary to C. albicans was diagnosed three weeks later. This case is noteworthy because of its clinical forensic context. In France, the public prosecutor orders a medico-legal assessment after an assault for all living victims in order to establish a causal relationship between the assault and its complications. In our case, the patient presented numerous risk factors for candidemia and the forensic specialist reasonably accepted that the causal relationship was certain but indirect. We have only found one published case of spondylodiscitis after an abdominal penetrating injury and the pathogenic agent was not mentioned. We have found no case reported in a forensic context. This unusual observation shows that it may be genuinely difficult to prove the causal relationship between an abdominal penetrating injury and an unusual infectious complication such as fungal spondylodiscitis. Copyright © 2014 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  16. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms. (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop


    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.


    Directory of Open Access Journals (Sweden)



    Full Text Available AIMS AND OBJECTIVES: The aim of this study was to , 1. E valuate the incidence of stab injuries admitting in our institute, t o know various parameter and its association with injuries such as age group, sex ratio, clinical presentation . 2. V arious surgical interventi on and conservative treatment according the nature of stab injuries and through examination. 3. M orbidity and mortality of these patients , settings and design . The study was conducted in a retrospective and prospec tive manner and included cases b etween January 2009 and September 2013. METHODS AND MATERIAL S: 424 patients admitted with stab injuries in ICU and ward . Various surgical interventions were done according to standard indication such as laparotomies , thoracotomies, vascular repair etc. , and resul ts reported. RESULTS: Out of 424 patients t here were over all 413 mal es (97.4% and 11 female s (2.6% highest number of cases (226 in the third decade(21 - 30 i.e. 53.3% maximum number of cases are from 11 - 40 yrs. Majority of the stab wounds were homicidal in nature comprising 386 cases i.e. 91 % followed by are accidental. Chaku ( knife were the most common weapon . The Chaku was used in 351 cases i.e. 82.8% maximun numbers of patients 309 were presented with localised tende r ness at site of stab injury i.e 72.87%. In the perspective of management 244 abdominal stab that there were total 189(44.6% exploratory laparotomy . In 189 laparotomies peritoneal breech in 114 patients on local examination / exploration was the major indic ation there were 114 i.e ., 60.3% CONCLUSION: Stab injuri es are becoming common now a days because people often try to settle interpersonal relationship and political problems by mean of stabbing, although its incidence more in our country as compare d to European studies because of overpopulation unemployment and poverty. Incidence of stab injury can be reduced by improving the social morale of people especially the younger generation by

  18. A combined experimental and numerical study of stab-penetration forces. (United States)

    Annaidh, Aisling Ní; Cassidy, Marie; Curtis, Michael; Destrade, Michel; Gilchrist, Michael D


    The magnitude of force used in a stabbing incident can be difficult to quantify, although the estimate given by forensic pathologists is often seen as 'critical' evidence in medico-legal situations. The main objective of this study is to develop a quantitative measure of the force associated with a knife stabbing biological tissue, using a combined experimental and numerical technique. A series of stab-penetration tests were performed to quantify the force required for a blade to penetrate skin at various speeds and using different 'sharp' instruments. A computational model of blade penetration was developed using ABAQUS/EXPLICIT, a non-linear finite element analysis (FEA) commercial package. This model, which incorporated element deletion along with a suitable failure criterion, is capable of systematically quantifying the effect of the many variables affecting a stab event. This quantitative data could, in time, lead to the development of a predictive model that could help indicate the level of force used in a particular stabbing incident. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab')2

    International Nuclear Information System (INIS)

    Anderson, C.J.; Schwarz, S.W.; Connett, J.M.


    Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab') 2 was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of 64 Cu-BAT-2IT-1A3-F(ab') 2 was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N double-prime,N'double-prime-tetraacetic acid (BAT) was conjugated to 1A3-F(ab') 2 using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab'. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab') 2 from TETA-1A3-Fab' after conjugation prior to labeling with 64 Cu. The biodistribution of 64 Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab') 2 , the immunoreactivity of 64 Cu-labeled 1A3-F(ab') 2 was significantly improved over that of non-FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 , and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 .The improved dosimetry of 64 Cu-labeled FPLC purified BAT-2IT-1A3-F(ab') 2 should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs

  20. Radiolabeled Antibody Fragment for Preparation of (177Lu-DOTAm-PAMAM G3.0-F(ab’2 trastuzumab as a Radiopharmaceutical for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    R.D. Haryuni


    Full Text Available Several radiolabeled monoclonal antibodies (mAbs have been used as radioimmunotherapy (RIT agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α particles, beta (β particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of (177Lu-DOTAm-PAMAM G3-F(ab'2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2. Due to its reduced molecular weight, the use of F(ab'2-trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH2 functional groups that are able to bind many DOTAs (£ 31 which in turn can bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab'2-trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab'2-trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC to produce SMCC-F(ab'2-trastuzumab. The next reaction was to conjugate SMCC-F(ab'2-trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab. Finally, the (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab was radiolabelled with 177Lu to produce (177Lu-DOTAm-PAMAM G3.0-F(ab'2-trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column.Received: 31 October 2015; Revised: 30 June 2016; Accepted: 25 September 2016

  1. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B


    Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage...

  2. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S


    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

  3. A Dynamic Stabbing-Max Data Structure with Sub-Logarithmic Query Time


    Nekrich, Yakov


    In this paper we describe a dynamic data structure that answers one-dimensional stabbing-max queries in optimal $O(\\log n/\\log\\log n)$ time. Our data structure uses linear space and supports insertions and deletions in $O(\\log n)$ and $O(\\log n/\\log \\log n)$ amortized time respectively. We also describe a $O(n(\\log n/\\log\\log n)^{d-1})$ space data structure that answers $d$-dimensional stabbing-max queries in $O((\\log n/\\log\\log n)^{d})$ time. Insertions and deletions are supported in $O((\\lo...

  4. Experimental study of thermal comfort on stab resistant body armor. (United States)

    Ji, Tingchao; Qian, Xinming; Yuan, Mengqi; Jiang, Jinhui


    This research aims to investigate the impacts of exercise intensity and sequence on human physiology parameters and subjective thermal sensation when wearing stab resistant body armor under daily working conditions in China [26 and 31 °C, 45-50 % relative humidity (RH)], and to investigate on the relationship between subjective judgments and objective parameters. Eight male volunteers were recruited to complete 3 terms of exercises with different velocity set on treadmill for 90 min at 26 °C and 31 °C, 45-50 % RH. In Exercise 1 volunteers were seated during the test. In Exercise 2, volunteers walked with the velocity of 3 km/h in the first 45 min and 6 km/h in the left 45 min. In Exercise 3, volunteers walked with the velocity of 6 km/h in the first 45 min and 3 km/h in the left 45 min. The body core temperature, skin temperature and subjective judgments were recorded during the whole process. Analysis of variance was performed among all the tests. Individual discrepancy of Exercise 1 is larger than that of Exercise 2 and 3. On the premise of the same walking distance and environmental conditions, core temperature in Exercise 3 is about 0.2 °C lower than that in Exercise 2 in the end; and with the velocity decrease from 6 km/h to 3 km/h in the end, thermal tolerance of Exercise 3 is about 1 degree lower than that in Exercise 2. Skin temperatures of human trunk were at least 1 °C higher than that of limbs. Activity narrows the individual discrepancy on core temperature. Within experimental conditions, decreasing of intensity at last stage makes the core temperature lower and the whole process much tolerable. The core temperature is more sensitive to the external disturbance on the balance of the whole body, and it can reflect the subjective thermal sensation and physical exertion.

  5. Development of a high-density nonwoven structure to improve the stab resistance of protective clothing material. (United States)

    Bao, Limin; Wang, Yanling; Baba, Takeichiro; Fukuda, Yasuhiro; Wakatsuki, Kaoru; Morikawa, Hideaki


    The purpose of this research was to enhance the stab resistance of protective clothing material by developing a new high-density nonwoven structure. Ice picks often injure Japanese police officers due to the strict regulation of swords in the country. Consequently, this study was designed to improve stab resistance against ice picks. Most existing anti-stab protective clothing research has focused on various fabrics impregnated with resin, an approach that brings with it problems of high cost and complicated processing. Seldom has research addressed the potential for improving stab resistance by using nonwoven structures, which exhibit better stab resistance than fabric. In this research, we prepared a series of nonwoven structures with densities ranging from about 0.14 g/cm 3 to 0.46 g/cm 3 by varying the number of stacked layers of Kevlar/polyester nonwoven under a hot press. We then proposed two methods for producing such hot-press nonwovens: the multilayer hot-press method and the monolayer hot-press method. Stab resistance was evaluated according to NIJ Standard-0115.00. We also investigated the relationship among nonwoven density, stab resistance, and flexural rigidity, and here we discuss the respective properties of the two proposed methods. Our results show that stab resistance and flexural rigidity increase with nonwoven density, but flexural rigidity of nonwovens prepared using the monolayer hot-press method only shows a slight change as nonwoven density increases. Though the two methods exhibit little difference in maximum load, the flexural rigidity of nonwovens prepared using the monolayer hot-press method is much lower, which contributes to superior wear comfort. Finally, we investigated the mechanism behind the stabbing process. Stabbing with an ice pick is a complicated process that involves many factors. Our findings indicate that nonwovens stop penetration primarily in two ways: nonwoven deformation and fiber fractures.

  6. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S


    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...... the development of lupus nephritis at an early stage....

  7. [Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25-35)]. (United States)

    Kolobov, V V; Davydova, T V; Fomina, V G


    Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp 1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ25-35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp 1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.

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

    Directory of Open Access Journals (Sweden)

    Xiaobo Xu


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

  9. Clinical patterns of primary stabbing headache: a single clinic-based prospective study. (United States)

    Kim, Dong Yeop; Lee, Mi Ji; Choi, Hyun Ah; Choi, Hanna; Chung, Chin-Sang


    The clinical features and disease courses of primary stabbing headache (PSH) are diverse. We aimed to identify distinct clinical patterns of PSH. We prospectively screened consecutive first-visit patients who presented with stabbing headache at the Samsung Medical Centre Headache Clinic from June 2015 to March 2016. Demographics, headache characteristics, and disease courses were prospectively evaluated. After discerning factors related to the chronicity at the time of presentation, clinical patterns were identified based on the frequency (daily vs. intermittent), clinical course (remitted or not), and total disease duration (3 months). In the 65 patients with PSH included in this study, monophasic (n = 31), intermittent (n = 17), and chronic daily (n = 12) patterns were identified. The median disease durations were 9 days for monophasic PSH, 9 months for chronic daily PSH, and 2 years for intermittent PSH. The features of monophasic PSH were greater severity, single and side-locked locations, more attacks per day, daily occurrence, and good treatment response. Chronic daily PSH was associated with female predominance, longer-lasting stabs, and multiple or migrating locations on bilateral or alternating sides. The characteristics of intermittent PSH included female predominance and sporadic stabs with less intensity. Our study demonstrated distinct clinical patterns of PSH. In addition to help early recognition of disease, our findings suggest different pathophysiologic mechanisms. Future prospective studies are required to reveal the etiologies of these different PSH patterns and their optimal treatment strategies.

  10. Influence of Chemical Surface Modification of Woven Fabrics on Ballistic and Stab Protection of Multilayer Packets

    Directory of Open Access Journals (Sweden)



    Full Text Available In order to achieve enhanced protective and wear (flexibility, less bulkiness properties of ballistic and stab protecting panels the investigation of chemical surface modification of woven p-aramid fabrics was performed applying different chemical composition shear thickening fluid (STF which improves friction inside fabric structure. For the chemical treatment silicic acid and acrylic dispersion water solutions were used and influence of their different concentrations on panels’ protective properties were investigated. Results of ballistic tests of multilayer protective panel have revealed that shear thickening effect was negligible when shooting at high energy range (E > 440 J. Determination of stab resistance of p-aramid panels has shown that different chemical composition of STFs had different influence on protective properties of the panels. Application of low concentrations of silicic acid determined higher stab resistance values comparing to higher concentrations of acrylic dispersion water solutions. At this stage of research stab tests results as ballistic ones determined that STF application for multilayer p-aramid fabrics protective panels is more efficient at low strike energy levels. DOI:

  11. Virtual casting of stab wounds in cartilage using micro-computed tomography. (United States)

    Pounder, Derrick J; Sim, Louise J


    In homicidal stabbings using a serrated knife, stab wounds involving costal cartilage leave striations on the cut surface of the cartilage from the serration points on the blade edge. Class characteristics of the serrated blade can be determined from the striation marks, and individualizing characteristics may be seen also. The traditional method for recording the striation marks involves the pernickety technique of casting with dental impression material. We assessed the potential utility of micro-computed tomography scanning of the stab track as a technique for nondestructive recording of striation patterns and virtual casting of them. Stab tracks in porcine cartilage, produced with a coarsely serrated blade, were scanned with a bench-top micro-computed tomography scanner. The typical irregularly regular striation pattern could be demonstrated, and the images manipulated, using proprietary software to produce a virtual cast. Whether the technology will have sufficient resolution to image not only class characteristic tool marks but also the much finer individualizing tool marks remains to be evaluated, but the technology shows considerable promise.


    DEFF Research Database (Denmark)

    Olsen, J. E.; Brown, D. J.; Baggesen, Dorte Lau


    Four strains of Salmonella berta and one of Salm. enteritidis were stored as stab cultures in sugar-free agar at 5 degrees, 22 degrees and 30 degrees C and in 15% glycerol at -80 degrees C. The stability of the plasmid profiles in each of the strains was monitored over a period of 2.5 years. Plas...

  13. Impact of body mass index on injury in abdominal stab wounds: implications for management. (United States)

    Bloom, Matthew B; Ley, Eric J; Liou, Douglas Z; Tran, Tri; Chung, Rex; Melo, Nicolas; Margulies, Daniel R


    Although it is assumed that obese patients are naturally protected against anterior abdominal stab wounds, the relationship has never been formally studied. We sought to examine the impact of body mass index (BMI) on severity of sustained injury, need for operation, and patient outcomes. We conducted a review of all patients presenting with abdominal stab wounds at an urban level I trauma center from January 2000-December 2012. Patients were divided into groups based on their BMI (35). Data abstracted included baseline demographics, physiologic data, and characterization of whether the stab wound had violated the peritoneum, caused intra-abdominal injury, or required an operation that was therapeutic. The one-sided Cochran-Armitage trend test was used for significance testing of the protective effect. Of 281 patients with abdominal stab wounds, 249 had complete data for evaluation. Chest and abdomen abbreviated injury scale trends decreased with increasing BMI, as did overall injury severity score, the percent of patients severely injured (injury severity score ≥ 25), and length of intensive care unit stay. Rates of peritoneal violation (100%, 84%, 77%, and 74%; P = 0.077), visceral injury (83%, 56%, 50%, and 30%; P = 0.022), and injury requiring a therapeutic operation (67%, 45%, 40%, and 20%; P = 0.034) all decreased with increasing BMI. Patients in the thinnest group required an operation three times more often than those in the most obese. Increased BMI protects patients with abdominal stab wounds and is associated with lower incidence of severe injury and need for operation. Heavier patients may be more suitable to observation and serial examinations, whereas very thin patients are more likely to require an operation and be critically injured. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; van der Graaf, W.T.; Oyen, W.J.G.; Boerman, O.C.


    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  15. Comparison of IgG and F(ab')2 fragments of bispecific anti-RCCxanti-DTIn-1 antibody for pretargeting purposes.

    NARCIS (Netherlands)

    Schaijk, F.G. van; Boerman, O.C.; Soede, A.C.; McBride, W.J.; Goldenberg, D.M.; Corstens, F.H.M.; Oosterwijk, E.


    PURPOSE: An effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCCxanti-DTPA(In) (bsMAb: G250xDTIn-1). Tumour uptake of a (111)In-labelled bivalent peptide after pretargeting with bsMAb G250xDTIn-1 was

  16. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik


    three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol...

  17. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability. (United States)

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold


    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  18. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  19. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody (United States)

    Shang, Yuqin; Mernaugh, Ray


    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  20. Complex Suicide by Self-stabbing and Drowning: A Case Report and a Review of Literature. (United States)

    Peyron, Pierre-Antoine; Casper, Thierry; Mathieu, Olivier; Musizzano, Yuri; Baccino, Eric


    Sharp force injuries and drowning are methods exceptionally combined in complex suicides. We report a challenging case of complex suicide by self-stabbing and drowning that illustrates the difficulty in discriminating between homicide and suicide in such circumstances of death. The corpse of a young man was found submerged in a river, stabbed nine times with two wounds that had penetrated the thorax and had caused lung injuries and a hemopneumothorax. The postmortem and histological examinations were consistent with a death caused by drowning, but the manner of death still remained undetermined. Police investigation finally concluded to a suicide, although no suicide note had been left and the victim had no underlying diagnosed mental disorder. The parameters that may help distinguish suicide from homicide at the autopsy should be interpreted in light of a thorough forensic investigation to determine the exact manner of death in such a case. © 2017 American Academy of Forensic Sciences.


    Directory of Open Access Journals (Sweden)

    Zaiton A


    Full Text Available This report describes a case of cortical blindness that followed successful surgical repair of two stab wounds in the heart in a 29-year old Libyan man. The patient presented in a state of pre cardiac arrest (shock and low cardiac output status, following multiple chest stab wounds. Chest tube was immediately inserted. Surgery was urgently performed suturing the two wounds; in the root of the aorta and in the left ventricle, and haemostasis was secured. Cardiac arrest was successfully prevented. The patient recovered smoothly, but 24 hours later he declared total blindness. Ophtalmic and neurological examinations and investigations that included fundoscopy, Electroencephalograms (EEGs and Computed Tomography Scans revealed no abnormalities, apart from absence of alpha waves in the EEGs. We diagnosed the case as cortical blindness and continued caring for the patient conservatively. Three days later, the patient regained his vision gradually and was discharged on the 7th postoperative day without any remarks.

  2. Serial knife stabbings with HIV exposure--implications for post-exposure prophylaxis. (United States)

    Jorgensen, Pernille; Marcus, Ulrich; Albrecht, Helmut; Suttorp, Norbert; Schürmann, Dirk


    Thirty-three persons became victims of a serial knife stabbing incident. One of the first victims one day later disclosed that he was HIV-infected. Thereafter thirty-one victims initiated HIV post-exposure prophylaxis (PEP), one exposed patient declined. None of the victims evaluated had seroconverted six months later. In most such incidents HIV exposure will be difficult to rule out as reliable information on the HIV serostatus of all serial victims will be lacking. It appears prudent, however, to inform serial stab victims about the potential risk of HIV transmission and to at least consider PEP in such scenarios. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

  3. Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake

    International Nuclear Information System (INIS)

    Rotman, Maarten; Welling, Mick M.; Boogaard, Marlinde L. van den; Moursel, Laure Grand; Graaf, Linda M. van der; Buchem, Mark A. van; Maarel, Silvère M. van der; Weerd, Louise van der


    Introduction: Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab′)2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. Methods: To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ( 111 In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 μg VHH-pa2H-Fc-DTPA- 111 In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. Results: We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50 mg/L growth medium. The fusion protein showed homodimerization – necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA- 111 In and the VHH-Fc-DTPA- 111 In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA- 111 In

  4. Astrocyte morphology after cortical stab wound revealed by single-cell confocal 3D morphometry

    Czech Academy of Sciences Publication Activity Database

    Chvátal, Alexandr; Anděrová, Miroslava; Petřík, David; Syková, Eva

    č. 2 (2003), s. 63 ISSN 0894-1491. [European Meeting on Glial Cell Function in Health and Disease /6./. Berlín, 03.09.2003-06.09.2003] R&D Projects: GA ČR GA305/02/1528; GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5039906; CEZ:MSM 111300004 Keywords : cortical stab wound * morphometry Subject RIV: FH - Neurology Impact factor: 4.677, year: 2003

  5. IP-telefon säästab oluliselt sidekulusid / Kristjan Otsmann

    Index Scriptorium Estoniae

    Otsmann, Kristjan, 1971-


    Telekomifirma Norby Telecomi poolt pakutavast IP-telefonist ehk helistamisest läbi Interneti, millega saab vähendada sidekulusid mitmeid kordi. Erinevatest internetipõhise kõnesidelahenduse lisateenustest. Vt. samas: Saaremaa Spa Hotellid säästab tuhandeid kroone kuus [intervjuu Saaremaa Spa Hotellide IT-juhiga Andri Võrguga]; Kuus olulist tööd enne IP-lahenduse tellimist

  6. Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice

    International Nuclear Information System (INIS)

    Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P.


    During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-( 131 I) labeled intact antibodies or 131 I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2

  7. Crystal structure of snake venom acetylcholinesterase in complex with inhibitory antibody fragment Fab410 bound at the peripheral site: evidence for open and closed states of a back door channel. (United States)

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale


    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.


    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  9. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    International Nuclear Information System (INIS)

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.


    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging

  10. The future of monoclonal antibody technology


    Zider, Alexander; Drakeman, Donald L


    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  11. Nuclear fragmentation

    International Nuclear Information System (INIS)

    Chung, K.C.


    An introduction to nuclear fragmentation, with emphasis in percolation ideas, is presented. The main theoretical models are discussed and as an application, the uniform expansion approximation is presented and the statistical multifragmentation model is used to calculate the fragment energy spectra. (L.C.)

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

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


    Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

  13. Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

    Energy Technology Data Exchange (ETDEWEB)

    Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Weisgraber, Karl [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Department of Pathology, University of California, San Francisco, CA 94143 (United States); Finkbeiner, Steven, E-mail: [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Departments of Neurology, Physiology and Neuroscience and Biomedical Sciences Program, University of California, San Francisco, CA 94143 (United States)


    Optimization of crystallization conditions and cryoprotectants decreased the anisotropy of the diffraction obtained from 3B5H10 Fab crystals. Dehydration improved the resolution of cryoprotected 3B5H10 crystals from 2.6 to 1.9 Å, but changed the space group of the crystals from P2{sub 1}2{sub 1}2 to P2{sub 1}. Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 Å resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 Å and space group P2{sub 1}2{sub 1}2. Dehydrated crystals diffracted isotropically to 1.9 Å with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 Å, β = 90.3° and space group P2{sub 1}.

