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Sample records for fab antigen-binding fragment

  1. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  2. Single chain Fab (scFab fragment

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    Brenneis Mariam

    2007-03-01

    Full Text Available Abstract Background The connection of the variable part of the heavy chain (VH and and the variable part of the light chain (VL by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd and the light chain (LC, resulting in the formation of a single chain Fab fragment (scFab, can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC connecting the constant part 1 of the heavy chain (CH1 and the constant part of the light chain (CL were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of

  3. Baculovirus display of functional antibody Fab fragments.

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    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  4. Production of single chain Fab (scFab fragments in Bacillus megaterium

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    Dübel Stefan

    2007-11-01

    Full Text Available Abstract Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab antibody format combining properties of single chain Fv (scFv and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.

  5. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

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    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  6. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN

    2006-01-01

    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  7. Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

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    Crivianu-Gaita, Victor; Thompson, Michael

    2015-08-15

    Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors.

  8. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

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    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  9. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

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    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

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

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

    1989-02-01

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

  11. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

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    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-01

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  12. Anti-fouling properties of Fab' fragments immobilized on silane-based adlayers

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    Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

    2015-12-01

    Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab') in biosensors. One Fab' linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab' fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab' fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab'-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.

  13. Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding

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    Silverman B David

    2007-11-01

    Full Text Available Abstract Background Comparison of different protein x-ray structures has previously been made in a number of different ways; for example, by visual examination, by differences in the locations of secondary structures, by explicit superposition of structural elements, e.g. α-carbon atom locations, or by procedures that utilize a common symmetry element or geometrical feature of the structures to be compared. Results A new approach is applied to determine the structural changes that an antibody protein domain experiences upon its interaction with an antigenic target. These changes are determined with the use of two different, however comparable, sets of principal axes that are obtained by diagonalizing the second-order tensors that yield the moments-of-geometry as well as an ellipsoidal characterization of domain shape, prior to and after interaction. Determination of these sets of axes for structural comparison requires no internal symmetry features of the domains, depending solely upon their representation in three-dimensional space. This representation may involve atomic, Cα, or residue centroid coordinates. The present analysis utilizes residue centroids. When the structural changes are minimal, the principal axes of the domains, prior to and after interaction, are essentially comparable and consequently may be used for structural comparison. When the differences of the axes cannot be neglected, but are nevertheless slight, a smaller relatively invariant substructure of the domains may be utilized for comparison. The procedure yields two distance metrics for structural comparison. First, the displacements of the residue centroids due to antigenic binding, referenced to the ellipsoidal principal axes, are noted. Second, changes in the ellipsoidal distances with respect to the non-interacting structure provide a direct measure of the spatial displacements of the residue centroids, towards either the interior or exterior of the domain

  14. Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids

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    Eleniste, Pierre P.; Hofstetter, Heike; Hofstetter, Oliver

    2013-01-01

    This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-L-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to L-amino acids but not to recognize the corresponding D-enantiomers. PMID:23827208

  15. Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells.

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    Wen, Yue-Jin; Mancino, Anne; Pashov, Anastas; Whitehead, Tracy; Stanley, Joseph; Kieber-Emmons, Thomas

    2005-02-01

    Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

  16. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    Science.gov (United States)

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  17. Strategies for fragment antigen binding expression in Escherichia coli%大肠杆菌表达抗原结合片段的策略

    Institute of Scientific and Technical Information of China (English)

    刘运洪; 侯宗柳

    2011-01-01

    BACKGROUND: Low-yields of antigen-binding fragment (Fab) restricts its full scale operation and application, which may be changed by remodeling or optimizing host cells, expression vectors, expression condition and purification conditions.OBJECTIVE: To summarize and introduce new strategies about expression and purification of Fab antibodies in Escherichia coli (E. coli).METHODS: CNKI and Medline databases were searched by the first author using key words of "Escherichia coli, Fab, Expression,antibody, purification" in Chinese or English from year 1990 to 2010. The Fab antibody expression and purification in E. coli were summed up and new researches concerning Fab antibody expression and purification in E. coli were introduced.RESULTS AND CONCLUSION: Totally 125 papers were found and 30 papers were included. The results show that the Fab antibodies are mainly by cytoplasmic expression and secreted expression in E. coli. Its purification methods are mainly by metal ions and chelating affinity purification and specific proteins affinity purification. The influence factors concerning Fab expression in E. coli are correlated to each other. A substantial increase in current research on the latest Fab antibodies in E. coli expression and purification strategies are not consistent optimal, there is no best, only the most suitable.%背景:抗原结合片段在大肠杆菌中表达量低是制约其大规模生产和应用的主要因素,通过对宿主细胞、表达载体、表达条件和纯化条件等进行改造与优化,将有可能获得基因工程抗体的高效表达.目的:总结并介绍抗原结合片断抗体在大肠杆菌中的表达和纯化的最新策略.方法:由第一作者用计算机检索中国期刊全文数据库(CNKI:1990/2010)和Medline数据库(1990/2010),检索词分别为"大肠杆菌,抗原结合片段,表达,纯化,抗体"和"Escherichia coli,Fab,Expression,antibody,purification",语言分别设定为中文和英文.从Fab抗体在大肠

  18