  14. In Vivo HER2-Targeted Magnetic Resonance Tumor Imaging Using Iron Oxide Nanoparticles Conjugated with Anti-HER2 Fragment Antibody. (United States)

    Ding, Ning; Sano, Kohei; Kanazaki, Kengo; Ohashi, Manami; Deguchi, Jun; Kanada, Yuko; Ono, Masahiro; Saji, Hideo


    The feasibility of iron oxide nanoparticles (IONPs) conjugated with anti-epidermal growth factor receptor 2 (HER2) single-chain antibody (scFv-IONPs) as novel HER2-targeted magnetic resonance (MR) contrast agents was investigated. The scFv-IONPs were prepared and identified. For in vitro MRI, NCI-N87 (HER2 high expression) and SUIT2 (low expression) cells were incubated with scFv-IONPs. For in vivo MRI, NCI-N87 and SUIT2 tumor-bearing mice were intravenously injected with scFv-IONPs and imaged before and 24 h post-injection. The scFv-IONPs demonstrated high transverse relaxivity (296.3 s -1  mM -1 ) and affinity toward HER2 (K D  = 11.7 nM). In the in vitro MRI, NCI-N87 cells treated with scFv-IONPs exhibited significant MR signal reduction (44.6 %) than SUIT2 cells (6.8 %). In the in vivo MRI, decrease of MR signals in NCI-N87 tumors (19.3 %) was more notable than that in SUIT2 tumors (6.2 %). The scFv-IONPs enabled HER2-specific tumor MR imaging, suggesting the potential of scFv-IONPs as a robust HER2-targeted MR contrast agent.

  15. Importance of diagnostic laparoscopy in the assessment of the diaphragm after left thoracoabdominal stab wound: A prospective cohort study. (United States)

    Yücel, Metin; Özpek, Adnan; Tolan, Hüseyin Kerem; Başak, Fatih; Baş, Gürhan; Ünal, Ethem; Alimoğlu, Orhan


    Stab wounds in the left thoracoabdominal region may cause diaphragmatic injury. The aim of the present study was to determine incidence of diaphragmatic injury and role of diagnostic laparoscopy in detection of injury in patients with left thoracoabdominal stab wound. Total of 81 patients (75 male, 6 female; mean age 27.5±9.8 years; range 14 to 60 years) who presented with left thoracoabdominal stab wound between April 2009 and September 2014 were evaluated. Laparotomy was performed on patients who had hemodynamic instability, signs of peritonitis, or organ evisceration. Remaining patients were followed conservatively. After 48 hours, diagnostic laparoscopy was performed on patients without laparotomy indication to examine the left diaphragm for injury. Follow-up and treatment findings were prospectively evaluated. Thirteen patients underwent laparotomy while diagnostic laparoscopy was performed on remaining 68 patients. Left diaphragmatic injury was observed in 19 patients (23.5%) in the study group. Four injuries were diagnosed by laparotomy and 15 were diagnosed by laparoscopy. Presence of hemopneumothorax did not yield difference in incidence of diaphragmatic injury (p=0.131). No significant difference was detected in terms of diaphragmatic injury with respect to entry site of stab wound in the thoracoabdominal region (p=0.929). It is important to evaluate the diaphragm in left thoracoabdominal stab injuries, and diagnostic laparoscopy is still the safest and most feasible method.

  16. Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Pfister, C.; Fournier, K.; Prevel, F.; Schreyer, M.; Carrel, S.; Mach, J.P.


    Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131 I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131 I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131 I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation

  17. 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.

  18. Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea. (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


    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.

  19. Cervical spondylodiscitis with epidural abscess after knife stab wounds to the neck: A case report. (United States)

    Voelker, Anna; von der Hoeh, Nicolas H; Gulow, Jens; Heyde, Christoph-Eckhard


    Cervical spondylodiscitis is usually caused by pyogenic infections, associated with retropharyngeal abscesses, or due to the swallowing of foreign bodies. No cases of cervical spondylodiscitis caused by a penetrating neck injury have been published in the literature. We describe a case of cervical spondylodiscitis after multiple knife stab wounds to the lateral soft tissue of the neck. Case report and review of the literature. A 54-year-old patient was brought to our clinic with destructive spondylodiscitis C3/4 with paravertebral and epidural abscesses. He had been involved in a fight and had suffered multiple stab wounds to his neck with a knife 1 month prior. The initial CT scan had revealed one deeper wound canal behind the sternocleidomastoid muscle on the left side without any injury to the vessels. The wound was cleaned and an antibiotic therapy with cefuroxime was given for 1 week. After an uneventful and complete healing of the wound the patient developed severe neck pain. Inflammatory laboratory parameters were elevated, and a MRI of the neck revealed a distinct spondylodiscitis C3/4 with paravertebral and epidural abscess formations. Surgery was performed and included debridement, abscess drainage, decompression of the spinal canal, fusion of the C3/4 segment using an autologous iliac crest bone graft and a plate osteosynthesis. A course of calculated antibiotic therapy was administered for 8 weeks. Normal laboratory parameters and no radiological signs of an ongoing inflammatory process were observed during follow-up examinations. The C3/4 segment was consolidated. Stab wound injuries to the neck not only bear the risk of injuries to the nerves, vessels and organs of the neck but also increase the risk of developing secondary spondylodiscitis. Specifically, cervical spondylodiscitis can result in distinct neurological symptoms, and surgical intervention should be performed in a timely manner. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Spinal stab injury with retained knife blades: 51 Consecutive patients managed at a regional referral unit. (United States)

    Enicker, Basil; Gonya, Sonwabile; Hardcastle, Timothy C


    Spinal stab wounds presenting with retained knife blades (RKB) are uncommon, often resulting in spinal cord injury (SCI) with catastrophic neurological consequences. The purpose of this study is to report a single unit's experience in management of this pattern of injury at this regional referral centre. Retrospective review of medical records identified 51 consecutive patients with spinal stabs presenting with a RKB at the Neurosurgery Department at Inkosi Albert Luthuli Central Hospital between January 2003 and February 2015. The data was analyzed for patient characteristics, level of the RKB, neurological status using the ASIA impairment scale, associated injuries, radiological investigations, management, hospital length of stay, complications and mortality. The mean age was 28±10.9 years (range 14-69), with 45 (88%) males (M: F=7.5:1). The median Injury Severity Score was 16 (range 4-26). RKB were located in the cervical [9,18%], thoracic [38,74%], lumbar [2,4%] and sacral [2,4%] spine. Twelve patients (24%) sustained complete SCI (ASIA A), while 21 (41%) had incomplete (ASIA B, C, D), of which 17 had features of Brown-Sequard syndrome. Eighteen (35%) patients were neurologically intact (ASIA E). There were 8 (16%) associated pneumothoraces and one vertebral artery injury. Length of hospital stay was 10±7.1 days (range 1-27). One patient (2%) died during this period. Stab injuries to the spine presenting with RKB are still prevalent in South Africa. Resources should be allocated to prevention strategies that decrease the incidence of inter-personal violence. All RKBs should be removed in the operating theatre by experienced surgeons to minimise complications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Laparoscopic diagnostic peritoneal lavage (L-DPL): A method for evaluation of penetrating abdominal stab wounds. (United States)

    Krausz, Michael M; Abbou, Benyamine; Hershko, Dan D; Mahajna, Ahmad; Duek, Daniel S; Bishara, Bishara; Israelit, Shlomo H


    The management of penetrating abdominal stab wounds has been the subject of continued reappraisal and controversy. In the present study a novel method which combines the use of diagnostic laparoscopy and DPL, termed laparoscopic diagnostic peritoneal lavage (L-DPL) is described Five trauma patients with penetrating injuries to the lower chest or abdomen were included. Standard videoscopic equipment is utilized for the laparoscopic trauma evaluation of the injured patient. When no significant injury is detected, the videoscope is withdrawn and 1000 mL of normal saline is infused through the abdominal trochar into the peritoneal cavity, and the effluent fluid studied for RBCs, WBC, amylase debry, bile as it is uced in regular diagnostic peritoneal lavage Laparoscopic peritoneal lavage (L-DPL) was then performed and proved to be negative in all 5 patients. RBC lavage counts above 100,000/mcrl were not considered as a positive lavage result, because the bleeding source was directly observed and controlled laparoscopically. All patients recovered uneventfully and were released within 3 days. This procedure combines the visual advantages of laparoscopy together with the sensitivity and specificty of DPL for the diagnosis of significant penetrating intra-abdominal injury, when the diagnostic strategy of selective consevatism for abdominal stab wounds is adopted. A method of laparoscopic diagnostic peritoneal lavage (L-DPL) in hemodynamically stable patients with penetrating lower thoracic or abdominal stab wounds is described. The method is especially applicable for trauma surgeons with only basic experience in laparoscopic technique. This procedure is used to obtain conclusive evidence of significant intra-abdominal injury, confirm peritoneal penetration, control intra-abdominal bleeding, and repair lacerations to the diaphragm and abdominal wall. The combination of laparoscopy and DPL afforded by the L-DPL method adds to the sensitivity and specificity of DPL, and

  2. Early MRI findings in stab wound of the cervical spine: two case reports

    Energy Technology Data Exchange (ETDEWEB)

    Alkan, A.; Baysal, T.; Sarac, K.; Sigirci, A.; Kutlu, R. [Inonu Universitesi Turgut Ozal Tip Merkezi, Radyoloji Anabilim Dali, Malatya (Turkey)


    MR imaging was found to be the most sensitive modality for the detection of spinal cord abnormalities in the acutely injured spine. Although it is reported that traumatic pneumomyelogram indicates a base-of-skull or middle cranial fossa fracture and is almost certainly associated with intracranial subarachnoid air, early MR imaging may demonstrate subarachnoid air in penetrating trauma of the spinal cord without head injury. We report two cervical-spine stab-wound cases, one of which had subarachnoid air on early MR findings. (orig.)

  3. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.


    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  4. Multiple stab wounds: understanding the manner of death through the psychological autopsy. (United States)

    Di Vella, G; Grattagliano, I; Curti, S; Catanesi, R; Sullivan, M K; Tattoli, L


    A 48-year-old physical therapist with no history of psychiatric disorders or suicidal tendencies was found dead by his relatives inside his apartment. Multiple stab wounds were present on the chest and the abdomen. Several more superficial cuts were also seen on the neck and the left wrist. At the death scene, there was a large pool of blood in the room, with secondary droplets and stains; a kitchen knife was found beside the body, and a suicide note on a table. Especially the number of wounds raised strong suspicion of a homicide in the first instance but some circumstances (the farewell letter, the absence of clothing injuries, no signs of third-party involvement) and autopsy findings (absence of defense or blunt injuries, the localization of the wounds, the presence of hesitation marks) pointed toward a suicide etiology. Because of the many stab injuries and the lack of a psychiatric history, further forensic investigations were required including a so-called psychological autopsy. This case report highlights that only a comprehensive interpretation of all the elements (circumstances, the scene of death, autopsy findings, and psychological autopsy) can lead to the correct solution of atypical cases of suicide.

  5. An analysis of predictors of morbidity after stab wounds of the pancreas in 78 consecutive injuries. (United States)

    Krige, J E J; Kotze, U K; Sayed, R; Navsaria, P H; Nicol, A J


    Penetrating injuries of the pancreas may result in serious complications. This study assessed the factors influencing morbidity after stab wounds of the pancreas. A retrospective univariate cohort analysis was carried out of 78 patients (74 men) with a median age of 26 years (range: 16-62 years) with stab wounds of the pancreas between 1982 and 2011. The median revised trauma score (RTS) was 7.8 (range: 2.0-7.8). Injuries involved the body (n=36), tail (n=24), head/uncinate process (n=16) and neck (n=2) of the pancreas. All 78 patients underwent a laparotomy. Sixty-five patients had AAST (American Association for the Surgery of Trauma) grade I or II pancreatic injuries and thirteen had grade III, IV or V injuries. Eight patients (10.3%) had an initial damage control operation. Sixty-nine patients (84.6%) had drainage of the pancreas only, six had a distal pancreatectomy and one had a pancreaticoduodenectomy. Most pancreas related complications occurred in patients with AAST grade III injuries; eight patients (10.2%) developed a pancreatic fistula. Four patients (5.1%) died. Grade of pancreatic injury (AAST grade I-II vs grade III-V injuries, pwound, morbidity was high. Increasing AAST grade of injury, high RTS, shock on admission to hospital, need for blood transfusion and repeat laparotomy were significant factors related to morbidity.

  6. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. (United States)

    Alanen, Heli I; Walker, Kelly L; Lourdes Velez Suberbie, M; Matos, Cristina F R O; Bönisch, Sarah; Freedman, Robert B; Keshavarz-Moore, Eli; Ruddock, Lloyd W; Robinson, Colin


    Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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


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

  8. Chameleon fragmentation

    International Nuclear Information System (INIS)

    Brax, Philippe; Upadhye, Amol


    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ 4 and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments

  9. Chameleon fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brax, Philippe [Institut de Physique Théorique, CEA, IPhT, CNRS, URA 2306, F-91191Gif/Yvette Cedex (France); Upadhye, Amol, E-mail:, E-mail: [Institute for the Early Universe, Ewha University, International Education, Building #601, 11-1, Daehyun-Dong Seodaemun-Gu, Seoul 120-750 (Korea, Republic of)


    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  10. Intermediate Fragment

    DEFF Research Database (Denmark)

    Kruse Aagaard, Anders


    ‘Engaging Through Architecture’ in 2015 by Aarhus School of Architecture as a part of the Ventura Lambrate Milan Design Week, where it was exhibited under the name Concrete. The fundamental pool of techniques and knowledge that set the agenda for the fragment was established before the intentions...

  11. Framing Fragmentation

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte


    to create architectural meaning and give character to an architecture of fragmentation. Layers are both seen as conceptual as well as material frames which define certain strong properties or meanings in the architectural work. Defining layers is a way of separating and organizing; it both defines...

  12. Rock fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brown, W.S.; Green, S.J.; Hakala, W.W.; Hustrulid, W.A.; Maurer, W.C. (eds.)


    Experts in rock mechanics, mining, excavation, drilling, tunneling and use of underground space met to discuss the relative merits of a wide variety of rock fragmentation schemes. Information is presented on novel rock fracturing techniques; tunneling using electron beams, thermocorer, electric spark drills, water jets, and diamond drills; and rock fracturing research needs for mining and underground construction. (LCL)

  13. [Gunshot and stab wounds in Germany--epidemiology and outcome: analysis from the TraumaRegister DGU®]. (United States)

    Bieler, D; Franke, A F; Hentsch, S; Paffrath, T; Willms, A; Lefering, R; Kollig, E W


    The management of gunshot wounds is a rare challenge for trauma surgeons in Germany and Central Europe as a result of the low incidence of this type of trauma. Penetrating injuries occur with an incidence of 5% in Germany. They are caused by gunshots or more commonly by knives or other objects, for example during accidents. Since even the number of patients who are treated at level 1 trauma centres is limited by the low incidence, the objective of this study was to assess the epidemiology and outcome of gunshot and stab wounds in Germany. Since 2009, the trauma registry of the German Trauma Society (TraumaRegister DGU®) has been used to assess not only whether a trauma was penetrating but also whether it was caused by a gunshot or a stabbing. On the basis of this registry, we identified relevant cases and defined the observation period. Data were taken from the standard documentation forms that participating German hospitals completed between 2009 and 2011. We did not specify exclusion criteria in order to obtain as comprehensive a picture as possible of the trauma entities investigated in this study. As a result of the high incidence of gunshot wounds to the head and the implications of this type of injury for the entire group, a subgroup of patients without head injuries was analysed. From 2009 to 2011, there were 305 patients with gunshot wounds and 871 patients with stab wounds. The high proportion of suicide-related gunshot wounds to the head resulted in a cumulative mortality rate of 39.7%. Stab wounds were associated with a lower mortality rate (6.2%). Every fourth patient with a gunshot or stab wound presented with haemorrhagic shock, which was considerably more frequently seen during the prehospital phase than during the inhospital phase of patient management. Of the patients with gunshot wounds, 26.9% required transfusions. This percentage was three times higher than that for patients with blunt trauma. In Germany, gunshot and stab wounds have a low

  14. An audit of failed non-operative management of abdominal stab wounds. (United States)

    Clarke, D L; Allorto, N L; Thomson, S R


    Selective non-operative management based on clinical assessment has been shown to be a generally safe approach in the management of penetrating stab wounds of the torso. However there will be a subset of patients who fail selective non-operative management. This audit focuses on the failures. The metropolitan surgical service in Pietermaritzburg covers 3 hospitals. At the weekly metropolitan morbidity and mortality meeting all trauma patients are reviewed. All cases of failed selective non-operative management of penetrating abdominal stab wounds are discussed. Failed non-operative management is usually defined as any patient who ultimately requires surgical exploration. We do not subscribe to this as we feel as long as the need for surgical intervention is recognised within a short period of time (wound were managed over the 2 year period under review. A total of 192 (56%) of these patients were subjected to mandatory laparotomy. Of these mandatory laparotomies 98% were positive. The remaining 148 (44%) patients were observed. Of the 148 observed patients a total of 30 (20%) subsequently underwent surgery. A total of 13 patients were only taken to surgery after 12h of observation. In this group of 13 patients the average delay between admission and recognition of injury was 40 h. There were six gastric injuries, one pyloric and pancreatic injury, two gallbladder injuries, one liver, one colon and two small bowel injuries. There were no deaths. 9 patients recovered with no additional morbidity. In the remainder, morbidity included, relaparotomy (1), open abdomen (1), renal failure (1) and prolonged stay in ICU (3). Clinical assessment accurately predicts the need for mandatory laparotomy following a stab wound to the torso. In patients who do not meet the indications for mandatory laparotomy and who are subjected to non-operative management 20% will come to surgery. A subgroup may only be recognised as requiring surgery after more than 12h. These patients are at

  15. Laparoscopic diagnostic peritoneal lavage (L-DPL: A method for evaluation of penetrating abdominal stab wounds

    Directory of Open Access Journals (Sweden)

    Krausz Michael M


    Full Text Available Abstract Background The management of penetrating abdominal stab wounds has been the subject of continued reappraisal and controversy. In the present study a novel method which combines the use of diagnostic laparoscopy and DPL, termed laparoscopic diagnostic peritoneal lavage (L-DPL is described Method Five trauma patients with penetrating injuries to the lower chest or abdomen were included. Standard videoscopic equipment is utilized for the laparoscopic trauma evaluation of the injured patient. When no significant injury is detected, the videoscope is withdrawn and 1000 mL of normal saline is infused through the abdominal trochar into the peritoneal cavity, and the effluent fluid studied for RBCs, WBC, amylase debry, bile as it is uced in regular diagnostic peritoneal lavage Results Laparoscopic peritoneal lavage (L-DPL was then performed and proved to be negative in all 5 patients. RBC lavage counts above 100,000/mcrl were not considered as a positive lavage result, because the bleeding source was directly observed and controlled laparoscopically. All patients recovered uneventfully and were released within 3 days. This procedure combines the visual advantages of laparoscopy together with the sensitivity and specificty of DPL for the diagnosis of significant penetrating intra-abdominal injury, when the diagnostic strategy of selective consevatism for abdominal stab wounds is adopted. Conclusion A method of laparoscopic diagnostic peritoneal lavage (L-DPL in hemodynamically stable patients with penetrating lower thoracic or abdominal stab wounds is described. The method is especially applicable for trauma surgeons with only basic experience in laparoscopic technique. This procedure is used to obtain conclusive evidence of significant intra-abdominal injury, confirm peritoneal penetration, control intra-abdominal bleeding, and repair lacerations to the diaphragm and abdominal wall. The combination of laparoscopy and DPL afforded by the L

  16. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    or from a position as business leader to a position in the state apparatus or in the Party and vice versa. To conceptualize the coexistence of the contradicting forces for further enterprise autonomy and continued central control that characterizes the evolving relationship between business groups...... and the Party-state, I suggest the notion of integrated fragmentation....

  17. Differences between postmortem computed tomography and conventional autopsy in a stabbing murder case

    Directory of Open Access Journals (Sweden)

    Talita Zerbini


    Full Text Available Objective: The aim of the present work is to analyze the differences and similarities between the elements of a conventional autopsy and images obtained from postmortem computed tomography in a case of a homicide stab wound. Method: Comparison between the findings of different methods: autopsy and postmortem computed tomography. Results: In some aspects, autopsy is still superior to imaging, especially in relation to external examination and the description of lesion vitality. However, the findings of gas embolism, pneumothorax and pulmonary emphysema and the relationship between the internal path of the instrument of aggression and the entry wound are better demonstrated by postmortem computed tomography. Conclusions: Although multislice computed tomography has greater accuracy than autopsy, we believe that the conventional autopsy method is fundamental for providing evidence in criminal investigations.

  18. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220. (United States)

    Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo


    A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples. (United States)

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Kowalewsky, Frank; Vogel, Rudolf; Heinrich, Julia


    Generic immunoassay formats in animal serum have been described for pharmacokinetic (PK) analysis of human full-length antibodies, but not of human antigen binding fragment (Fab) proteins. Here we characterize two murine monoclonal antibodies (mAb) raised against human immunoglobulin G (IgG) which bind to unique epitopes in the Fab region of human IgG. mAb M-1.7.10 is directed against the constant domain of the kappa light chain and mAb M-1.19.31 binds to the constant domain 1 (CH1) of the heavy chain. Surface plasmon resonance analysis showed that mAb M-1.7.10 does not cross-react with sera from mouse, rat, rabbit, dog, marmoset, rhesus macaque, baboon and cynomolgus monkey, but binds to human and chimpanzee serum (dissociation constant K(D) of 6.8 × 10(-12) and 3.1 × 10(-11)M, respectively). mAb M-1.19.31 shows a higher K(D) for human and chimpanzee IgG (2.0 × 10(-9)M and 5.8 × 10(-10)M, respectively), but also does not bind to serum of the other species. Therefore, mAb M-1.7.10 was used as capture and mAb M-1.19.31 as detection reagent in a generic enzyme linked immunosorbent assay (ELISA) to quantify the human anti-IGF-1R Fab in mouse serum. The generic human Fab assay showed a limit of detection of 31.5 ng/mL anti-IGF-1R Fab. Intra- and inter-assay precision was less than 12% and the accuracy range for all controls was within ±20% of the target concentration. The generic human Fab ELISA was applied to determine serum levels of human anti-IGF-1R Fab after intravenous (iv) administration of 10mg/kg to mice. The resulting concentration-time profile was nearly identical to that obtained by analysis with a validated specific ELISA for anti-IGF-1R Fab. The mean relative concentration of anti-IGF-1R Fab analyzed by the generic assay was 82-118% of that of the specific assay. This equivalence was confirmed in a cynomolgus monkey study with the full length human mAb anti-TROP-2 IgG. Both specific ELISAs used mAb M-1.7.10 as detection reagent and their targets for

  20. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide......-antibody interface and the antibody intraface.the microenvironment and ecology of Acaryochloris and Prochloron, and in this thesis we attempted to further describe the distribution, growth characteristics and adaptive/regulatory mechanisms of these two cyanobacteria, both in their natural habitat and under defined...

  1. Fragmentation based

    Directory of Open Access Journals (Sweden)

    Shashank Srivastava


    Gaining the understanding of mobile agent architecture and the security concerns, in this paper, we proposed a security protocol which addresses security with mitigated computational cost. The protocol is a combination of self decryption, co-operation and obfuscation technique. To circumvent the risk of malicious code execution in attacking environment, we have proposed fragmentation based encryption technique. Our encryption technique suits the general mobile agent size and provides hard and thorny obfuscation increasing attacker’s challenge on the same plane providing better performance with respect to computational cost as compared to existing AES encryption.

  2. Architectural fragments

    DEFF Research Database (Denmark)

    Bang, Jacob Sebastian


    the photographs as a starting point for a series of paintings. This way of creating representations of something that already exists is for me to see a way forward in the "decoding" of my own models into other depictions. The models are analyzed through a series of representations in different types of drawings....... I try to invent the ways of drawing the models - that decode and unfold them into architectural fragments- into future buildings or constructions in the landscape. [1] Luigi Moretti: Italian architect, 1907 - 1973 [2] Man Ray: American artist, 1890 - 1976. in 2015, I saw the wonderful exhibition...

  3. 19th Biannual Symposium of the German Aerospace Aerodynamics Association (STAB) and the German Society for Aeronautics and Astronautics (DGLR)

    CERN Document Server

    Heller, Gerd; Krämer, Ewald; Wagner, Claus; Breitsamter, Christian


    This book presents contributions to the 19th biannual symposium of the German Aerospace Aerodynamics Association (STAB) and the German Society for Aeronautics and Astronautics (DGLR). The individual chapters reflect ongoing research conducted by the STAB members in the field of numerical and experimental fluid mechanics and aerodynamics, mainly for (but not limited to) aerospace applications, and cover both nationally and EC-funded projects. Special emphasis is given to collaborative research projects conducted by German scientists and engineers from universities, research-establishments and industries. By addressing a number of cutting-edge applications, together with the relevant physical and mathematics fundamentals, the book provides readers with a comprehensive overview of the current research work in the field. Though the book’s primary emphasis is on the aerospace context, it also addresses further important applications, e.g. in ground transportation and energy. .

  4. Multiple self-inflicted stab wounds to neck, chest and abdomen as a unique manner of suicide. (United States)

    Kaliszan, Michał; Kernbach-Wighton, Gerhard; Bouhaidar, Ralph


    This is a case report of a 30-year-old man found dead in his flat lying on the floor with multiple stab wounds over the body, surrounded by an extensive volume of blood. Examination of the scene of death showed a secure flat, locked from inside. A blood-stained knife was present close to the body and two unstained notes left on the sofa at the locus. A small plastic bag containing white powder (which following toxicological examinations appeared to be cocaine) and an almost full bottle of beer were present on a table. Autopsy revealed more than 40 stab wounds to neck, chest, and abdomen arranged in isolated groups within which the wounds showed similar directions and had a transverse orientation. Together with hesitation marks located on the neck and wrists these characteristics allowed to interpret this case as a suicide.

  5. The direct cost of traumatic secretion transfer in hermaphroditic land snails: individuals stabbed with a love dart decrease lifetime fecundity


    Kimura, Kazuki; Chiba, Satoshi


    Several taxa of simultaneously hermaphroditic land snails exhibit a conspicuous mating behaviour, the so-called shooting of love darts. During mating, such land snail species transfer a specific secretion by stabbing a mating partner's body with the love dart. It has been shown that sperm donors benefit from this traumatic secretion transfer, because the secretions manipulate the physiology of a sperm recipient and increase the donors' fertilization success. However, it is unclear whether rec...

  6. Comparison between isolated serial clinical examination and computed tomography for stab wounds in the anterior abdominal wall. (United States)

    Breigeiron, Ricardo; Breitenbach, Tiago Cataldo; Zanini, Lucas Adalberto Geraldi; Corso, Carlos Otavio


    to compare abdominal computer tomography (CT) with isolated serial clinical exam (SCE) in the management of anterior abdominal stab wounds. randomized prospective study performed at Hospital de Pronto Socorro de Porto Alegre involving patients with anterior abdominal stab wounds without indication of immediate laparotomy; patients were divided in two groups: CT group and SCE group, In the SCE group, patients were followed up with serial clinical exam every 6 hours, Patients of CT group were submitted to abdominal computer tomography after initial evaluation. 66 patients were studied and 33 were included in each group, Of total, six were submitted to surgery, three of each group, In the SCE group, patients submitted to surgery in media waited 12 hours from arrival to diagnosis without any non-therapeutic surgeries, The remaining 30 patients of this group were discharged from hospital after 24 hours of observation, In the CT group, three patients showed alteration at CT and were submitted to laparotomy, one non-therapeutic, The others were discharged from hospital after 24 hours of observation, Abdominal computer tomography had a positive predictive value (PPV) of 67% and negative predictive value (NPV) of 100%, with 96% of accuracy, Isolated serial clinical exam showed PPV and NPV of 100% and 100% of accuracy. selective management of anterior abdominal stabs is safe, when a rigorous selection of patients is observed, Isolated serial clinical exam may be performed without computer tomography, without increase of hospitalization time or morbidity, reducing costs, exposure to radiation, mortality and morbidity and non-therapeutic laparotomies.

  7. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    proved their influence by obstructing the creation of new ministries and regulatory commissions that would limit their powers. The heads of these business groups often outrank their counterparts in state administrative organs and bureaus that are supposed to regulate their activities. The increased role...... of these business leaders prompts the question of whether we are seeing the development of distinct interest groups that could challenge Party and state authority and create a fragmented polity. However, through the nomenklatura system the Party has an important instrument of control to wield over business groups....... Through this system the Party controls the appointment and promotion of the heads of the most important state-owned enterprises. The nomenklatura system also enables the Party to rotate leaders in big business from a position as CEO in one company to a similar position in another state-owned company...

  8. A new method of ergonomic testing of gloves protecting against cuts and stabs during knife use. (United States)

    Irzmańska, Emilia; Tokarski, Tomasz


    The paper presents a new method of ergonomic evaluation of gloves protecting against cuts and stabs during knife use, consisting of five manual dexterity tests. Two of them were selected based on the available literature and relevant safety standards, and three were developed by the authors. All of the tests were designed to simulate occupational tasks associated with meat processing as performed by the gloved hand in actual workplaces. The tests involved the three most common types of protective gloves (knitted gloves made of a coverspun yarn, metal mesh gloves, and metal mesh gloves with an ergonomic polyurethane tightener) and were conducted on a group of 20 males. The loading on the muscles of the upper limb (adductor pollicis, flexor carpi ulnaris, flexor carpi radialis, and biceps brachii) was measured using surface electromyography. For the obtained muscle activity values, correlations were found between the glove type and loading of the upper limb. ANOVA showed that the activity of all muscles differed significantly between the five tests. A relationship between glove types and electromyographic results was confirmed at a significance level of α = 0.05. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. [Forensic medical expertise of the stab wounds: the current state-of-the art]. (United States)

    Sarkisian, B A; Karpov, D A


    This review of special literature encompasses the publications on the injuries inflicted by piercing objects. The results of analysis of these materials indicate that both the mechanisms of formation and the morphological features of stab wounds and damages to the clothes are studied perfectly well. Their shape and morphological patterns are shown to be dependent on the size, shape, and cross-section area of the great variety of the piercing objects. However, investigations carried out thus far did not take into consideration the formation of the signs of skin plastic deformation, such as edge portions of borderline thickening, stretching of epidermis over the walls, transformation of the epidermal network pattern into longitudinal folding, etc. It is concluded that further studies are necessary to better characterize injuries inflicted by piercing objects differing in the sharpness and the shape of cross section and thereby to obtain a deeper insight in the morphological features. It can be expected that such studies will provide a basis for the development of criteria for the individual and intra-group expert identification of the traumatic agents.

  10. The spectrum of injuries resulting from posterior abdominal stab wounds: a South African experience. (United States)

    Kong, V Y; Oosthuizen, G V; Clarke, D L


    The spectrum of injury associated with anterior abdominal stab wounds (SWs) is well established. The literature on the spectrum of organ injury associated with SWs to the posterior abdomen, however, is limited. We reviewed our experience of 105 consecutive patients who had established indications for laparotomy managed over a 4-year period in a high volume trauma service in South Africa. Of the 105 patients, 97 (92%) were male and the overall mean age was 24 years. Fifty-seven patients (54%) had immediate indications for laparotomy. The remaining 48 patients (46%) initially underwent active clinical observation and the indications for laparotomy became apparent during the observation period. Of the 105 laparotomies performed, 94 (90%) were positive and 11 (10%) were negative. Of the 94 positive laparotomies, 92 were therapeutic and 2 were non-therapeutic. A total of 176 organ injuries were identified: 50 (53%) of the 94 patients sustained a single organ injury while the remaining 44 (47%) sustained multiple organ injuries. The most commonly injured organs were the colon (n=63), spleen (n=21) and kidney (n=19). The pattern of intra-abdominal injuries secondary to SWs to the posterior abdomen is different to that seen with the anterior abdomen. Colonic injury is most commonly encountered, followed by injuries to the spleen and kidney. Clinicians must remain vigilant because of the potential for occult injuries.

  11. Determining injuries from posterior and flank stab wounds using computed tomography tractography. (United States)

    Bansal, Vishal; Reid, Chris M; Fortlage, Dale; Lee, Jeanne; Kobayashi, Leslie; Doucet, Jay; Coimbra, Raul


    Unlike anterior stab wounds (SW), in which local exploration may direct management, posterior SW can be challenging to evaluate. Traditional triple contrast computed tomography (CT) imaging is cumbersome and technician-dependent. The present study examines the role of CT tractography as a strategy to manage select patients with back and flank SW. Hemodynamically stable patients with back and flank SW were studied. After resuscitation, Betadine- or Visipaque®-soaked sterile sponges were inserted into each SW for the estimated depth of the wound. Patients underwent abdominal helical CT scanning, including intravenous contrast, as the sole abdominal imaging study. Images were reviewed by an attending radiologist and trauma surgeon. The tractogram was evaluated to determine SW trajectory and injury to intra- or retroperitoneal organs, vascular structures, the diaphragm, and the urinary tract. Complete patient demographics including operative management and injuries were collected. Forty-one patients underwent CT tractography. In 11 patients, tractography detected violation of the intra- or retroperitoneal cavity leading to operative exploration. Injuries detected included: the spleen (two), colon (one), colonic mesentery (one), kidney (kidney), diaphragm (kidney), pneumothorax (seven), hemothorax (two), iliac artery (one), and traumatic abdominal wall hernia (two). In all patients, none had negative CT findings that failed observation. In this series, CT tractography is a safe and effective imaging strategy to evaluate posterior torso SW. It is unknown whether CT tractography is superior to traditional imaging modalities. Other uses for CT tractography may include determining trajectory from missile wounds and tangential penetrating injuries.

  12. Anti-sulfotyrosine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM


    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  13. Bifunctional antibodies for radioimmunotherapy. (United States)

    Chatal, J F; Faivre-Chauvet, A; Bardies, M; Peltier, P; Gautherot, E; Barbet, J


    In two-step targeting technique using bifunctional antibodies, a nonradiolabeled immunoconjugate with slow uptake kinetics (several days) is initially injected, followed by a small radiolabeled hapten with fast kinetics (several hours) that binds to the bispecific immunoconjugate already taken up by the tumor target. In patients with colorectal or medullary thyroid cancer, clinical studies performed with an anti-CEA/anti-DTPA-indium bifunctional antibody and an indium-111-labeled di-DTPA-TL bivalent hapten showed that tumor uptake was not modified compared to results for F(ab')2 fragments of the same anti-CEA antibody directly labeled with indium-111, whereas the radioactivity of normal tissues was significantly reduced (3- to 6-fold). The fast tumor uptake kinetics (several hours) and high or very high tumor-to-normal tissue ratios obtained with the bifunctional antibody technique are favorable parameters for efficient radioimmunotherapy.

  14. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro


    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  15. NG2 cells are not a major source of reactive astrocytes after neocortical stab wound injury. (United States)

    Komitova, Mila; Serwanski, David R; Lu, Q Richard; Nishiyama, Akiko


    NG2 cells are an abundant glial cell type in the adult brain. They are distinct from astrocytes, mature oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes and a subpopulation of protoplasmic astrocytes in the ventral forebrain during development. To determine whether NG2 cells generate reactive astrocytes in the lesioned brain, stab wound injury was created in adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in NG2 cells and their progeny, and the phenotype of the EGFP(+) cells was analyzed at 10 and 30 days post lesion (dpl). The majority (>90%) of the reactive astrocytes surrounding the lesion that expressed glial fibrillary acidic protein (GFAP) lacked EGFP expression, and conversely the majority (>90%) of EGFP(+) cells were GFAP-negative. However, 8% of EGFP(+) cells co-expressed GFAP at 10 dpl. Most of these EGFP(+) GFAP(+) cells were morphologically distinct from hypertrophic reactive astrocytes and exhibited weak GFAP expression. NG2 was detected in a fraction of the EGFP(+) GFAP(+) cells found at 10 dpl. By 30 dpl the number of EGFP(+) GFAP(+) cells had decreased more than four-fold from 10 dpl. A similar transient appearance of EGFP(+) GFAP(+) cells with simple morphology was observed in NG2creER™:ZEG double transgenic mice in which EGFP expression had been induced in NG2 cells prior to injury. NG2 cell-specific deletion of the oligodendrocyte lineage transcription factor Olig2 using NG2creER™:Olig2(fl/fl) :ZEG triple transgenic mice did not increase the number of EGFP(+) reactive astrocytes. These findings suggest that NG2 cells are not a major source of reactive astrocytes in the neocortex. Copyright © 2011 Wiley-Liss, Inc.

  16. Applications of recombinant antibodies in plant pathology. (United States)

    Ziegler, Angelika; Torrance, Lesley


    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.


    NARCIS (Netherlands)


    A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass

  18. Thermally Bonded PET–Basalt Sandwich Composites for Heat Pipeline Protection: Preparation, Stab Resisting, and Thermal-Insulating Properties

    Directory of Open Access Journals (Sweden)

    Ting-Ting Li


    Full Text Available In order to solve the cost and bulky problems of buried thermal pipeline insulating materials, this study adopts basalt fabric and low-melting PET nonwoven to construct low-cost and light-weight pipeline thermal-insulating composites after needle punching and thermal bonding processes. Research result shows that thermal-bonded temperature affected the stab resistance and burst energy more significantly. As thermal-bonded temperature increased, knife resistance and spike resistance presented the upward and then downward trends, but the burst energy gradually decreased. Yarn pull-out result shows that the enhancement of stab resistance of intra-/inter-thermal-bonded structure resulted from the increment in the coefficient of friction between yarns. When PET–basalt sandwich composites were thermal-bonded at 140 °C for 5 min, the maximum knife and spike resistance were 147.00 N (1.99 J and 196.30 N (1.11 J, respectively, and burst energy was 4.79 J, thermal conductivity reduced to 0.0073 W/(m∙K. The resultant thermally bonded sandwich composites can be used as thermal-insulating protection for buried thermal pipeline.

  19. Penetrating Stab Injuries to the Anterior Abdomen: Use of Multi-Detector Computed Tomography to Predict the Need for Laparotomy

    Energy Technology Data Exchange (ETDEWEB)

    Park, Dae Hong; Choi, Seung Joon; Jeong, Yu Mi; Kim, Hyung Sik; Choi, Hye Young [Dept. of Radiology, Gachon University, Gil Hospital, Incheon (Korea, Republic of)


    The aim of this study was to determine how well multi-detector computed tomography (MDCT) scans predict the need for a laparotomy in patients with anterior abdominal stab injuries. Eighty patients with abdominal stab injuries who underwent MDCT scans were enrolled. MDCT was performed to identify active bleeding and injured organs and to assess the accuracy between MDCT and the laparotomy findings. MDCT was considered positive or negative with respect to the need for an exploratory laparotomy. The diagnostic performance of MDCT for identifying the need for laparotomy was estimated. MDCT predicted bowel and mesentery injuries in 31 of 80 patients and 28 patients were truly positive. MDCT predicted active bleeding in 23 of 80 patients and 19 patients had active bleeding. MDCT predicted the need for laparotomy in 43 of 80 patients. A laparotomy was performed in 55 of the 80 patients and 42 were therapeutic. Overall, a MDCT scan predicted the need for laparotomy with 95% sensitivity, 92% specificity, and 93% accuracy. MDCT scans can be used to identify active bleeding and injured organs and are an effective tool for determining the need for surgical exploration.

  20. Fatal tiger attack: a case report with emphasis on typical tiger injuries characterized by partially resembling stab-like wounds. (United States)

    Pathak, Hrishikesh; Borkar, Jaydeo; Dixit, Pradeep; Dhawane, Shailendra; Shrigiriwar, Manish; Dingre, Niraj


    Fatalities due to attacks by tigers on humans are uncommon and are rarely described in the medico-legal literature. We herein present a forensic investigation in a unique case of a fatal tiger attack in the wild on a 35 year old female in India by an Indian Bengal tiger (Panthera tigris tigris). The attack resulted in two pairs of puncture wounds over the nape area with occult cervical spine injuries resulting from transfixing of spine due to the tiger canines; multiple puncture wounds, numerous scratches and abrasions consistent with the tiger claw injuries and injury to the right jugulocarotid vessels. This case outlines the characteristic injury pattern from such an attack along with the multiple sources of the tiger injuries. The analysis of these injuries might reveal the motivation behind the attack and the big cat species involved in the attack. A tiger injury is sometimes compared with a stab injury, as the patterned injuries due to a tiger bite are characterized by multiple penetrating, stab-like wounds. So, a special attention is paid toward establishment of the cause of death from bites by the animal teeth under unknown circumstances of trauma and to exclude the possibility of a homicide beyond reasonable doubt in such cases. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  1. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  2. scFv Antibody: Principles and Clinical Application


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


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

  3. Universal elements of fragmentation

    International Nuclear Information System (INIS)

    Yanovsky, V. V.; Tur, A. V.; Kuklina, O. V.


    A fragmentation theory is proposed that explains the universal asymptotic behavior of the fragment-size distribution in the large-size range, based on simple physical principles. The basic principles of the theory are the total mass conservation in a fragmentation process and a balance condition for the energy expended in increasing the surface of fragments during their breakup. A flux-based approach is used that makes it possible to supplement the basic principles and develop a minimal theory of fragmentation. Such a supplementary principle is that of decreasing fragment-volume flux with increasing energy expended in fragmentation. It is shown that the behavior of the decreasing flux is directly related to the form of a power-law fragment-size distribution. The minimal theory is used to find universal asymptotic fragment-size distributions and to develop a natural physical classification of fragmentation models. A more general, nonlinear theory of strong fragmentation is also developed. It is demonstrated that solutions to a nonlinear kinetic equation consistent with both basic principles approach a universal asymptotic size distribution. Agreement between the predicted asymptotic fragment-size distributions and experimental observations is discussed.

  4. Diagnostic Accuracy of Abdominal wall Ultrasonography and Local Wound Exploration in Predicting the Need for Laparotomy following Stab Wound

    Directory of Open Access Journals (Sweden)

    Ali Vafaei


    Full Text Available Introduction: Screening of patients with anterior abdominal penetrating trauma in need for laparotomy is an important issue in management of these cases. This study aimed to compare the accuracy of abdominal wall ultrasonography (AWU and local wound exploration (LWE in this regard.Methods: This diagnostic accuracy study was conducted on ≥ 18 year-old patients presenting to emergency department with anterior abdominal stab wound and stable hemodynamics, to compare the characteristics of AWU and LWE in screening of patients in need of laparotomy.Results: 50 cases with the mean age of 28.44 ± 7.14 years were included (80% male. Sensitivity, specificity and area under the receiver operating characteristic (ROC curve of AWU were 70.58 (95% CI: 44.04 – 88.62, 93.33 (95% CI: 76.49 – 98.83, and 81.96 (95% CI: 69.91 – 94.01, respectively. These measures were 88.23 (62.25 – 97.93, 93.33 (76.49 – 98.83, and 90.78 (95% CI: 81.67 – 99.89 for LWE, respectively. The difference in overall accuracy of the two methods was not statistically significant (p = 0.0641.Conclusion: Based on the findings of the present study, AWU and LWE had the same specificity but different sensitivities in screening of anterior abdominal stab wound patients in need of laparotomy. The overall accuracy of LWE was slightly higher (91.48% versus 85.1%.

  5. Poly(I:C) adjuvant strongly enhances parasite-inhibitory antibodies and Th1 response against Plasmodium falciparum merozoite surface protein-1 (42-kDa fragment) in BALB/c mice. (United States)

    Mehrizi, Akram Abouie; Rezvani, Niloufar; Zakeri, Sedigheh; Gholami, Atefeh; Babaeekhou, Laleh


    Malaria vaccine development has been confronted with various challenges such as poor immunogenicity of malaria vaccine candidate antigens, which is considered as the main challenge. However, this problem can be managed using appropriate formulations of antigens and adjuvants. Poly(I:C) is a potent Th1 inducer and a human compatible adjuvant capable of stimulating both B- and T-cell immunity. Plasmodium falciparum merozoite surface protein 1 42 (PfMSP-1 42 ) is a promising vaccine candidate for blood stage of malaria that has faced several difficulties in clinical trials, mainly due to improper adjuvants. Therefore, in the current study, poly(I:C), as a potent Th1 inducer adjuvant, was evaluated to improve the immunogenicity of recombinant PfMSP-1 42 , when compared to CFA/IFA, as reference adjuvant. Poly(I:C) produced high level and titers of anti-PfMSP-1 42 IgG antibodies in which was comparable to CFA/IFA adjuvant. In addition, PfMSP-1 42 formulated with poly(I:C) elicited a higher ratio of IFN-γ/IL-4 (23.9) and IgG2a/IgG1 (3.77) with more persistent, higher avidity, and titer of IgG2a relative to CFA/IFA, indicating a potent Th1 immune response. Poly(I:C) could also help to induce anti-PfMSP-1 42 antibodies with higher growth-inhibitory activity than CFA/IFA. Altogether, the results of the current study demonstrated that poly(I:C) is a potent adjuvant that can be appropriate for being used in PfMSP-1 42 -based vaccine formulations.

  6. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon


    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  7. Double jeopardy revisited: clinical decision making in unstable patients with, thoraco-abdominal stab wounds and, potential injuries in multiple body cavities. (United States)

    Clarke, Damian L; Gall, Tamara M H; Thomson, Sandie R


    In the setting of the hypovolaemic patient with a thoraco-abdominal stab wound and potential injuries in both the chest and abdomen, deciding which cavity to explore first may be difficult.Opening the incorrect body cavity can delay control of tamponade or haemorrhage and exacerbate hypothermia and fluid shifts. This situation has been described as one of double jeopardy. All stab victims from July 2007 to July 2009 requiring a thoracotomy and laparotomy at the same operation were identified from a database. Demographics, site and nature of injuries, admission observations and investigations as well as operative sequence were recorded. Correct sequencing was defined as first opening the cavity with most lethal injury. Incorrect sequencing was defined as opening a cavity and finding either no injury or an injury of less severity than a simultaneous injury in the unopened cavity. The primary outcome was survival or death. Sixteen stab victims underwent thoracotomy and laparotomy during the same operation. All were male with an age range of 18–40 (mean/median 27). Median systolic blood pressure on presentation was 90 mm Hg. (quartile range 80–90 mm Hg). Median base excess was 6.5 (quartile range 12 to 2.2). All the deaths were the result of cardiac injuries. Incorrect sequencing occurred in four patients (25%). In this group there were four negative abdominal explorations prior to thoracotomy with two deaths. There was one death in the correct sequencing group. Incorrect sequencing in stab victims who require both thoracotomy and laparotomy at the same sitting is associated with a high mortality. This is especially true when the abdomen is incorrectly entered first whilst the life threatening pathology is in the chest. Clinical signs may be confusing, leading to incorrect sequencing of exploration. The common causes for confusion include failure to appreciate that cardiac tamponade does not present with bleeding and difficulty in assessing peritonism in an

  8. Fission fragment angular momentum

    International Nuclear Information System (INIS)

    Frenne, D. De


    Most of the energy released in fission is converted into translational kinetic energy of the fragments. The remaining excitation energy will be distributed among neutrons and gammas. An important parameter characterizing the scission configuration is the primary angular momentum of the nascent fragments. Neutron emission is not expected to decrease the spin of the fragments by more than one unit of angular momentum and is as such of less importance in the determination of the initial fragment spins. Gamma emission is a suitable tool in studying initial fragment spins because the emission time, number, energy, and multipolarity of the gammas strongly depend on the value of the primary angular momentum. The main conclusions of experiments on gamma emission were that the initial angular momentum of the fragments is large compared to the ground state spin and oriented perpendicular to the fission axis. Most of the recent information concerning initial fragment spin distributions comes from the measurement of isomeric ratios for isomeric pairs produced in fission. Although in nearly every mass chain isomers are known, only a small number are suitable for initial fission fragment spin studies. Yield and half-life considerations strongly limit the number of candidates. This has the advantage that the behavior of a specific isomeric pair can be investigated for a number of fissioning systems at different excitation energies of the fragments and fissioning nuclei. Because most of the recent information on primary angular momenta comes from measurements of isomeric ratios, the global deexcitation process of the fragments and the calculation of the initial fragment spin distribution from measured isomeric ratios are discussed here. The most important results on primary angular momentum determinations are reviewed and some theoretical approaches are given. 45 refs., 7 figs., 2 tabs

  9. A stab wound to the axilla illustrating the importance of brachial plexus anatomy in an emergency context: a case report. (United States)

    Casal, Diogo; Cunha, Teresa; Pais, Diogo; Iria, Inês; Angélica-Almeida, Maria; Millan, Gerardo; Videira-Castro, José; Goyri-O'Neill, João


    Although open injuries involving the brachial plexus are relatively uncommon, they can lead to permanent disability and even be life threatening if accompanied by vascular damage. We present a case report of a brachial plexus injury in which the urgency of the situation precluded the use of any ancillary diagnostic examinations and forced a rapid clinical assessment. We report a case of a Portuguese man who had a stabbing injury at the base of his left axilla. On observation in our emergency room an acute venous type of bleeding was present at the wound site and, as a result of refractory hypotension after initial management with fluids administered intravenously, he was immediately carried to our operating room. During the course of transportation, we observed that he presented hypoesthesia of the medial aspect of his arm and forearm, as well as of the ulnar side of his hand and of the palmar aspect of the last three digits and of the dorsal aspect of the last two digits. Moreover, he was not able to actively flex the joints of his middle, ring, and small fingers or to adduct or abduct all fingers. Exclusively relying on our anatomical knowledge of the axillary region, the site of the stabbing wound, and the physical neurologic examination, we were able to unequivocally pinpoint the place of the injury between the anterior division of the lower trunk of his brachial plexus and the proximal portion of the following nerves: ulnar, medial cutaneous of his arm and forearm, and the medial aspect of his median nerve. Surgery revealed a longitudinal laceration of the posterior aspect of his axillary vein, and confirmed a complete section of his ulnar nerve, his medial brachial and antebrachial cutaneous nerves, and an incomplete section of the ulnar aspect of his median nerve. All structures were repaired microsurgically. Three years after the surgery he showed a good functional outcome. We believe that this case report illustrates the relevance of a sound anatomical

  10. Radioimmunotherapy with Tenarad, a {sup 131}I-labelled antibody fragment targeting the extra-domain A1 of tenascin-C, in patients with refractory Hodgkin's lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Aloj, Luigi [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Medicina Nucleare, Napoli (Italy); D' Ambrosio, Laura; Aurilio, Michela; Morisco, Anna; Caraco' , Corradina; Di Gennaro, Francesca; Lastoria, Secondo [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa Medicina Nucleare, Napoli (Italy); Frigeri, Ferdinando; Capobianco, Gaetana; Pinto, Antonio [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Ematologia Oncologica, Napoli (Italy); Giovannoni, Leonardo; Menssen, Hans D. [Philogen, SpA, Siena (Italy); Neri, Dario [Institute of Pharmaceutical Sciences, ETH, Zurich (Switzerland)


    The extra-domain A1 of tenascin-C (TC-A1) is highly expressed in the extracellular matrix of tumours and on newly formed blood vessels and is thus a valuable target for radionuclide therapy. Tenarad is a fully human miniantibody or small immunoprotein (SIP, molecular weight 80 kDa) labelled with {sup 131}I that is derived from a TC-A1-binding antibody. Previous phase I/II studies with a similar compound ({sup 131}I-L19SIP) used for radioimmunotherapy (RIT) have shown preliminary efficacy in a variety of cancer types. In this ongoing phase I/II trial, Tenarad was administered to patients with recurrent Hodgkin's lymphoma (HL) refractory to conventional treatments. Eight patients (four men, four women; age range 19 - 41) were enrolled between April 2010 and March 2011. All patients had received a median of three previous lines of chemotherapy (range three to six) and seven had also undergone autologous stem cell transplantation (ASCT) or bone marrow transplantation. In addition, seven patients received external beam radiation. All patients had nodal disease, constitutional B symptoms and some showed extranodal disease in skeletal bone (four patients), lung (three), liver (two) and spleen (one). Baseline assessments included whole-body FDG PET with contrast-enhanced CT and diagnostic Tenarad planar and SPECT studies. Patients were considered eligible to receive a therapeutic dose of Tenarad (2.05 GBq/m{sup 2}) if tumour uptake was more than four times higher than that of muscle. All patients were eligible and received the therapeutic dose of Tenarad. Only one patient developed grade 4 thrombocytopenia and leucocytopenia, requiring hospitalization and therapeutic intervention. All other patients had haematological toxicity of grade 3 or lower, which resolved spontaneously. At the first response assessment (4 - 6 weeks after therapy), one patient showed a complete response, one showed a partial response (PR) and five had disease stabilization (SD). Five patients

  11. Fragments of protein A eluted during protein A affinity chromatography. (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert


    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  12. The direct cost of traumatic secretion transfer in hermaphroditic land snails: individuals stabbed with a love dart decrease lifetime fecundity. (United States)

    Kimura, Kazuki; Chiba, Satoshi


    Several taxa of simultaneously hermaphroditic land snails exhibit a conspicuous mating behaviour, the so-called shooting of love darts. During mating, such land snail species transfer a specific secretion by stabbing a mating partner's body with the love dart. It has been shown that sperm donors benefit from this traumatic secretion transfer, because the secretions manipulate the physiology of a sperm recipient and increase the donors' fertilization success. However, it is unclear whether reception of dart shooting is costly to the recipients. Therefore, the effect of sexual conflict and antagonistic arms races on the evolution of traumatic secretion transfer in land snails is still controversial. To examine this effect, we compared lifetime fecundity and longevity between the individuals that received and did not receive dart shooting from mating partners in Bradybaena pellucida. Our experiments showed that the dart-receiving snails suffered reduction in lifetime fecundity and longevity. These results suggest that the costly mating behaviour, dart shooting, generates conflict between sperm donors and recipients and that sexually antagonistic arms races have contributed to the diversification of the morphological and behavioural traits relevant to dart shooting. Our findings also support theories suggesting a violent escalation of sexual conflict in hermaphroditic animals. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  13. Selective non-operative management of stab wounds to the posterior abdomen is safe: the Pietermaritzburg experience. (United States)

    Kong, Victor; Oosthuizen, George; Sartorius, Benn; Clarke, Damian


    The selective non-operative management (SNOM) of stab injuries of the anterior abdomen is well established, but its application to the posterior abdomen remains controversial. A retrospective review of 1013 patients was undertaken at a major trauma service in South Africa over a five-year period. Ninety per cent of patients were males, and the mean age was 25 years. The mean time from injury to presentation was 4h and 73% of all injuries were inflicted by knives. A total of 9% (93) of patients required a laparotomy [Group A] and 82% (833) were successfully observed without the need for operative intervention [Group B]. CT imaging was performed on 52 patients (5%) who had haematuria [Group C], 25 (3%) who had neurological deficits [Group D], and 10 (1%) with retained weapon injuries [Group E]. The accuracy of physical examination for identifying the presence of organ injury was 88%. All observed patients who required laparotomy declared themselves within 24h. There were no mortalities as direct result of our current management protocol. Selective management based on active clinical observation and serial physical examination is safe, and when coupled with the judicious use of advanced imaging, is a prudent and reliable approach in a resource constrained environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The future of antibodies as cancer drugs. (United States)

    Reichert, Janice M; Dhimolea, Eugen


    Targeted therapeutics such as monoclonal antibodies (mAbs) have proven successful as cancer drugs. To profile products that could be marketed in the future, we examined the current commercial clinical pipeline of mAb candidates for cancer. Our analysis revealed trends toward development of a variety of noncanonical mAbs, including antibody-drug conjugates (ADCs), bispecific antibodies, engineered antibodies and antibody fragments and/or domains. We found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets and mechanisms of action (MOA). Although well-validated targets, such as epidermal growth factor receptor (EGFR) and CD20, continue to provide opportunities for companies, we found notable trends toward targeting less-well-validated antigens and exploration of innovative MOA such as the generation of anticancer immune responses or recruitment of cytotoxic T cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Profile of sequential determinants in tissue polypeptide antigen BrCN:B fragment. (United States)

    Chersi, A; Camera, M; Trinca, M L; Castelli, M


    A synthetic approach has been applied to determine the profile of sequential determinants of one immunodominant region of Tissue Polypeptide Antigen (TPA). Five overlapping peptides, covering 30 of the 32 amino acid residues of this fragment, were chemically synthesized, and their antibody-binding activities for rabbit anti-TPA antibodies determined by enzyme-linked immunoadsorbant assays. Anti-TPA reacted with two overlapping fragments at the COOH-terminal end of the fragment, but not with peptides that include Arg 15 considered as essential for the antigenicity of the whole fragment. This might suggest that this critical residue is involved in the formation of a complex conformational determinant.

  16. Do monoclonal antibodies recognize linear sequential determinants? (United States)

    Camera, M; Muratti, E; Trinca, M L; Chersi, A


    A group of 19 anti-class II monoclonal antibodies produced in different laboratories were tested in ELISA for their ability to bind to a panel of synthetic peptides selected from HLA-DQ alpha and beta chains. No one of the antibodies tested was found to react with the synthetic fragments, thus confirming the common finding that MoAbs generally fail to recognize fragments of the native antigen. The possibility that this result might be partly due to the procedure used for screening hybridoma supernatants is discussed.

  17. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Glaessner, H.


    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

  18. Embedded Fragments Registry (EFR) (United States)

    Department of Veterans Affairs — In 2009, the Department of Defense estimated that approximately 40,000 service members who served in OEF/OIF may have embedded fragment wounds as the result of small...

  19. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J


    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  20. Fragmented Work Stories

    DEFF Research Database (Denmark)

    Humle, Didde Maria; Reff Pedersen, Anne


    , edited and performed by the storyteller in an ongoing process allowing tensions, discontinuities and editing between failures and achievements, between dreams and work realities and between home and work life. We argue that by including different types of fragmentation, we offer a new type......Following a strand of narrative studies pointing to the living conditions of storytelling and the micro-level implications of working within fragmented narrative perspectives, this article contributes to narrative research on work stories by focusing on how meaning is created from fragmented...... stories. We argue that meaning by story making is not always created by coherence and causality; meaning is created by different types of fragmentation: discontinuities, tensions and editing. The objective of this article is to develop and advance antenarrative practice analysis of work stories...

  1. Fragmentation Main Model (United States)

    Earth Data Analysis Center, University of New Mexico — The fragmentation model combines patch size and patch continuity with diversity of vegetation types per patch and rarity of vegetation types per patch. A patch was...

  2. Physics of projectile fragments

    International Nuclear Information System (INIS)

    Minamisono, Tadanori


    This is a study report on the polarization phenomena of the projectile fragments produced by heavy ion reactions, and the beta decay of fragments. The experimental project by using heavy ions with the energy from 50 MeV/amu to 250 MeV/amu was designed. Construction of an angle-dispersion spectrograph for projectile fragments was proposed. This is a two-stage spectrograph. The first stage is a QQDQQ type separator, and the second stage is QDQD type. Estimation shows that Co-66 may be separated from the nuclei with mass of 65 and 67. The orientation of fragments can be measured by detecting beta-ray. The apparatus consists of a uniform field magnet, an energy absorber, a stopper, a RF coil and a beta-ray hodoscope. This system can be used for not only this purpose but also for the measurement of hyperfine structure. (Kato, T.)

  3. Fragment Impact Toolkit (FIT)

    Energy Technology Data Exchange (ETDEWEB)

    Shevitz, Daniel Wolf [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Key, Brian P. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Garcia, Daniel B. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)


    The Fragment Impact Toolkit (FIT) is a software package used for probabilistic consequence evaluation of fragmenting sources. The typical use case for FIT is to simulate an exploding shell and evaluate the consequence on nearby objects. FIT is written in the programming language Python and is designed as a collection of interacting software modules. Each module has a function that interacts with the other modules to produce desired results.

  4. Antibody biotechnology

    African Journals Online (AJOL)



    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  5. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  6. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  7. A survey on spinal cord injuries resulting from stabbings: a case series study of 12 years' experience. (United States)

    Saeidiborojeni, Hamid Reza; Moradinazar, Mehdi; Saeidiborojeni, Sepehr; Ahmadi, Alireza


    Penetrating spinal cord injuries (SCIs) are an uncommon injury and not reported very frequently. SCIs cause sensory, motor and genitourinary system problems or a combination of sensorimotor dysfunctions. These are among the most debilitating kinds of disorders and negatively affect quality of life, not only for the patient, but also for their family members. Therefore, the present study aims to evaluate complete or incomplete SCIs and the course of the injury and the prognosis for SCIs caused by stab wounds. This case-series design study was performed on 57 patients attending the emergency department of Taleqani Trauma Center (Kermanshah, Iran) due to SCIs caused by violent encounters involving sharp objects such as a knife, dagger, whittle and Bowie-knife between 1999 and 2011. An assessment of sensory and motor functions was performed as part of the neurological examination on admission, and during the treatment, using the Frankel Classification grading system, and the results were recorded. The average age of patients was 27 years (SD= 7.9, Range=17 to 46 years). The results of the study showed a proportion of cervical, thoracic and lumbar injuries of 23 (40%), 24 (42%) and 10 (18%), respectively. There was no case of cerebrospinal fluid leakage (CSF) or infection at the wound site in the subjects. Regarding the extent of the SCI, the combined neurological assessment showed that several patients (43%) had a complete SCI with no sensory and motor functions in the sacral segments and the segments below the site of injury. In 32 patients (57%) incomplete injuries were observed; i.e. they showed only some degrees of sensory-motor functions that were below the neurological level. Both complete and incomplete SCIs are of great importance because the prognosis of SCI is directly associated with the location and extent of injury. It should be considered that partial recovery from SCIs is possible in few cases of complete injuries. Therefore, all the patients should be

  8. Hybrid IgG4/IgG4 Fc antibodies form upon 'Fab-arm' exchange as demonstrated by SDS-PAGE or size-exclusion chromatography

    NARCIS (Netherlands)

    Rispens, Theo; den Bleker, Tamara H.; Aalberse, Rob C.


    Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab + Fc) with a molecular weight of ca. 100 kDa. These antibodies can

  9. [A study of the predictive value of the primary review and complementary examinations in assessing the need for surgery in patients with stab wounds in the torso]. (United States)

    Martínez Casas, Isidro; Sancho Insenser, Joan; Climent Agustín, Marta; Membrilla Fernández, Estela; Pons Fragero, María José; Guzmán Ahumada, Juan; Grande Posa, Luis


    Observation is the gold standard for stable patients with stab wounds. The aim of the study was to analyse the value of the primary review and complementary examinations to predict the need for surgery in stab wound patients in order to decrease observation times. A retrospective study of stab wound patients recorded in a database. Clinical and diagnostic workup parameters were analysed. The main variable was the need for surgery. A total of 198 patients were included between 2006 and 2009, with a mean injury severity score (ISS) of 7.8±7, and 0.5% mortality. More than half (52%) of the patients suffered multiple wounds. Wound distribution was 23% neck, 46% thorax and 31% abdomen. Surgery was required in 73 (37%) patients (59% immediate, 27% delayed and 14% delayed). The need for surgery was associated with a lower revised trauma score (RTS), evisceration, active bleeding, and fascial penetration. Initial and control haemoglobin levels were significantly lower in patients who required surgery. A positive computerised tomography (CT) scan was associated with surgery. There were complications in 18% of patients, and they were more frequent in those who underwent surgery. There was no difference in complication rates between immediate and delayed (P=.72). Surgery was finally required in 10% of the patients with no abnormalities in the primary review and diagnostic workup, and 6% of those developed complications. None of the parameters studied could individually assess the need for surgery. Primary and secondary reviews were the most important diagnostic tool, but CT scan should be used more often. An observation period of 24 hours is recommended in torso penetrating wounds. Copyright © 2012 AEC. Published by Elsevier Espana. All rights reserved.

  10. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner


    fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...... small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271(+), as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer...

  11. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech


    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  12. Functionally fused antibodies--a novel adjuvant fusion system

    DEFF Research Database (Denmark)

    Larsen, Martin; Jensen, Kim Bak; Christensen, Peter Astrup


    Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radio...

  13. Fragmentation of kidney stones

    International Nuclear Information System (INIS)

    Kovacs, K.; Kun, F.; Vertse, T.


    Complete text of publication follows. Fragmentation, i.e. the breaking of particulate materials into smaller pieces is abundant in nature and underlies several industrial processes, which attracted a continuous interest in scientific and engineering research over the past decades. In industrial applications, fragmentation processes are mostly used for the comminution of ores in various types of mills. Kidney stone is a well known human dis- ease which embitters the life of many people (in a country like the USA about 10 6 cases are registered yearly). In order to extract large kidney stones (diameter ≥ 1 cm) from the human body without operation, one of the most efficient treatment is the fragmentation of kidney stones by the so-called extracorporal shock wave lithography method: a shock wave penetrating the human body is generated by an electric pulse. The repeated application of the shock wave gradually fragments the stones into pieces of size ≤ 2 mm which then leave the body through the urine system. Recently, a novel type of lithographic method has been suggested by using widely focused shock waves which fragment the stones by a squeezing mechanism. Laboratory experiments showed that the widely focused squeezing waves achieve a higher fragmentation efficiency than the frequently used shock waves of sharp focus. Based on this method a novel medical treatment can be introduced which is less demanding for the patients. Before the application of the method in the clinical practice a detailed understanding of the fragmentation mechanism of kidney stones due to shock waves is required. Since analytic theoretical methods have serious limitations in this field, we develop a realistic model of the mechanical behavior of kidney stones and a simulation code which makes possible to study the mechanism of breakup under various external conditions. Computer simulations in two dimensions have revealed a peculiar way of crack formation, i.e. the crack which finally breaks

  14. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt


    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  15. Fragments of the Past


    Peter Szende; Annie Holcombe


    With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  16. Picking Up (On) Fragments

    NARCIS (Netherlands)

    Ellis, Phil


    abstractThis article discusses the implications for archival and media archaeological research and reenactment artwork relating to a recent arts practice project: reenacttv: 30 lines / 60 seconds. It proposes that archival material is unstable but has traces and fragments that are full of creative

  17. Fragments of Time

    DEFF Research Database (Denmark)

    Christiansen, Steen Ledet

    Time travel films necessarily fragment linear narratives, as scenes are revisited with differences from the first time we saw it. Popular films such as Back to the Future mine comedy from these visitations, but there are many different approaches. One extreme is Chris Marker's La Jetée - a film...

  18. Wildlife habitat fragmentation. (United States)

    John. Lehmkuhl


    A primary issue in forest wildlife management is habitat fragmentation and its effects on viability, which is the "bottom line" for plant and animal species of conservation concern. Population viability is the likelihood that a population will be able to maintain itself (remain viable) over a long period of time-usually 100 years or more. Though it is true...

  19. Fragments of the Past

    Directory of Open Access Journals (Sweden)

    Peter Szende


    Full Text Available With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  20. Stone fragmentation by ultrasound

    Indian Academy of Sciences (India)

    Some delicate nerves and fibres in the surrounding areas of the stones present in the kidney are also damaged by high ultrasonic intensity used in such systems. In the present work, enhancement of the kidney stone fragmentation by using ultrasound is studied. The cavitation bubbles are found to implode faster, with more ...

  1. Synthesis of arabinoxylan fragments

    DEFF Research Database (Denmark)

    Underlin, Emilie Nørmølle; Böhm, Maximilian F.; Madsen, Robert

    , or production of commercial chemicals which are mainly obtained from fossil fuels today.The arbinoxylan fragments have a backbone of β-1,4-linked xylans with α-L-arabinose units attached at specific positions. The synthesis ultilises an efficient synthetic route, where all the xylan units can be derived from D...

  2. A comparative audit of gunshot wounds and stab wounds to the neck in a South African metropolitan trauma service. (United States)

    Madsen, A S; Laing, G L; Bruce, J L; Clarke, D L


    Introduction The aim of this comparative study of gunshot wounds (GSWs) and stab wounds (SWs) to the neck was to quantify the impact of the mechanism of injury on the outcome and management of penetrating neck injury (PNI). Methods A prospective trauma registry was interrogated retrospectively. Data were analysed pertaining to demographics and injury severity score (ISS), physiology on presentation, anatomical site of wounds and injuries sustained, investigations, management, outcome and complications. Results There were 452 SW and 58 GSW cases over the 46 months of the study. Patients with GSWs were more likely to have extracervical injuries than those with SWs (69% vs 63%). The incidence of a 'significant cervical injury' was almost twice as high in the GSW cohort (55% vs 31%). For patients with transcervical GSWs, this increased to 80%. The mean ISS was 17 for GSW and 11 for SW patients. Those in the GSW cohort presented with threatened airways and a requirement for an emergency airway three times as often as patients with SWs (24% vs 7% and 14% vs 5% respectively). The incidence among GSW and SW patients respectively was 5% and 6% for airway injuries, 12% and 8% for injuries to the digestive tract, 21% and 16% for vascular injuries, 59% and 10% for associated cervical injuries, 36% and 14% for maxillofacial injuries, 16% and 9% for injuries to the head, and 35% and 45% for injuries to the chest. In the GSW group, 91% underwent computed tomography angiography (CTA), with 23% of these being positive for a vascular injury. For SWs, 74% of patients underwent CTA, with 17% positive for a vascular injury. Slightly more patients with GSWs required operative intervention than those with SWs (29% vs 26%). Conclusions Patients with GSWs to the neck have a worse outcome than those with injuries secondary to SWs. However, the proportion of neck injuries actually requiring direct surgical intervention is not increased and most cases with PNI secondary to GSWs can be managed

  3. scFv Antibody: Principles and Clinical Application

    Directory of Open Access Journals (Sweden)

    Zuhaida Asra Ahmad


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

  4. scFv antibody: principles and clinical application. (United States)

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


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

  5. Construction of phage antibody repertoires from the blood of West Nile virus-infected donors. (United States)

    Throsby, Mark; de Kruif, John


    A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a repertoire that can be used in panning procedures to identify many unique monoclonal antibodies.

  6. Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment


    Syvänen, Stina; Edén, Desireé; Sehlin, Dag


    Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to d...

  7. Subcloning of DNA fragments. (United States)

    Struhl, K


    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

  8. The Serendipity of Fragmentation

    DEFF Research Database (Denmark)

    Leixnering, Stephan; Meyer, Renate E.

    , it was the central government’s task to coordinate, steer and control the newly emerged decentralized organizations. This raises questions about the overall design of the public sector at present. Our paper engages with the prevalent public governance phenomenon of fragmentation from a design perspective in order......Reform approaches in the public sector led to significant changes in the sector’s design. Especially NPM-inspired reform measures which had largely aimed at organizational disaggregation created pluriform landscapes of public sector organizations (PSOs). Following a core public governance principle...... form of organizing between networks and formal organization: lacking a single center and featuring multiplex and multifaceted relations within the politico-administrative apparatus and between government and PSOs, high fragmentation, local and robust action, but latent structures of significant formal...

  9. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    Energy Technology Data Exchange (ETDEWEB)

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue


    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  10. Fragment-fragment correlations in near-binary fragmentation of C60

    International Nuclear Information System (INIS)

    Vandenbosch, R.; Henry, B.; Cooper, C.H.; Liang, J.F.; Will, D.I.


    The collision dynamics of C 60 with H 2 and He gas has been studied using reverse kinematics. A beam of C 60 - is obtained by electron attachment to neutral molecules exiting an oven and then accelerated to energies between 75 and 150 keV. The collisions take place in a windowless gas cell. We energy analyze the products in a pair of electrostatic analyzers which are oriented so that coincidences between light and heavy fragments can be observed. Our energy, and hence mass, resolution is sufficient to uniquely identify all C n clusters between n=1 and 60. The principal question we are addressing is whether heavy products in the mass range C 34 to C 56 are produced solely by sequential emission of C 2 fragments, or whether longer chains (and possibly rings) compete with C 2 emission. From our coincidence studies we have conclusive evidence that fragments such as C 8 are fragmentation partners to heavy fragments. In general we find that the products of a binary fragmentation are sufficiently excited that sequential decay follows the initial fragmentation. Although only even-n heavy fragments are observed, the coincident light fragments include both odd and even-n fragments due to subsequent fragmentation of the excited lighter partner of the initial binary fragmentation. This scenario has been confirmed by studying coincidences between two light fragments. copyright 1997 American Institute of Physics


    NARCIS (Netherlands)



    The conditions for a scaling behaviour from the fragmentation process leading to slow protons are discussed- The scaling referred to implies that the fragmentation functions depend on the light-cone momentum fraction only. It is shown that differences in the fragmentation functions for valence- and

  12. Polynucleotides encoding anti-sulfotyrosine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM


    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  13. The Serendipity of Fragmentation

    DEFF Research Database (Denmark)

    Leixnering, Stephan; Meyer, Renate E.

    Reform approaches in the public sector led to significant changes in the sector’s design. Especially NPM-inspired reform measures which had largely aimed at organizational disaggregation created pluriform landscapes of public sector organizations (PSOs). Following a core public governance principle...... form of organizing between networks and formal organization: lacking a single center and featuring multiplex and multifaceted relations within the politico-administrative apparatus and between government and PSOs, high fragmentation, local and robust action, but latent structures of significant formal...

  14. Generic behaviours in impact fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Sator, N.; Mechkov, S.; Sausset, F. [Paris-6 Univ. Pierre et Marie Curie, Lab. de Physique Theorique de la Matiere Condensee, UMR CNRS 7600, 75 - Paris (France); Mechkov, S. [Ecole Normale Superieure, Lab. de Physique Statistique, 75 - Paris (France)


    From atomic nuclei to supernovae, including plates and rocks, every cohesive system can be broken into fragments, provided that the deposited energy is sufficiently large compared to its cohesive energy. We present a simple numerical model for investigating the general properties of fragmentation. By use of molecular dynamics simulations, we study the impact fragmentation of a solid disk of interacting particles with a wall. Regardless of the particular form of the interaction potential, the fragment size distribution exhibits a power law behaviour with an exponent that increases logarithmically with the energy deposited in the system, in agreement with experiments. We expect this behaviour to be generic in fragmentation phenomena. (authors)

  15. Fragmentation of Chitosan by Acids

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Kasaai


    Full Text Available Fragmentation of chitosan in aqueous solution by hydrochloric acid was investigated. The kinetics of fragmentation, the number of chain scissions, and polydispersity of the fragments were followed by viscometry and size exclusion chromatography. The chemical structure and the degree of N-acetylation (DA of the original chitosan and its fragments were examined by 1H NMR spectroscopy and elemental analysis. The kinetic data indicates that the reaction was of first order. The results of polydispersity and the DA suggest that the selected experimental conditions (temperature and concentration of acid were appropriate to obtain the fragments having the polydispersity and the DA similar to or slightly different from those of the original one. A procedure to estimate molecular weight of fragments as well as the number of chain scissions of the fragments under the experimental conditions was also proposed.

  16. Detection of thrombi using a Tc-99m labelled antifibrin monoclonal antibody (MoAb)

    International Nuclear Information System (INIS)

    Wasser, M.N.J.M.


    This thesis presents an investigation into the possibility of immunoscintigraphic detection of thrombi using an antifibrin monoclonal antibody, and fragments of the latter. The antifibrin antibody and tis fragments were labelled with Ec-99m, which has excellent characteristics for imaging with a gamma camera. The characterization of the antifibrin antibody and its fragments, the assessment of quality of labelling with Tc-99m, and results of experiments in vitro and in animals, which show the potential of immunoscintigraphic detection, are described. (author). 142 refs.; 44 figs.; 5 tabs

  17. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene


    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.

  18. Antibody proteases: induction of catalytic response. (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D


    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  19. Intermediate mass fragments emission in binary fragmentation model

    International Nuclear Information System (INIS)

    Bhattacharya, C.; Bhattacharya, S.


    Intermediate mass fragments emission in intermediate-energy nucleus-nucleus collisions has been studied in the framework of a generalized model where the fragments are assumed to be emitted from binary fissionlike decay of the fully equilibrated compound nucleus. The present formulation, with a schematic exit channel shape configuration and simple rotating liquid-drop nuclear potential, has been found to explain most of the intermediate mass fragments emission cross sections reasonably well without incorporating any free parameters in the calculation

  20. Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro. (United States)

    Griot-Wenk, M; Griot, C; Pfister, H; Vandevelde, M


    Treatment of dissociated murine brain cell cultures with an antibody recognizing galactocerebroside (GalC) led to degeneration of oligodendrocytes with loss of their cell processes. F(ab')2 fragments prepared from this antibody showed no effect. The anti-GalC antibody--but not its F(ab')2 fragments b2 was able to stimulate macrophages in these cultures as seen in a chemiluminescence assay. Therefore, antibodies bound to oligodendrocytes stimulated nearby macrophages by interacting with their Fc receptors. The oligodendroglial damage coincided with the release of toxic compounds by the stimulated macrophages, since treatment of the cultures with the anti-GalC antibody and a variety of other macrophage stimulating agents led to secretion of reactive oxygen species and--in some experiments--tumor necrosis factor, both known to be toxic for oligodendrocytes. These in vitro experiments show evidence that antibody-dependent cellular cytotoxicity may be an important mechanism of tissue destruction in inflammatory demyelinating diseases.

  1. Fragmented medial coronoid process

    International Nuclear Information System (INIS)

    Juhasz, Cs.; Juhasz, T.


    Fragmented medial coronoid process: (FCP) is often considered to be part of the osteochondrosis dissecans complex, but trauma and growth discrepancies between the radius and ulna are proposed as causes. There is little to clinically differentiate FCP, from osteochondrosis dissecans (OCD) of the elbow. Pain on, flexion-extension of the elbow and lateral rotation of the paw is a little more consistent in FCP. Radiographic examination of the elbow is important despite the, fact that radiographic signs of the FCP are often nonspecific. Excessive osteoarthrosis and superimposition of the radial head and coronoid process make identification of the FCP difficult. Craniocaudal, flexed mediolateral and 25 degree craniocaudal-lateromedial views are necessary for diagnosis. Osteophyte production is more dramatic with FCP than with OCD and suggests therefore the occurrence of OCP in many cases. Although the detached process may be seen on any view, the oblique projection offers the least obstructed view. Exposure of the joint is identical to that for OCD, that means a medial approach with osteotomy of the epicondyle. In most cases the process is loose enough to be readily apparent, but in some it is necessary to exert force on the process in order to find the cleavage plane. It is necessary to remove the osteophytes as well and to inspect and irrigate the joint carefully to remove cartilage fragments before closure. Confinement is advisable for 4 weeks before returning the dog to normal activity. The outlook for function is good if the FCP is removed before secondary degenerative joint disease is well established

  2. Sortilin Fragments Deposit at Senile Plaques in Human Cerebrum

    Directory of Open Access Journals (Sweden)

    Xia Hu


    Full Text Available Genetic variations in the vacuolar protein sorting 10 protein (Vps10p family have been linked to Alzheimer’s disease (AD. Here we demonstrate deposition of fragments from the Vps10p member sortilin at senile plaques (SPs in aged and AD human cerebrum. Sortilin changes were characterized in postmortem brains with antibodies against the extracellular and intracellular C-terminal domains. The two antibodies exhibited identical labeling in normal human cerebrum, occurring in the somata and dendrites of cortical and hippocampal neurons. The C-terminal antibody also marked extracellular lesions in some aged and all AD cases, appearing as isolated fibrils, mini-plaques, dense-packing or circular mature-looking plaques. Sortilin and β-amyloid (Aβ deposition were correlated overtly in a region/lamina- and case-dependent manner as analyzed in the temporal lobe structures, with co-localized immunofluorescence seen at individual SPs. However, sortilin deposition rarely occurred around the pia, at vascular wall or in areas with typical diffuse Aβ deposition, with the labeling not enhanced by section pretreatment with heating or formic acid. Levels of a major sortilin fragment ~15 kDa, predicted to derive from the C-terminal region, were dramatically elevated in AD relative to control cortical lysates. Thus, sortilin fragments are a prominent constituent of the extracellularly deposited protein products at SPs in human cerebrum.

  3. Fluctuations in the fragmentation process

    International Nuclear Information System (INIS)

    Botet, R.; Ploszajczak, M.


    Some general framework of sequential fragmentation is presented, as provided by the newly proposed Fragmentation - Inactivation - Binary model, and to study briefly its basic and universal features. This model includes as particular cases most of the previous kinetic fragmentation models. In particular it is discussed how one arrives in this framework to the critical behaviour, called the shattering transition. This model is then compared to recent data on gold multifragmentation at 600 MeV/nucl. (authors) 20 refs., 5 figs

  4. An Algebra for Program Fragments

    DEFF Research Database (Denmark)

    Kristensen, Bent Bruun; Madsen, Ole Lehrmann; Møller-Pedersen, Birger


    Program fragments are described either by strings in the concrete syntax or by constructor applications in the abstract syntax. By defining conversions between these forms, both may be intermixed. Program fragments are constructed by terminal and nonterminal symbols from the grammar and by variab......Program fragments are described either by strings in the concrete syntax or by constructor applications in the abstract syntax. By defining conversions between these forms, both may be intermixed. Program fragments are constructed by terminal and nonterminal symbols from the grammar...... and by variables having program fragments as values. Basic operations such as valuetransfer, composition and decomposition are defined for program fragments allowing more complicated operations to be implemented. Usual operations such as testing for equality are defined, and in addition more specialized operations...... such as testing that a program fragment is derivable from another and converting program fragments in concrete form to abstract form are defined. By introducing regular expressions in the grammar these may be used in program fragments in concrete form. By defining constructors for regular expressions these may...

  5. MRI of displaced meniscal fragments

    International Nuclear Information System (INIS)

    Dunoski, Brian; Zbojniewicz, Andrew M.; Laor, Tal


    A torn meniscus frequently requires surgical fixation or debridement as definitive treatment. Meniscal tears with associated fragment displacement, such as bucket handle and flap tears, can be difficult to recognize and accurately describe on MRI, and displaced fragments can be challenging to identify at surgery. A displaced meniscal fragment can be obscured by synovium or be in a location not usually evaluated at arthroscopy. We present a pictorial essay of meniscal tears with displaced fragments in patients referred to a pediatric hospital in order to increase recognition and accurate interpretation by the radiologist, who in turn can help assist the surgeon in planning appropriate therapy. (orig.)

  6. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement. (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan


    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  7. Principles for computational design of binding antibodies. (United States)

    Baran, Dror; Pszolla, M Gabriele; Lapidoth, Gideon D; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D; Fleishman, Sarel J


    Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called "ideal" folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we ( i ) used sequence-design constraints derived from antibody multiple-sequence alignments, and ( ii ) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes.

  8. Polymer fragmentation in extensional flow

    International Nuclear Information System (INIS)

    Maroja, Armando M.; Oliveira, Fernando A.; Ciesla, Michal; Longa, Lech


    In this paper we present an analysis of fragmentation of dilute polymer solutions in extensional flow. The transition rate is investigated both from theoretical and computational approaches, where the existence of a Gaussian distribution for the breaking bonds has been controversial. We give as well an explanation for the low fragmentation frequency found in DNA experiments

  9. Fracture mechanics model of fragmentation

    International Nuclear Information System (INIS)

    Glenn, L.A.; Gommerstadt, B.Y.; Chudnovsky, A.


    A model of the fragmentation process is developed, based on the theory of linear elastic fracture mechanics, which predicts the average fragment size as a function of strain rate and material properties. This approach permits a unification of previous results, yielding Griffith's solution in the low-strain-rate limit and Grady's solution at high strain rates

  10. Polymer fragmentation in extensional flow

    Energy Technology Data Exchange (ETDEWEB)

    Maroja, Armando M.; Oliveira, Fernando A.; Ciesla, Michal; Longa, Lech


    In this paper we present an analysis of fragmentation of dilute polymer solutions in extensional flow. The transition rate is investigated both from theoretical and computational approaches, where the existence of a Gaussian distribution for the breaking bonds has been controversial. We give as well an explanation for the low fragmentation frequency found in DNA experiments.

  11. Improvement of the method of obtaining human IgA Fc-fragments

    Directory of Open Access Journals (Sweden)

    O. Y. Galkin


    Full Text Available To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies. The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C. As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine. Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.


    Directory of Open Access Journals (Sweden)

    O.V. Galkin


    Full Text Available Obtaining single-chain variable fragments (scFv of recombinant antibodies in E. coli cells is often associated with numerous problems causing low yields or inactive conformation of the product. The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins. Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

  13. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function. (United States)

    Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit


    To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

  14. Making Recombinant Monoclonal Antibody And Radiolabelling For Medical Purpose

    International Nuclear Information System (INIS)

    Nguyen Thi Thu; Duong Van Dong; Vo Thi Cam Hoa; Bui Van Cuong; Chu Van Khoa; Vu Bich Huong; Le Quang Huan


    Recombinant monoclonal antibody labeling with 131 I specific to tumor cell has been studied and prepared for treatment of Hodgkin lymphoma. In this study, a recombinant monoclonal antibody with two specific properties is a hybrid molecule created by coupling an antibody variable fragments with peptide melittin. The gene coding the antibody fragment has been obtained from human synthetic Fv libraries using for panning and screening on populations of lymphocytes fragmented from human blood cells with Hodgkin diseases. The gene encoding peptit melittin has been cloned from honeybee Apis cerana DNA. The gene coding recombinant monoclonal antibody has been expressed in E.coli BL21 (DE3) at 37 o C and was induced with 0.6 mM IPTG. The recombinant compound has been purified by affinity chromatography with HiTrap affinity column. The obtained recombinant monoclonal antibody has showed cytolytic activities when added to cell culture medium for LU cancer cell line with the amount of 100 - 200 mg/ml. This monoclonal antibody is labeled with 131 I using chloramine T procedure. ChT mass for the oxidation of 50 μg monoclonal antibody in 76 MBq was 10 μg. Sodium metabisulfite was used as a reducing agent. Reaction time was above 3 mins. The radiochemical purity was determined using electrophoresis and TLC methods. Radiochemical yield was > 97%. Radiochemical purity after purification was > 99%. Nuclear purity was > 99%. Stability of the label antibody was 12 days. This is the product promise potential used in the diagnostic and therapeutic of Hodgkin lymphoma. (author)

  15. Acetylcholine receptor antibody (United States)

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

  16. Platelet antibodies blood test (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  17. Standard NIJ 0115.00 za testiranje otpornosti zaštitnih prsluka na ubode nožem i predmetima sa oštrim vrhom / NIJ 0115.00 standard for testing stab resistance of body armor

    Directory of Open Access Journals (Sweden)

    Đuro Jovanić


    Full Text Available Standard NIJ 0115.00 (prema Nacionalnom institutu pravde SAD, namenjen je za utvrđivanje minimuma tehničkih zahteva i metoda ispitivanja otpornosti zaštitnih prsluka, koji treba da zaštite gornji deo tela od uboda nožem i predmetima sa oštrim vrhom. Područje primene ovog standarda ograničeno je samo na pretnje ubodima, a ne na balističke pretnje, poput onih koje su obuhvaćene standardom NIJ 0101.04 za testiranje balističke otpornosti zaštitnih prsluka. / In this work made presentation of NIJ 0115.04. (National Institute of Justice standard, witch purpose is to establish minimum performance requirements and methods of testing for the stab resistance of body armor intended to protect the torso against slash and stab threats. The scope of the standard is limited to stab resistance only; the standard does not address ballistic threats, as those are covered by NIJ Standard 0101.04. Ballistic Resistance of Personal Body Armor.

  18. The spectroscopy of fission fragments

    International Nuclear Information System (INIS)

    Phillips, W.R.


    High-resolution measurements on γ rays from fission fragments have provided a rich source of information, unobtainable at the moment in any other way, on the spectroscopy of neutron-rich nuclei. In recent years important data have been obtained on the yrast- and near yrast-structure of neutron-rich fission fragments. We discuss the scope of measurements which can be made on prompt gamma rays from secondary fission fragments, the techniques used in the experiments and some results recently obtained. (author)

  19. The spectroscopy of fission fragments

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, W.R. [Department of Physics and Astronomy, University of Manchester, Manchester, M13 9PL (United Kingdom); Collaboration: La Direction des Sciences de la Matiere du CEA (FR); Le Fonds National de la Recherche Scientifique de Belgique (BE)


    High-resolution measurements on {gamma} rays from fission fragments have provided a rich source of information, unobtainable at the moment in any other way, on the spectroscopy of neutron-rich nuclei. In recent years important data have been obtained on the yrast- and near yrast-structure of neutron-rich fission fragments. We discuss the scope of measurements which can be made on prompt gamma rays from secondary fission fragments, the techniques used in the experiments and some results recently obtained. (author) 24 refs., 8 figs., 1 tab.

  20. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    International Nuclear Information System (INIS)

    Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Lurilli, A.P.; Ponzo, F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A.C.


    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99m Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  1. Human IgG subclass antibodies to the 19 kilodalton carboxy ...

    African Journals Online (AJOL)

    Human IgG subclass antibodies to the 19 kilodalton carboxy terminal fragment of Plasmodium Falciparum merozoite surface protein 1 (MSP1 19 ) and predominance of the MAD20 allelic ... Results: Both the prevalence and the mean concentration of serum IgG1 , and to a lesser extent IgG3, antibodies increased with age.

  2. Immuno-PET : A navigator in monoclonal antibody development and applications

    NARCIS (Netherlands)

    van Dongen, Guus A. M. S.; Visser, Gerard W. M.; Hooge, Marjolijn N. Lub-de; Perk, Lars R.; de Vries, Elisabeth G. E.


    Monoclonal antibodies (mAbs) have been approved for use as diagnostics and therapeutics in a broad range of medical indications, but especially in oncology. In addition, hundreds of new mAbs, engineered mAb fragments, and nontraditional antibody-like scaffolds directed against either validated or

  3. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.


    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  4. Specificity of rabbit antibodies elicited by related synthetic peptides. (United States)

    Chersi, A; Houghten, R A; Chillemi, F; Zito, R; Centis, D


    Three 17-residue peptides, presenting from 65% to 70% sequence homology, and one endecapeptide, with no apparent homology with the first three, were chemically synthesized and investigated in their ability to elicit rabbit antipeptide antibodies. The complex cross reactivities of the antisera were investigated by testing the binding of the antibodies to the intact peptides, to their enzymatic fragments, and by the use of specific immunoadsorbents. Antipeptide antibodies may or may not crossreact with related "parent" peptides, this depending upon number, distribution, and localization of amino acid differences in low or high antigenicity regions of the immunogen. Related peptides may elicit antibodies that crossreact almost completely, and therefore not specific for one or the other "parent" peptide. Those antibodies may therefore be of little use for the selective recognition of closely related structures.

  5. QGP and Modified Jet Fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xin-Nian


    Recent progresses in the study of jet modification in hotmedium and their consequences in high-energy heavy-ion collisions are reviewed. In particular, I will discuss energy loss for propagating heavy quarks and the resulting modified fragmentation function. Medium modification of the parton fragmentation function due to quark recombination are formulated within finite temperature field theory and their implication on the search for deconfined quark-gluon plasma is also discussed.

  6. Robust Object Tracking Using Valid Fragments Selection. (United States)

    Zheng, Jin; Li, Bo; Tian, Peng; Luo, Gang

    Local features are widely used in visual tracking to improve robustness in cases of partial occlusion, deformation and rotation. This paper proposes a local fragment-based object tracking algorithm. Unlike many existing fragment-based algorithms that allocate the weights to each fragment, this method firstly defines discrimination and uniqueness for local fragment, and builds an automatic pre-selection of useful fragments for tracking. Then, a Harris-SIFT filter is used to choose the current valid fragments, excluding occluded or highly deformed fragments. Based on those valid fragments, fragment-based color histogram provides a structured and effective description for the object. Finally, the object is tracked using a valid fragment template combining the displacement constraint and similarity of each valid fragment. The object template is updated by fusing feature similarity and valid fragments, which is scale-adaptive and robust to partial occlusion. The experimental results show that the proposed algorithm is accurate and robust in challenging scenarios.

  7. Targeting Malignant Brain Tumors with Antibodies

    Directory of Open Access Journals (Sweden)

    Rok Razpotnik


    Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

  8. Human anti-animal antibody interferences in immunological assays. (United States)

    Kricka, L J


    The scope and significance of human anti-animal antibody interference in immunological assays is reviewed with an emphasis on human anti-animal immunoglobulins, particularly human anti-mouse antibodies (HAMAs). Anti-animal antibodies (IgG, IgA, IgM, IgE class, anti-isotype, and anti-idiotype specificity) arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years. Prevalence estimates for anti-animal antibodies in the general population vary widely and range from HAMA, which causes both positive and negative interferences in two-site mouse monoclonal antibody-based assays. Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized, polyethylene glycolylated, or Fab fragments of antibody agents. Sample pretreatment or assay redesign can eliminate immunoassay interferences caused by anti-animal antibodies. Enzyme immunoassays, immunoradiometric assays, immunofluorescence, and HPLC assays have been designed to detect HAMA and other anti-animal antibodies, but intermethod comparability is complicated by differences in assay specificity and lack of standardization. Human anti-animal antibodies often go unnoticed, to the detriment of patient care. A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable. Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference.

  9. Recent progress on perturbative QCD fragmentation functions

    International Nuclear Information System (INIS)

    Cheung, K.


    The recent development of perturbative QCD (PQCD) fragmentation functions has strong impact on quarkonium production. I shall summarize B c meson production based on these PQCD fragmentation functions, as well as, the highlights of some recent activities on applying these PQCD fragmentation functions to explain anomalous J/ψ and ψ' production at the Tevatron. Finally, I discuss a fragmentation model based on the PQCD fragmentation functions for heavy quarks fragmenting into heavy-light mesons

  10. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

    DEFF Research Database (Denmark)

    Shilova, N V; Navakouski, M J; Huflejt, M


    The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted...... pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization....

  11. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs. (United States)

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


    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries (United States)

    Tullila, Antti; Nevanen, Tarja K.


    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  13. Fragmentation properties of 6Li

    International Nuclear Information System (INIS)

    Lovas, R.G.; Kruppa, A.T.; Beck, R.; Dickmann, F.


    The α+d and t+τ cluster structure of 6 Li is described in a microscopic α+d cluster model through quantities that enter into the description of cluster fragmentation processes. The states of the separate clusters α, d, t and τ are described as superpositions of Os Slater determinants belonging to different potential size parameters. To describe both the 6 Li and fragment state realistically, nucleon-nucleon forces optimized for the used model state spaces were constructed. The fragmentation properties predicted by them slightly differ from those calculated with some forces of common use provided the latter are modified so as to reproduce the α, d and 6 Li energies. (author) 61 refs.; 9 figs

  14. Incomplete Antibodies May Reduce ABO Cross-Match Incompatibility: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Mehmet Özen


    Full Text Available Objective: Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab part of the antibody (incomplete antibody may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests. Materials and Methods: Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2 fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2 solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2 solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2 antibodies and their effects on the agglutination normally observed with complete antibodies were then measured. Results: No agglutination for the purified incomplete anti-A Fab (2 with A Rh+ erythrocyte and anti-B Fab (2 with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies. Conclusion: We determined that the Fab (2 fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an

  15. Hands as markers of fragmentation

    Directory of Open Access Journals (Sweden)

    A. Barnard


    Full Text Available Margaret Atwood is an internationally read, translated, and critiqued writer whose novels have established her as one of the most esteemed authors in English (McCombs & Palmer, 1991:1. Critical studies of her work deal mainly with notions of identity from psychoanalytical perspectives. This study has identified a gap in current critical studies on Atwood’s works, namely the challenging of textual unity which is paralleled in the challenging of the traditional (single narrative voice. The challenging of textual unity and the single narrative voice brings about the fragmentation of both. This article will focus on the role that hands play as markers of fragmentation in “The Blind Assassin” (2000. In the novel, the writing hand destabilises the narrative voice, since it is not connected to the voice of a single author. If the author of the text – the final signified – is eliminated, the text becomes fragmentary and open, inviting the reader to contribute to the creation of meaning. Hands play a signficant role in foregrounding the narrator’s fragmented identity, and consequently, the fragmentation of the text. We will investigate this concept in the light of Roland Barthes’ notion of the scriptor, whose hand is metaphorically severed from his or her “voice”. Instead of the text being a unified entity, it becomes unstable and it displays the absence of hierarchical textual levels. Based mainly on Barthes’ writings, this article concludes that hands foreground the narrator’s fragmented identity, which is paralleled in the fragmented text.

  16. Possibilities in improvement of the specific biodistribution by means of radiolabelled antibodies after complexation

    International Nuclear Information System (INIS)

    Heinrich, H.


    The applicability of labeled by different nuclides poly- or monoclonal antibodies in nuclear medicine diagnostics is restricted by incidental consequences. Overhigh background activities and/or high (additional) binding reactivities in the liver are troublesome. Hence we examined if the lokalizing biodistribution of labeled antibodies regarding the target organ can be improved by complexation with metal ions. The investigations were carried out with the pancreas of four Beagle dogs. Xenogenic polyclonal antibodies were refined by passing down the gammaglobulin fraction of rabbit serum a sephadex G 200 - column three times and hence radiolabeled by Na 131 I. Then the antibodies were injected i.p. either as complete molecules or as partially digested by pepsin fragments F(ab) 2 -fragments in order to avoid nonspecific Fc interactions. In preparing the i.p. injection another part of the antibody preparations both complete and fragments had been complexed with copper ions in an alkaline buffer giving a stable copper-protein-complex (Biuret-complex). Undergoing scanning and after killing the dogs the organ deposition of the radiolabel had been observed and noticed. The complexation with copper of radiolabeled antibodies improves strikingly the specific target organ uptake. Because of inflammatory and other incidental consequences the Fc-fragments should be removed by digestion with enzymes. Higher specific organ uptake should be obtainable if the poyclonal antibody fraction is more refined specifically by immunosorption with respect to its affinity to the specific antigenic structures. (orig.) [de

  17. Effects of genetic engineering on the pharmacokinetics of antibodies

    International Nuclear Information System (INIS)

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


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

  18. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang


    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  19. [Stab wounds of the hand and forearm due to Kuluna in Kinshasa (Democratic Republic of Congo): types of injuries and treatment]. (United States)

    Kibadi, K; Portaels, F; Pichot, Y; Kapinga, M; Moutet, F


    Democratic Republic of Congo (DRC), a particular form of juvenile delinquency and insecurity intensifies in the city of Kinshasa. This is the phenomenon Kuluna. It is organized gangs equipped with machetes and other weapons. The main objective of this study is to know the phenomenon Kuluna and describe the upper limb injuries caused by machetes, while insisting on the specifics of the management of these lesions in our communities. This retrospective descriptive study examines 14 cases of wounds of the hand and forearm due to stab phenomenon Kuluna, in Kinshasa. It covers the period from 1 November 2010 to 1 November 2013. Among the 14 patients with lesions in the hand and forearm admitted and treated at the Unit of Plastic Reconstructive and Aesthetic Surgery, Hand Surgery and Burns, University Clinics of Kinshasa to attacks due to the phenomenon Kuluna. We have 11 men and 3 women. The average age was 33, 5 years (extremes of 21 and 56 years). The right upper limb is reached that the left upper limb, respectively 12 patients and 2 patients. The lesions are localized to the wrist in the majority of cases (10 patients) in the palm of hand and in 3 patients in the fingers in 1 patient. The palmar surface is reached (10 cases) and the dorsal (4 cases). Zone 5 of the International Classification of flexor and Zone 8 topographic classification extensors at hand are the predilection sites of lesions respectively the palmar surface (6 out of 10) and the dorsal (2 case 4). The median nerve at the wrist is cut in half the cases. On bone lesions localized to the forearm, we observed a high incidence of fracture of the ulna (62.5%). The treatment begins with the stabilization of bone pieces, gestures revascularization and nerve sutures and suture tendon and finally skin coverage. Rehabilitation was mandatory, she supervises the actions of repair and it continues until the recovery of function.

  20. Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression. (United States)

    Mayrhofer, Patrick; Kunert, Renate


    Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

  1. Human antibody production in transgenic animals. (United States)

    Brüggemann, Marianne; Osborn, Michael J; Ma, Biao; Hayre, Jasvinder; Avis, Suzanne; Lundstrom, Brian; Buelow, Roland


    Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

  2. Fragmented nature: consequences for biodiversity

    NARCIS (Netherlands)

    Olff, H.; Ritchie, M.E.


    We discuss how fragmentation of resources and habitat operate differently on species diversity across spatial scales, ranging from positive effects on local species coexistence to negative effect on intermediate spatial scales, to again positive effects on large spatial and temporal scales. Species

  3. Fragmented nature : consequences for biodiversity

    NARCIS (Netherlands)

    Olff, Han; Ritchie, Mark E.


    We discuss how fragmentation of resources and habitat operate differently on species diversity across spatial scales, ranging from positive effects on local species coexistence to negative effect on intermediate spatial scales, to again positive effects on large spatial and temporal scales. Species

  4. Nuclear energy release from fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Cheng [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Souza, S.R. [Instituto de Física, Universidade Federal do Rio de Janeiro Cidade Universitária, Caixa Postal 68528, 21945-970 Rio de Janeiro (Brazil); Tsang, M.B. [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); National Superconducting Cyclotron Laboratory and Physics and Astronomy Department, Michigan State University, East Lansing, MI 48824 (United States); Zhang, Feng-Shou, E-mail: [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Center of Theoretical Nuclear Physics, National Laboratory of Heavy Ion Accelerator of Lanzhou, Lanzhou 730000 (China)


    It is well known that binary fission occurs with positive energy gain. In this article we examine the energetics of splitting uranium and thorium isotopes into various numbers of fragments (from two to eight) with nearly equal size. We find that the energy released by splitting {sup 230,232}Th and {sup 235,238}U into three equal size fragments is largest. The statistical multifragmentation model (SMM) is applied to calculate the probability of different breakup channels for excited nuclei. By weighing the probability distributions of fragment multiplicity at different excitation energies, we find the peaks of energy release for {sup 230,232}Th and {sup 235,238}U are around 0.7–0.75 MeV/u at excitation energy between 1.2 and 2 MeV/u in the primary breakup process. Taking into account the secondary de-excitation processes of primary fragments with the GEMINI code, these energy peaks fall to about 0.45 MeV/u.

  5. Phthalocyanides sensitized fragmentation of proteins

    Czech Academy of Sciences Publication Activity Database

    Klementová, S.; Tothová, D.; Revaková, R.; Kasková, M.; Wagnerová, Dana Marie


    Roč. 5, č. 1 (2001), s. 13-18 ISSN 0972-0626 R&D Projects: GA ČR GA203/96/1322 Institutional research plan: CEZ:AV0Z4032918 Keywords : phthalocyanides * photosensitied fragmentation of proteins Subject RIV: CA - Inorganic Chemistry

  6. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

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


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

  7. Radioimmunoimaging of tumors with a pantumor antibody

    International Nuclear Information System (INIS)

    Chen, D.C.P.; Siegel, M.E.; Chen, F.; Taylor, O.R.; Epstein, A.L.


    The TNT-1 antibody was developed to bind intracellular nuclear antigens that are accessible only in degenerative or necrotic cells. Since about 50% of tumor cells are in various stages of cell degeneration or death, this antibody could serve as a pantumor antibody for tumor detection. After intravenous injection of 10 μg of TNT-1F(ab')2 fragments labeled with 20 μCi of I-131, serial images were obtained at 1 and 4 hours and daily for 6 days in mice bearing various human tumors. Accumulation of TNT-1 was imaged in a necrotic tumor as early as 4 hours after injection and because more intense at 48 hours. The tumor-muscle ratio was as high as 29:1. Intense accumulation was noted in the necrotic tumor, about nine times that of healthy tumor. In conclusion, TNT-1, a pantumor antibody, can detect necrotic tumors in animal models. It may be an ideal imaging agent for cancer detection


    Directory of Open Access Journals (Sweden)

    A A. Jafari


    Full Text Available After using 3 different generations of antibodies including human and non-human hyperimmune sera, monoclonal antibodies and chimeric antibodies, more recently a newer approach has been developed in which the antibody genes are cloned directly from a patient peripheral B-lymphocytes and expressed in a host like E. coli. In this study the Candida albicans serotype A (NCTC 3153 mannan was purified using a modified Fehling method and used for selection of human recombinant antibody from a C. albicans phage antibody library. After four rounds of affinity selecting (panning, 2 predominant clones were chosen by DNA fingerprinting and ELISA. A 248 amino acid DNA fragment coding for anti-C. albicans mannan scFv was sequenced and cloned in a pBAD-TOPO cloning vector to produce a soluble and phage free antibody. The analysis of antibody sequences by V base Index (DNAPLOT confirmed the human antibody origin with the VH4 family in V segment of heavy variable chain and VL3 (Lambda 3 in J segment of the light variable chain. This antibody fragment was purified using immobilized metal affinity chromatography and inmmunoblotted as a 31kDa recombinant protein.

  9. Experimental Volcanology: Fragmentation and Permeability (United States)

    Spieler, O.


    An increasing number of scientists design new experiments to analyse processes that control the dynamics of explosive eruptions. There research is mostly coupled to numerical models and aims toward its controlling parameters. The fragmentation process, its threshold and the speed of the fragmentation wave as well as the energy consumed by the fragmentation are some hot spots of the experimental volcanology. Analysing the fragmentation behaviour of volcaniclastics as close to the natural system as possible, we found a number of physical constrains. Identifying the porosity as determining factor of the threshold, we realised that neither threshold nor the speed of the fragmentation process are solely controlled by the rock density. The later results of the shock tube type apparatus lead to the analysis of the specific surface area and permeability as direct links to textural features. Permeability analysis performed in a modified shock tube type apparatus, show two clear, distinct trends for dome rock and pyroclastic samples. The specific surface determined by Argon sorbtion (BET) as well as textural features of pumices from Campi Flegrei, Montserrat and Krakatoa (1883) give in contrary evidence of a more complex story. Large spherical, or ellipsoidal bubbles around fractured crystals prove that the high permeability of the pumice has partially developed after the fixing of the bubble size distribution. This puts up the question, if permeability measurements on pyroclastic samples reveal relevant numbers! The surface tension controlled 'self sealing' behaviour of surfaces from foaming obsidian hinders in situ measurements. Close textural investigations will have to clarify how the 'post process' samples deviate from the syneruptive conduit filling.

  10. The VERDI fission fragment spectrometer

    International Nuclear Information System (INIS)

    Fregeau, M. O.; Brys, T.; Gamboni, T.; Geerts, W.; Oberstedt, S.; Oberstedt, A.; Borcea, R.


    The VERDI time-of-flight spectrometer is dedicated to measurements of fission product yields and of prompt neutron emission data. Pre-neutron fission-fragment masses will be determined by the double time-of-flight (TOF) technique. For this purpose an excellent time resolution is required. The time of flight of the fragments will be measured by electrostatic mirrors located near the target and the time signal coming from silicon detectors located at 50 cm on both sides of the target. This configuration, where the stop detector will provide us simultaneously with the kinetic energy of the fragment and timing information, significantly limits energy straggling in comparison to legacy experimental setup where a thin foil was usually used as a stop detector. In order to improve timing resolution, neutron transmutation doped silicon will be used. The high resistivity homogeneity of this material should significantly improve resolution in comparison to standard silicon detectors. Post-neutron fission fragment masses are obtained form the time-of-flight and the energy signal in the silicon detector. As an intermediary step a diamond detector will also be used as start detector located very close to the target. Previous tests have shown that poly-crystalline chemical vapour deposition (pCVD) diamonds provides a coincidence time resolution of 150 ps not allowing complete separation between very low-energy fission fragments, alpha particles and noise. New results from using artificial single-crystal diamonds (sCVD) show similar time resolution as from pCVD diamonds but also sufficiently good energy resolution. (authors)

  11. Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab' fragment

    DEFF Research Database (Denmark)

    Spangfort, M D; Mirza, Osman Asghar; Gajhede, M


    of the clinical symptoms of allergy. In order to study the structural basis of allergen-antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab' fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1...

  12. Antibodies Against Melanin

    African Journals Online (AJOL)


    Jan 6, 1973 ... Departments of Internal Medicine and Anatomical Pathology, University of Stellenbosch and MRC. Pigment Metabolism Research Unit, ... at the production of antibodies against natural melanoprotein. and a consideration of our negative .... the random polymerization of several monomers, antibody formed ...

  13. Recombinant renewable polyclonal antibodies. (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


    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.

  14. New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness

    Directory of Open Access Journals (Sweden)

    Isabel Corraliza-Gorjón


    Full Text Available Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2+ breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin and bevacizumab (Avastin, respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient’s own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.

  15. Screening Phage-Display Antibody Libraries Using Protein Arrays. (United States)

    Jara-Acevedo, Ricardo; Díez, Paula; González-González, María; Dégano, Rosa María; Ibarrola, Nieves; Góngora, Rafael; Orfao, Alberto; Fuentes, Manuel


    Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation. Here, we describe a new method based on an array technology that allows the deposition of hundreds to thousands of phages by micro-contact on a unique nitrocellulose surface. This setup comes in combination with bioinformatic approaches that enables simultaneous affinity screening in a HT format of antibody-displaying phages.

  16. Antibody engineering: methods and protocols

    National Research Council Canada - National Science Library

    Chames, Patrick


    "Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient...

  17. Anti-insulin antibody test (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

  18. Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens

    International Nuclear Information System (INIS)

    Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.


    Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125 I. The 125 I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography

  19. Fragmentation of suddenly heated liquids

    International Nuclear Information System (INIS)

    Blink, J.A.


    Fragmentation of free liquids in Inertial Confinement Fusion reactors could determine the upper bound on reactor pulse rate. The x-ray ablated materials must cool and recondense to allow driver beam propagation. The increased surface area caused by fragmentation will enhance the cooling and condensation rates. Relaxation from the suddenly heated state will move a liquid into the negative pressure region under the liquid-vapor P-V dome. The lithium equation of state was used to demonstrate that neutron-induced vaporization uses only a minor fraction of the added heat, much less than would be required to drive the expansion. A 77% expansion of the lithium is required before the rapid vaporization process of spinodal decomposition could begin, and nucleation and growth are too slow to contribute to the expansion

  20. Fragmentering og korridorer i landskabet

    DEFF Research Database (Denmark)

    Hammershøj, M.; Madsen, A. B.

    , at fragmentering af habitater resulterer i en reduktion og isolering af mange plante- og dyrepopulationer. Det er desuden vist, at korridorer har en funktion som habitater, hvilket er medvirkende til, at et område med korridorer kan huse flere arter og individer end et tilsvarende område uden korridorer. Der......Rapporten indeholder en litteraturudredning, der er baseret på en bearbejdning af den tilgængelige nationale og internationale litteratur omhandlende fragmentering og korridorer på det botaniske og zoologiske område. I alt 1.063 titler ligger til grund for udredningen. Udredningen har vist...... mangler dog entydige beviser for, at korridorer kan være af afgørende betydning for rekolonisering af habitater, i hvilke en given art er forsvundet. Afslutningsvis gives en liste med forskningsbehov samt en række anbefalinger....

  1. Fragmentation of percolation cluster perimeters (United States)

    Debierre, Jean-Marc; Bradley, R. Mark


    We introduce a model for the fragmentation of porous random solids under the action of an external agent. In our model, the solid is represented by a bond percolation cluster on the square lattice and bonds are removed only at the external perimeter (or `hull') of the cluster. This model is shown to be related to the self-avoiding walk on the Manhattan lattice and to the disconnection events at a diffusion front. These correspondences are used to predict the leading and the first correction-to-scaling exponents for several quantities defined for hull fragmentation. Our numerical results support these predictions. In addition, the algorithm used to construct the perimeters reveals itself to be a very efficient tool for detecting subtle correlations in the pseudo-random number generator used. We present a quantitative test of two generators which supports recent results reported in more systematic studies.

  2. Fragmented nature: consequences for biodiversity


    Olff, Han; Ritchie, Mark E.


    We discuss how fragmentation of resources and habitat operate differently on species diversity across spatial scales, ranging from positive effects on local species coexistence to negative effect on intermediate spatial scales, to again positive effects on large spatial and temporal scales. Species with different size and mobility can be regulated by different processes at the same spatial scale, a principle that may contribute to diversity. Differences in species richness between local commu...

  3. Virtual reunification of papyrus fragments


    Vannini, Lucia


    Many Greek and Latin papyri, originally belonging to only one book (be it in roll or codex form), are currently scattered among different libraries. While it is not possible to physically rejoin these fragments as they cannot be moved from their institutions, they may be virtually reunited thanks to the techniques of digitisation, image processing and electronic publishing. This paper focuses on some issues – emerged from the work of my MA dissertation – that virtual reunification of Greek an...

  4. Monoclonal antibody "gold rush". (United States)

    Maggon, Krishan


    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.

  5. Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies

    Directory of Open Access Journals (Sweden)

    Fa Yang


    Full Text Available With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

  6. Fragmentation measurement using image processing

    Directory of Open Access Journals (Sweden)

    Farhang Sereshki


    Full Text Available In this research, first of all, the existing problems in fragmentation measurement are reviewed for the sake of its fast and reliable evaluation. Then, the available methods used for evaluation of blast results are mentioned. The produced errors especially in recognizing the rock fragments in computer-aided methods, and also, the importance of determination of their sizes in the image analysis methods are described. After reviewing the previous work done, an algorithm is proposed for the automated determination of rock particles’ boundary in the Matlab software. This method can determinate automatically the particles boundary in the minimum time. The results of proposed method are compared with those of Split Desktop and GoldSize software in two automated and manual states. Comparing the curves extracted from different methods reveals that the proposed approach is accurately applicable in measuring the size distribution of laboratory samples, while the manual determination of boundaries in the conventional software is very time-consuming, and the results of automated netting of fragments are very different with the real value due to the error in separation of the objects.

  7. Residual Fragments after Percutaneous Nephrolithotomy

    Directory of Open Access Journals (Sweden)

    Kaan Özdedeli


    Full Text Available Clinically insignificant residual fragments (CIRFs are described as asymptomatic, noninfectious and nonobstructive stone fragments (≤4 mm remaining in the urinary system after the last session of any intervention (ESWL, URS or PCNL for urinary stones. Their insignificance is questionable since CIRFs could eventually become significant, as their presence may result in recurrent stone growth and they may cause pain and infection due to urinary obstruction. They may become the source of persistent infections and a significant portion of the patients will have a stone-related event, requiring auxilliary interventions. CT seems to be the ultimate choice of assessment. Although there is no concensus about the timing, recent data suggests that it may be performed one month after the procedure. However, imaging can be done in the immediate postoperative period, if there are no tubes blurring the assessment. There is some evidence indicating that selective medical therapy may have an impact on decreasing stone formation rates. Retrograde intrarenal surgery, with its minimally invasive nature, seems to be the best way to deal with residual fragments.

  8. Accuracy of Computed Tomography in Diagnosis of Intra-abdominal Injuries in Stable Patients with Anterior Abdominal Stab Wounds: A Systematic Review and Meta-Analysis. (United States)

    Baron, Bonny J; Benabbas, Roshanak; Kohler, Casey; Biggs, Carina; Roudnitsky, Valery; Paladino, Lorenzo; Sinert, Richard


    Work-up for patients presenting to the Emergency Department (ED) following an anterior abdominal stab wound (AASW) has been debated since the 1960s. Experts agree that patients with peritonitis, evisceration, or hemodynamic instability should undergo immediate laparotomy (LAP), however, workup of stable, asymptomatic or non-peritoneal, patients is not clearly defined. To evaluate the accuracy of computed tomography of abdomen and pelvis (CTAP) for diagnosis of intra-abdominal injuries requiring Therapeutic Laparotomy (THER-LAP) in ED patients with AASW. Is a negative CT scan without a period of observation sufficient to safely discharge a hemodynamically stable, asymptomatic AASW patient? We searched PUBMED, EMBASE, and Scopus from their inception until May 2017 for studies on ED patients with AASW. We defined the reference standard test as LAP for patients who were managed surgically and those with THER-LAP were considered as disease-positive. In those who were managed nonsurgically, inpatient observation was considered the reference standard. We used the Quality Assessment Tool for Diagnostic Accuracy Studies (QUADAS-2) to evaluate the risk of bias and applicability of the included studies. We attempted to compute the pooled sensitivity, specificity, Likelihood Ratios (LR+, LR-) using a random-effects model with MetaDiSc software and calculate testing and treatment thresholds for CT scan applying the Pauker and Kassirer model. Seven studies were included encompassing 575 patients. The weighted prevalence of THER-LAP was 34.3% (95% CI 30.5-38.2%). Studies had variable quality and the inclusion criteria were not uniform. The operating characteristics of CT scan were: sensitivity 50%-100%, specificity 39%-97%, LR+ 1.0-15.7 and LR- 0.07-1.0. The high heterogeneity (I-square>75%) of the operating characteristics of CT scan prevented pooling of the data and therefore the testing and treatment thresholds could not be estimated. The articles revealed a high prevalence (8

  9. Iodine 123 radiolabeling of monoclonal antibodies for in vivo procedures

    International Nuclear Information System (INIS)

    Mills, S.L.; Denardo, S.J.; Denardo, G.L.; Epstein, A.L.; Peng, J.S.; Colcher, D.


    When labeled to monoclonal antibodies (MAbs) or their fragments, 123 I can be used for imaging or for predicting the treatment potential and radiation dosimetry of 131 I labeled to the same molecular species. Because 123 I (p,5n) from the Crocker Nuclear Laboratory is in dilute solution, when compared with commercial 125 I of labeling grade, we have evaluated labeling parameters using Chloramine-T as the oxidant and derived an optimum set of labeling conditions that provide a 60-80% radiochemical yield of highly immunoreactive antibody. When Lym-1, an IgG-2a murine antibody against human lymphoma, was used, yields of labeled immunoglobulin were decreased by protein or Chloramine-T concentrations less than 0.4 microgram/microliter and 0.8 microgram/microliter, respectively; denaturation of the immunoglobulin occurred when the Chloramine-T concentration was greater than 1.0 microgram/microliter. Optimum labeling occurred at pH 7-8 with deleterious effects when the pH was below 5 or above 10. An optimum method for labeling antibodies with multimillicurie amounts of 123 I (less than one iodine atom per 100 antibody molecules) is described. Some of the observations derived from this study are also applicable to the preparation of treatment doses of 131 I-labeled antibodies, wherein the amount of antibody can be a restrictive factor

  10. Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics. (United States)

    Bumbaca, Daniela; Boswell, C Andrew; Fielder, Paul J; Khawli, Leslie A


    Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.

  11. Fragmentation during primordial star formation (United States)

    Dutta, Jayanta

    Understanding the physics of the very first stars in the universe, the so-called Population III (or Pop III) stars, is crucial in determining how the universe evolved into what we observe today. In the standard model of Pop III star formation, the baryonic matter, mainly atomic hydrogen, collapses gravitationally into small Dark Matter (DM) minihalos. However, so far there is little understanding on how the thermal, dynamical and chemical evolution of the primordial gas depend on the initial configuration of the minihalos (for example, rotation of the unstable clumps inside minihalos, turbulence, formation of molecular hydrogen and cosmic variance of the minihalos). We use the modified version of the Gadget-2 code, a three-dimensional smoothed particle hydrodynamics (SPH) simulations, to follow the evolution of the collapsing gas in both idealized as well as more realistic minihalos. Unlike some earlier cosmological calculations, the implementation of sink particles allows us to follow the evolution of the accretion disk that builds up in the centre of each minihalo and fragments. We find that the fragmentation behavior depends on the adopted choice of three-body H2 formation rate coefficient. The increasing cooling rate during rapid conversion of the atomic to molecular hydrogen is offset by the heating due to gas contraction. We propose that the H2 cooling, the heating due to H2 formation and compressional heating together set a density and temperature structure in the disk that favors fragmentation. We also find that the cloud's initial degree of rotation has a significant effect on the thermal and dynamical evolution of the collapsing gas. Clouds with higher rotation exhibit spiral-arm-like structures that become gravitationally unstable to fragmentation on several scales. These type of clouds tend to fragment more and have lower accretion rates compared to their slowly rotating counterparts. In addition, we find that the distribution of specific angular

  12. Kinetics of intralymphatically delivered monoclonal antibodies

    International Nuclear Information System (INIS)

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.


    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations

  13. Detection and differentiation of cytomegalovirus antibodies by radioimmunoassay

    International Nuclear Information System (INIS)

    Jankowski, M.A.; Gut, W.; Nawrocka, E.; Kantoch, M.


    A sensitive radioimmunological method was adapted for the assay of anticytomegalovirus IgG and IgM antibodies. Binding of immunoglobulins G with cytomegalovirus (CMV) antigens was greatly reduced when an antigen prepared by sonication of the infected cells in glycine buffer was used in place of the antigen obtained by freezing and thawing of the cells. The application of labelled serum against the Fc fragment of IgG made it possible to identify selectively the antibodies bound with the virus antigen by antigenic determinants. (Auth.)

  14. Identification of amplified fragment length polymorphism (AFLP ...

    African Journals Online (AJOL)

    Identification of amplified fragment length polymorphism (AFLP) fragments linked to soybean mosaic virus resistance gene in Glycine soja and conversion to a sequence characterized amplified regions (SCAR) marker for rapid selection.

  15. Serum herpes simplex antibodies (United States)

    ... causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test is Performed A blood sample ... person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  16. Antibody Blood Tests (United States)

    Antibody Blood Tests Researchers have discovered that people with celiac disease who eat gluten have higher than normal levels of ... do I do if I have a negative blood test (or panel) but I’m still having symptoms? ...

  17. Polarization and alignment of nucleus fission fragments

    International Nuclear Information System (INIS)

    Barabanov, A.L.; Grechukhin, D.P.


    Correlation of fragment orientation with orientation axis of fissile nucleus and with n-vector f vector of fragment divergence is considered. Estimations of polarization and alignment of fission fragments of preliminarily oriented nuclei in correlation (with n-vector f recording) and integral (with n-vector f averaging) experiments were conducted. It is shown that high sensitivity of polarization and fragment alignment to the character of nucleus movement at the stage of descent from barrier to rupture point exists

  18. A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line. (United States)

    Yu, Jie; Wang, Xiaoxiao; Xu, Tao; Jin, Qiuheng; Duan, Jinyuan; Wu, Jie; Wu, Haiyan; Xu, Ting; Ye, Sheng


    Combinations of different antibodies have been shown to be more effective for managing certain diseases than monotherapy. Co-expression of the antibody mixture in a single cell line is key to reducing complexity during antibody development and manufacturing. However, co-transfection of multiple light and heavy chains into cells often leads to production of mismatched, heterodimeric by-products that are inactive, making the development of co-expression systems that robustly and efficiently produce highly active antibody mixtures a high priority. In this study, we modified the CH3 domain interface of the antibody fragment crystallizable (Fc) region by changing several charge pairs to create electrostatic interactions favoring Fc homodimer formation and disfavoring Fc heterodimer formation. When co-expressed, these modified antibodies with altered charge polarity across the Fc dimer interface preferentially formed homodimers that fully preserved the functions of each component, rather than inactive heterodimers whose formation was reduced because of rationally designed repulsive interactions. We designed eight different combinations and experimentally screened the best one, which enabled us to produce a binary antibody mixture against the EGF receptor with a minimal heterodimer contaminant. We further determined the crystal structure of a triple-mutated Fc variant in the best combination, and we elucidated the molecular interactions favoring Fc homodimer over heterodimer formation, which provided a structural basis for further optimization. The approach presented here demonstrates the feasibility of rational antibody modification for efficient and consistent production of monoclonal antibody mixtures in a single cell line and thus broadens our options for manufacturing more effective antibody-based therapeutic agents. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Scaling and four-quark fragmentation

    NARCIS (Netherlands)

    Scholten, O.; Bosveld, G. D.


    The conditions for a scaling behaviour from the fragmentation process leading to slow protons are discussed. The scaling referred to implies that the fragmentation functions depend on the light-cone momentum fraction only. It is shown that differences in the fragmentation functions for valence- and

  20. Quark fragmentation in e+e- collisions

    International Nuclear Information System (INIS)

    Oddone, P.


    This brief review of new results in quark and gluon fragmentation observed in e + e - collisions concentrates mostly on PEP results and, within PEP, mostly on TPC results. The new PETRA results have been reported at this conference by M. Davier. It is restricted to results on light quark fragmentation since the results on heavy quark fragmentation have been reported by J. Chapman

  1. Remarks about the hypothesis of limiting fragmentation

    International Nuclear Information System (INIS)

    Chou, T.T.; Yang, C.N.


    Remarks are made about the hypothesis of limiting fragmentation. In particular, the concept of favored and disfavored fragment distribution is introduced. Also, a sum rule is proved leading to a useful quantity called energy-fragmentation fraction. (author). 11 refs, 1 fig., 2 tabs

  2. Scaling and critical behaviour in nuclear fragmentation

    International Nuclear Information System (INIS)

    Campi, X.


    These notes review recent results on nuclear fragmentation. An analysis of experimental data from exclusive experiments is made in the framework of modern theories of fragmentation of finite size objects. We discuss the existence of a critical regime of fragmentation and the relevance of scaling and finite size scaling

  3. Self-organized criticality in fragmenting

    DEFF Research Database (Denmark)

    Oddershede, L.; Dimon, P.; Bohr, J.


    The measured mass distributions of fragments from 26 fractured objects of gypsum, soap, stearic paraffin, and potato show evidence of obeying scaling laws; this suggests the possibility of self-organized criticality in fragmenting. The probability of finding a fragment scales inversely to a power...

  4. A Molecular Dynamics Simulation of the Human Lysozyme –Camelid VHH HL6 Antibody System

    Directory of Open Access Journals (Sweden)

    Zhi-Yuan Su


    Full Text Available Amyloid diseases such as Alzheimer’s and thrombosis are characterized by an aberrant assembly of specific proteins or protein fragments into fibrils and plaques that are deposited in various tissues and organs. The single-domain fragment of a camelid antibody was reported to be able to combat against wild-type human lysozyme for inhibiting in-vitro aggregations of the amyloidogenic variant (D67H. The present study is aimed at elucidating the unbinding mechanics between the D67H lysozyme and VHH HL6 antibody fragment by using steered molecular dynamics (SMD simulations on a nanosecond scale with different pulling velocities. The results of the simulation indicated that stretching forces of more than two nano Newton (nN were required to dissociate the protein-antibody system, and the hydrogen bond dissociation pathways were computed.

  5. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov


    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  6. Towards crystal structures of antibodies and transcription factors

    Czech Academy of Sciences Publication Activity Database

    Písačková, Jana; Procházková, Kateřina; Král, Vlastimil; Fábry, Milan; Řezáčová, Pavlína


    Roč. 20, č. 2 (2013), s. 121-123 ISSN 1211-5894 R&D Projects: GA MŠk ME08016; GA MŠk 1M0505; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : scFv antibody fragment * DeoR repressor protein * thermofluor assay * protein crystallization Subject RIV: FR - Pharmacology ; Medidal Chemistry

  7. Cancer Imaging and Therapy with Bispecific Antibody Pretargeting


    Goldenberg, David M.; Chatal, Jean-Francois; Barbet, Jacques; Boerman, Otto; Sharkey, Robert M.


    This article reviews recent preclinical and clinical advances in the use of pretargeting methods for the radioimmunodetection and radioimmunotherapy of cancer. Whereas directly-labeled antibodies, fragments, and subfragments (minibodies and other constructs) have shown promise in both imaging and therapy applications over the past 25 years, their clinical adoption has not fulfilled the original expectations due to either poor image resolution and contrast in scanning or insufficient radiation...

  8. Positron emission tomographic imaging of tumors using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Zalutsky, M.R.


    This research project is developing methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). This report describes the development of methods for labeling MAbs and their fragments with positron-emitting halogen nuclides, fluorine-18 and iodine-124. These nulides were selected because of the widespread availability of F-18 and because of our extensive experience in the development of new protein radiohalogenation methods.

  9. Reframing landscape fragmentation's effects on ecosystem services. (United States)

    Mitchell, Matthew G E; Suarez-Castro, Andrés F; Martinez-Harms, Maria; Maron, Martine; McAlpine, Clive; Gaston, Kevin J; Johansen, Kasper; Rhodes, Jonathan R


    Landscape structure and fragmentation have important effects on ecosystem services, with a common assumption being that fragmentation reduces service provision. This is based on fragmentation's expected effects on ecosystem service supply, but ignores how fragmentation influences the flow of services to people. Here we develop a new conceptual framework that explicitly considers the links between landscape fragmentation, the supply of services, and the flow of services to people. We argue that fragmentation's effects on ecosystem service flow can be positive or negative, and use our framework to construct testable hypotheses about the effects of fragmentation on final ecosystem service provision. Empirical efforts to apply and test this framework are critical to improving landscape management for multiple ecosystem services. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The formation of planets by disc fragmentation

    Directory of Open Access Journals (Sweden)

    Stamatellos Dimitris


    Full Text Available I discuss the role that disc fragmentation plays in the formation of gas giant and terrestrial planets, and how this relates to the formation of brown dwarfs and low-mass stars, and ultimately to the process of star formation. Protostellar discs may fragment, if they are massive enough and can cool fast enough, but most of the objects that form by fragmentation are brown dwarfs. It may be possible that planets also form, if the mass growth of a proto-fragment is stopped (e.g. if this fragment is ejected from the disc, or suppressed and even reversed (e.g by tidal stripping. I will discuss if it is possible to distinguish whether a planet has formed by disc fragmentation or core accretion, and mention of a few examples of observed exoplanets that are suggestive of formation by disc fragmentation.

  11. Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

    Directory of Open Access Journals (Sweden)

    María Elena Iezzi


    Full Text Available Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs, in particular those engineered from the variable heavy-chain fragment (VHH gene found in Camelidae heavy-chain antibodies (or IgG2 and IgG3, are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

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

    Directory of Open Access Journals (Sweden)

    Juan C eAlmagro


    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.

  13. Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Petersen, E


    This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

  14. A High Through-put Platform for Recombinant Antibodies to Folded Proteins*


    Hornsby, Michael; Paduch, Marcin; Miersch, Shane; Sääf, Annika; Matsuguchi, Tet; Lee, Brian; Wypisniak, Karolina; Doak, Allison; King, Daniel; Usatyuk, Svitlana; Perry, Kimberly; Lu, Vince; Thomas, William; Luke, Judy; Goodman, Jay


    Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had ...

  15. Neutralization of feline immunodeficiency virus by polyclonal cat antibody: Simultaneous involvement of hypervariable regions 4 and 5 of the surface glycoprotein.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); W. Huisman (Willem); J.A. Karlas (Jos); G.F. Rimmelzwaan (Guus); M.L. Bosch (Marnix); A.D.M.E. Osterhaus (Albert)


    textabstractSites involved in antibody-mediated neutralization of feline immunodeficiency virus were mapped by reciprocal exchange of envelope fragments or amino acids between molecular clones of feline immunodeficiency virus with different susceptibilities to neutralization by a polyclonal cat

  16. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi


    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...... 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...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  17. Fragmentation of Ceramics in Rapid Expansion Mode (United States)

    Maiti, Spandan; Geubelle, Philippe H.; Rangaswamy, Krishnan

    The study of the fragmentation process goes back to more than a century, motivated primarily by problems related to mining and ore handling (Grady and Kipp, 1985). Various theories have been proposed to predict the fragmentation stress and the fragment size and distribution. But the investigations are generally case specific and relate to only a narrow set of fragmentation processes. A number of theoretical studies of dynamic fragmentation in a rapidly expanding body can be found in the literature. For example, the study summarized in (Grady, 1982) presents a model based on a simple energy balance concept between the surface energy released due to fracture and the kinetic energy of the fragments. Subsequent refinements of the energy balance model have been proposed by (Glenn and Chudnovsky, 1986), which take into account the strain energy of the fragments and specify a threshold stress below which no fragmentation occurs. These models assume that the fracture events are instantaneous and occur simultaneously. Evidently, these assumptions are quite restrictive and these models can not take into account the transient nature of the fragmentation process after the onset of fracture in the material. A more recent model proposed by (Miller et al., 1999) however takes into account this time-dependent nature of the fragmentation event and the distribution of flaws of various strengths in the original material.

  18. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    International Nuclear Information System (INIS)

    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


    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. Rabbit antipeptide antibodies against restricted domains of the histocompatibility complex. (United States)

    Chersi, A; Houghten, R A


    The work reported here concerns the specificity of four antibodies elicited by synthetic peptides corresponding to three domains of the HLA-B7 glycoprotein, and one of the beta 2 microglobulin. One of the four peptides had been previously investigated, but the data obtained by its elicited antibodies were uncomplete. Of the three HLA-B7 peptides assayed, one was unable to raise an immune response. The other two produced antibodies in good titer that reacted with the antigen peptide, but were unable to recognize antigenic determinants when tested on membrane glycoproteins solubilized from human lymphoblastoid cells. In contract, the antibody elicited by the only fragment from the beta 2microglobulin recognized the native beta 2microglobulin molecule as well as HLA/beta 2m complexes, and reacted with intact human cells, as determined by ELISA and by FACS analysis. This peptide, which contains one of the most hydrophylic fragment of the whole molecule, is likely an important antigenic site, since it is also recognized by traditional antisera raised against native beta 2microglobulin.

  20. Development of Antibody-Coated Magnetite Nanoparticles for Biomarker Immobilization

    Directory of Open Access Journals (Sweden)

    Christian Chapa Gonzalez


    Full Text Available Magnetic nanoparticles (MNPs have great potential in biomedical applications because of their magnetic response offers the possibility to direct them to specific areas and target biological entities. Magnetic separation of biomolecules is one of the most important applications of MNPs because their versatility in detecting cancer biomarkers. However, the effectiveness of this method depends on many factors, including the type of functionalization onto MNPs. Therefore, in this study, magnetite nanoparticles have been developed in order to separate the 5′-nucleotidase enzyme (5eNT. The 5eNT is used as a bio-indicator for diagnosing diseases such as hepatic ischaemia, liver tumor, and hepatotoxic drugs damage. Magnetic nanoparticles were covered in a core/shell type with silica, aminosilane, and a double shell of silica-aminosilane. A ScFv (fragment antibody and anti-CD73 antibody were attached to the coated nanoparticles in order to separate the enzyme. The magnetic separation of this enzyme with fragment antibody was found to be 28% higher than anti-CD73 antibody and the enzyme adsorption was improved with the double shell due to the increased length of the polymeric chain. Magnetite nanoparticles with a double shell (silica-aminosilane were also found to be more sensitive than magnetite with a single shell in the detection of biomarkers.

  1. Identification and evaluation of nextgeneration PTM-specific antibodies

    DEFF Research Database (Denmark)

    Persson, Nina Emilia

    selectionprocess needs to exclude cross-reactive or suboptimal binders at an early stage to avoid problemsin the downstream evaluation process.In this thesis, a microarray screening assay is described. This assay combines an antibody and anantigen microarray for high content screening of multiple single......-chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection.......In subsequent project, a new antibody library was constructed from the heavy chain variabledomain of G2-D11 and random light chains domains from immunized mice. Using this antibodylibrary and our screening assay, we could select a number of antibodies specific for the Tnantigenon a peptide from the CD43 protein...

  2. Immunoscintigraphy of ovarian carcinoma using OC 125 monoclonal antibody

    International Nuclear Information System (INIS)

    Park, Sang Yoon


    Immunoscintigraphy (ISG) with I-131 labeled OC 125 F (ab')2 fragments was studied in 7 patients for primary diagnosis and follow up of ovarian cancer. Total body planar photoscans with a scintillation camera were performed three to seven days after antibody application and results were compared with operation and/or computed tomography (CT) examination. By the region of interest technique, the tumor to background ratio was calaulated in vivo. Results are as follows. 1) The sensitivity of ISG and CT for detection of 14 tumor sites which were confirmed with histopathology were 100 % and 57.1 % and the sensitivity for the detection of omental metastasis were 100 % and 20 % respectively. 2) There were no correlation between the serum CA 125 levels and tumor to background antibody uptake ratio. 3) Tumor to background antibody uptake ratio were progressively increased from day 3 to day 7. (author)

  3. Fragmentation in Carbon Therapy Beams

    CERN Document Server

    Charara, Y M


    The state of the art Monte Carlo code HETC-HEDS was used to simulate spallation products, secondary neutron, and secondary proton production in A-150 Tissue Equivalent Plastic phantoms to investigate fragmentation of carbon therapy beams. For a 356 MeV/Nucleon carbon ion beam, production of charged particles heavier than protons was 0.24 spallation products per incident carbon ion with atomic numbers ranging from 1 through 5 (hydrogen to boron). In addition, there were 4.73 neutrons and 2.95 protons produced per incident carbon ion. Furthermore, as the incident energy increases, the neutron production rate increases at a rate of 20% per 10 MeV/nucleon. Secondary protons were created at a rate between 2.62-2.87 per carbon ion, while spallation products were created at a rate between 0.20-0.24 per carbon ion.

  4. Dynamic effects in fragmentation reactions

    International Nuclear Information System (INIS)

    Bertsch, G. F.; Esbensen, H.


    Fragmentation reactions offer a useful tool to study the spectroscopy of halo nuclei, but the large extent of the halo wave function makes the reaction theory more difficult. The simple reaction models based on the eikonal approximation for the nuclear interaction or first-order perturbation theory for the Coulomb interaction have systematic errors that they investigate here, comparing to the predictions of complete dynamical calculations. They find that stripping probabilities are underpredicted by the eikonal model, leading to extracted spectroscopy strengths that are two large. In contrast, the Coulomb excitation is overpredicted by the simple theory. They attribute this to a screening effect, as is well known in the Barkas effect on stopping powers. The errors decrease with beam energy as E(sub beam)(sup -1), and are not significant at beam energies above 50 MeV/u. At lower beam energies, the effects should be taken into account when extracting quantitative spectroscopic strengths

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

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.


    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.

  6. Heavy chain only antibodies

    DEFF Research Database (Denmark)

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


    -binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...... for combating HER2+ breast cancer....

  7. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation. (United States)

    Ong, E K; Knox, R B; Singh, M B


    The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

  8. Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae. (United States)

    McLaughlin, G L; Brandt, F H; Visvesvara, G S


    Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

  9. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J


    reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples...... isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always...... with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm...

  10. Impact failure and fragmentation properties of metals

    Energy Technology Data Exchange (ETDEWEB)

    Grady, D.E. [Applied Research Associates, Albuquerque, NM (United States); Kipp, M.E. [Sandia National Labs., Albuquerque, NM (United States)


    In the present study we describe the development of an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

  11. Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1. (United States)

    Lomakin, Ya A; Shmidt, A A; Bobik, T V; Chernov, A S; Pyrkov, A Yu; Aleksandrova, N M; Okunola, D O; Vaskina, M I; Ponomarenko, N A; Telegin, G B; Dubina, M V; Belogurov, A A


    Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

  12. Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Juan C. Almagro


    Full Text Available The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life, which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

  13. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells. (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M


    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  14. Fragment Size Distribution of Blasted Rock Mass (United States)

    Jug, Jasmin; Strelec, Stjepan; Gazdek, Mario; Kavur, Boris


    Rock mass is a heterogeneous material, and the heterogeneity of rock causes sizes distribution of fragmented rocks in blasting. Prediction of blasted rock mass fragmentation has a significant role in the overall economics of opencast mines. Blasting as primary fragmentation can significantly decrease the cost of loading, transport, crushing and milling operations. Blast fragmentation chiefly depends on the specific blast design (geometry of blast holes drilling, the quantity and class of explosive, the blasting form, the timing and partition, etc.) and on the properties of the rock mass (including the uniaxial compressive strength, the rock mass elastic Young modulus, the rock discontinuity characteristics and the rock density). Prediction and processing of blasting results researchers can accomplish by a variety of existing software’s and models, one of them is the Kuz-Ram model, which is possibly the most widely used approach to estimating fragmentation from blasting. This paper shows the estimation of fragmentation using the "SB" program, which was created by the authors. Mentioned program includes the Kuz-Ram model. Models of fragmentation are confirmed and calibrated by comparing the estimated fragmentation with actual post-blast fragmentation from image processing techniques. In this study, the Kuz-Ram fragmentation model has been used for an open-pit limestone quarry in Dalmatia, southern Croatia. The resulting calibrated value of the rock factor enables the quality prognosis of fragmentation in further blasting works, with changed drilling geometry and blast design parameters. It also facilitates simulation in the program to optimize blasting works and get the desired fragmentations of the blasted rock mass.

  15. Antithyroid microsomal antibody (United States)

    ... that you have a higher chance of developing thyroid disease in the future. Antithyroid microsomal antibodies may be ... PA: Elsevier; 2016:chap 11. Weiss RE, Refetoff S. Thyroid function testing. In: Jameson JL, De Groot LJ, eds. Endocrinology: Adult and ... Lupus Read more ...

  16. Antibodies Targeting EMT (United States)


    determine their targets on the cell. The newly discovered antibodies will then be engineered for utility as new highly specific drugs and diagnostics in...are from the aldo-keto reductase family (AKRs). Remarkably, 3 of the top 10 genes with induction in the mesenchymal TES2b cells Figure 1. Amino

  17. Monoclonal antibodies in haematopathology

    Energy Technology Data Exchange (ETDEWEB)

    Grignani, F.; Martelli, M.F.; Mason, D.Y.


    This book contains over 40 selections. Some of the titles are: Oncogene (c-myc, c-myb) amplification in acute myelogenous leukaemia; Ultrastructural characterization of leukaemic cells with monoloclonal antibodies; Origin of B-cell malignancies; Immunohistology of gut lymphomas; and Spurious evidence of lineage infidelity in monocytic leukaemia.

  18. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

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


    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...

  19. Evading pre-existing anti-hinge antibody binding by hinge engineering (United States)

    Kim, Hok Seon; Kim, Ingrid; Zheng, Linda; Vernes, Jean-Michel; Meng, Y. Gloria; Spiess, Christoph


    ABSTRACT Antigen-binding fragments (Fab) and F(ab′)2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA. PMID:27606571

  20. Gluon fragmentation in T(1S) decays

    International Nuclear Information System (INIS)

    Bienlein, J.K.


    In T(1S) decays most observables (sphericity, charged multiplicity, photonic energy fraction, inclusive spectra) can be understood assuming that gluons fragment like quarks. New results from LENA use the (axis-independent) Fox-Wolfram moments for the photonic energy deposition. Continuum reactions show 'standard' Field-Feynman fragmentation. T(1S) decays show a significant difference in the photonic energy topology. It is more isotropic than with the Field-Feynman fragmentation scheme. Gluon fragmentation into isoscalar mesons (a la Peterson and Walsh) is excluded. But if one forces the leading particle to be isoscalar, one gets good agreement with the data. (orig.)

  1. Measuring the temperature of hot nuclear fragments

    International Nuclear Information System (INIS)

    Wuenschel, S.; Bonasera, A.; May, L.W.; Souliotis, G.A.; Tripathi, R.; Galanopoulos, S.; Kohley, Z.; Hagel, K.; Shetty, D.V.; Huseman, K.; Soisson, S.N.; Stein, B.C.; Yennello, S.J.


    A new thermometer based on fragment momentum fluctuations is presented. This thermometer exhibited residual contamination from the collective motion of the fragments along the beam axis. For this reason, the transverse direction has been explored. Additionally, a mass dependence was observed for this thermometer. This mass dependence may be the result of the Fermi momentum of nucleons or the different properties of the fragments (binding energy, spin, etc.) which might be more sensitive to different densities and temperatures of the exploding fragments. We expect some of these aspects to be smaller for protons (and/or neutrons); consequently, the proton transverse momentum fluctuations were used to investigate the temperature dependence of the source.

  2. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J


    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  3. Humanized Antibodies for Antiviral Therapy (United States)

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


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

  4. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo


    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...

  5. Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody

    DEFF Research Database (Denmark)

    Lassen, K.S.; Bradbury, A.R.; Heegaard, N.H.


    complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO(3)H) antibody) using CE and a panel of sulfated and nonsulfated...... peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide...

  6. Mechanisms Affecting Population Density in Fragmented Habitat

    Directory of Open Access Journals (Sweden)

    Lutz Tischendorf


    Full Text Available We conducted a factorial simulation experiment to analyze the relative importance of movement pattern, boundary-crossing probability, and mortality in habitat and matrix on population density, and its dependency on habitat fragmentation, as well as inter-patch distance. We also examined how the initial response of a species to a fragmentation event may affect our observations of population density in post-fragmentation experiments. We found that the boundary-crossing probability from habitat to matrix, which partly determines the emigration rate, is the most important determinant for population density within habitat patches. The probability of crossing a boundary from matrix to habitat had a weaker, but positive, effect on population density. Movement behavior in habitat had a stronger effect on population density than movement behavior in matrix. Habitat fragmentation and inter-patch distance may have a positive or negative effect on population density. The direction of both effects depends on two factors. First, when the boundary-crossing probability from habitat to matrix is high, population density may decline with increasing habitat fragmentation. Conversely, for species with a high matrix-to-habitat boundary-crossing probability, population density may increase with increasing habitat fragmentation. Second, the initial distribution of individuals across the landscape: we found that habitat fragmentation and inter-patch distance were positively correlated with population density when individuals were distributed across matrix and habitat at the beginning of our simulation experiments. The direction of these relationships changed to negative when individuals were initially distributed across habitat only. Our findings imply that the speed of the initial response of organisms to habitat fragmentation events may determine the direction of observed relationships between habitat fragmentation and population density. The time scale of post-fragmentation

  7. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)


    textabstractSynthetic 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

  8. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.


    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

  9. Targeting human prostate cancer with (111) In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, S.; Rij, C.M. van; Franssen, G.M.; Fracasso, G.; Helfrich, W.; Eek, A.; Oyen, W.J.G.; Colombatti, M.; Boerman, O.C.


    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing

  10. Targeting human prostate cancer with In-111-labeled D2B IgG, F(ab ')(2) and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.


    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing

  11. The fragmentation of prestellar clouds

    International Nuclear Information System (INIS)

    Ibanez S, M.H.


    The radiative heating problem has been solved analytically, in first approximation, for a semi-infinite cloud model with transverse fluctuations in extinction. The radiative heating problem has been solved with the help of a numerical (approximated) method for the following models: a) semi-infinite cloud with transverse fluctuations in extinction; b) finite cloud with mean optical thickness tau 0 and transverse fluctuations in extinction. Assuming isobaricity as a first approximation, the chemical equation for H 2 formation (in non-equilibrium condition) and the energy equation were solved numerically like a two boundary-value problem. The conditions under which H 2 formation can induce fragmentation in a contracting cloud are examined. A study (in orders of magnitude) of the turbulence as a mechanism generator of density fluctuations has been done. If the Kolmogorov spectral law is assumed, subsonic turbulence is enough to provide any prestellar cloud with the elemental fluctuations which are effectively amplified by molecule formation in a time shorter than one free-fall. (author)

  12. Capturing Biological Activity in Natural Product Fragments by Chemical Synthesis. (United States)

    Crane, Erika A; Gademann, Karl


    Natural products have had an immense influence on science and have directly led to the introduction of many drugs. Organic chemistry, and its unique ability to tailor natural products through synthesis, provides an extraordinary approach to unlock the full potential of natural products. In this Review, an approach based on natural product derived fragments is presented that can successfully address some of the current challenges in drug discovery. These fragments often display significantly reduced molecular weights, reduced structural complexity, a reduced number of synthetic steps, while retaining or even improving key biological parameters such as potency or selectivity. Examples from various stages of the drug development process up to the clinic are presented. In addition, this process can be leveraged by recent developments such as genome mining, antibody-drug conjugates, and computational approaches. All these concepts have the potential to identify the next generation of drug candidates inspired by natural products. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  13. Decoupling habitat fragmentation from habitat loss: butterfly species mobility obscures fragmentation effects in a naturally fragmented landscape of lake islands. (United States)

    MacDonald, Zachary G; Anderson, Iraleigh D; Acorn, John H; Nielsen, Scott E


    Since the publication of the theory of island biogeography, ecologists have postulated that fragmentation of continuous habitat presents a prominent threat to species diversity. However, negative fragmentation effects may be artifacts; the result of species diversity declining with habitat loss, and habitat loss correlating positively with degree of fragmentation. In this study, we used butterfly assemblages on islands of Lake of the Woods, Ontario, Canada to decouple habitat fragmentation from habitat loss and test two competing hypotheses: (1) the island effect hypothesis, which predicts that decreasing fragment size and increasing fragment isolation reduces species diversity beyond the effects of habitat loss, and (2) the habitat amount hypothesis, which negates fragmentation effects and predicts that only total habitat area determines the diversity of species persisting on fragmented landscapes. Using eight independent size classes of islands (ranging from 0.1 to 8.0 ha) that varied in number of islands while holding total area constant, species diversity comparisons, species accumulation curves, and species-area relationship extrapolations demonstrated that smaller insular habitats contained at least as many butterfly species as continuous habitat. However, when highly mobile species occurring on islands without their larval food plants were excluded from analyses, island effects on potentially reproducing species became apparent. Similarily, generalized linear models suggested that effects of island isolation and vascular plant richness on insular butterfly richness were confounded by species of high mobility. We conclude that inter-fragment movements of highly mobile species may obscure important fragmentation effects on potentially reproducing populations, questioning support for the habitat amount hypothesis.

  14. Magnetic Purification of Antibodies (United States)

    Dhadge, Vijaykumar Laxman

    This work aimed at the development of magnetic nanoparticles for antibody purification and at the evaluation of their performance in Magnetic fishing and in a newly developed hybrid technology Magnetic Aqueous Two Phase Systems. Magnetic materials were produced by coprecipitation and solvothermal approaches. Natural polymers such as dextran, extracellular polysaccharide and gum Arabic were employed for coating of iron oxide magnetic supports. Polymer coated magnetic supports were then modified with synthetic antibody specific ligands,namely boronic acid, a triazine ligand (named 22/8) and an Ugi ligand (named A2C7I1). To optimize the efficacy of magnetic nanoparticles for antibody magnetic fishing, various solutions of pure and crude antibody solutions along with BSA as a non-specific binding protein were tested. The selectivity of magnetic nanoparticle for antibody, IgG, was found effective with boronic acid and ligand 22/8. Magnetic supports were then studied for their performance in high gradient magnetic separator for effective separation capability as well as higher volume handling capability. The magnetic materials were also supplemented to aqueous two phase systems, devising a new purification technology. For this purpose, magnetic particles modified with boronic acid were more effective. This alternative strategy reduced the time of operation,maximized separation capability (yield and purity), while reducing the amount of salt required. Boronic acid coated magnetic particles bound 170 +/- 10 mg hIgG/g MP and eluted 160 +/- 5 mg hIgG/g MP, while binding only 15 +/- 5 mg BSA/g MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 x 105 M-1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed/g MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely

  15. Distribution and pharmacokinetics of radiolabeled monoclonal antibody OC 125 after intravenous and intraperitoneal administration in gynecologic tumors

    International Nuclear Information System (INIS)

    Haisma, H.J.; Moseley, K.R.; Battaile, A.; Griffiths, T.C.; Knapp, R.C.


    Radiolabeled monoclonal antibodies may be useful for radioimmunotherapy of gynecologic tumors. Iodine 131-labeled F(ab')2 fragments of a monoclonal antibody, OC 125, with specificity for ovarian carcinoma, were used to study the distribution and pharmacokinetics of this antibody in patients with gynecologic tumors. The radiolabeled antibody was injected intravenously or intraperitoneally into 10 patients suspected of having ovarian cancer. Blood and urine samples were used for pharmacokinetic studies, and biopsy specimens were examined for the uptake of antibody. The serum half-life of the labeled antibody was 30 hours after intravenous administration, with 20% of the injected dose per liter detected at 24 hours. After intraperitoneal injection, the appearance of antibody in serum was slow, with a maximum level of 1.4% of the injected dose per liter at 24 hours. Urinary excretion of the radiolabeled antibody was similar for intravenous and intraperitoneal administration, with approximately 50% of the injected dose excreted after 48 hours. Intraperitoneal administration of the radiolabeled antibody resulted in a higher uptake of antibody in the tumor and a lower uptake of antibody in normal tissues. On the basis of this limited study, intraperitoneal administration of radiolabeled antibody is preferred over intravenous administration for radioimmunotherapy of ovarian cancer

  16. Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Seismann, H.


    and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain...

  17. A note on convex renorming and fragmentability

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Abstract. Using the game approach to fragmentability, we give new and simpler proofs of the following known results: (a) If the Banach space admits an equivalent. Kadec norm, then its weak topology is fragmented by a metric which is stronger than the norm topology. (b) If the Banach space admits an equivalent rotund ...

  18. Fragmentation of eastern United States forest types (United States)

    Kurt H. Riitters; John W. Coulston


    Fragmentation is a continuing threat to the sustainability of forests in the Eastern United States, where land use changes supporting a growing human population are the primary driver of forest fragmentation (Stein and others 2009). While once mostly forested, approximately 40 percent of the original forest area has been converted to other land uses, and most of the...

  19. Thermodynamics of the fuel fragmentation gas

    International Nuclear Information System (INIS)

    Perez, R.B.; Alsmiller, R.G. Jr.


    In the context of nuclear reactor safety studies, a program is in progress at ORNL whereby fuel-fragmentation situations are mocked up by the application of high-current capacitor discharges through solid UO 2 samples. The goal of the present work is to predict such quantities as the number of gas and liquid fragments and their energy distributions. The point of view adopted is that upon fragmentation, a cloud of UO 2 vapor is formed containing ''primeval'' liquid fragments which act as condensation centers. In the evolution of time, fragment growth is controlled by nucleation, coagulation and evaporation processes. Eventually, the vapor-droplet system will reach a situation in which clusters (fragments) of various sizes and UO 2 vapor will coexist in an ''association-disassociation'' equilibrium. Thus, the physical model considered here consists of the identification of the fragmentation gas with an ''imperfect'' vapor, made up of interacting UO 2 vapor and liquid fragments. The results of the study are presented

  20. Radioimmunodetection of prostate cancer by 111In-labeled monoclonal antibody against prostatic acid phosphatase

    International Nuclear Information System (INIS)

    Ahonen, A.; Karnani, P.; Heikkilae, J.; Nurmi, M.


    Purified human prostate acid phosphatase (PAP) was used to generate a specific monoclonal antibody (FC 3001) for detection of PAP expressed by some prostatic carcinomas. DTPA derivatives of MoAb-F(ab')2-fragments were labeled with indium-111 chloride. This labeled antibody was tested in 15 prostate cancer patients who underwent staging pelvic lymphadenectomy; 9 of them received labeled antibody alone whereas 6 received simultaneous injections of labeled and unlabeled antibody with two dose levels (40 or 80 mg). Biodistribution data obtained by direct blood measurements and imaging procedures indicated that simultaneous injection of unlabeled antibody reduced both the blood elimination rate and the accumulation in the liver. Accumulation of the radionuclide in pelvic lymph node metastases was observed in some patients but in a couple of patients accumulation was noted also in normal lymph nodes. The method cannot in its present design replace staging pelvic lymphadenectomy and further studies are needed for elaboration of clinically useful radioimmunodetection methods. (orig.)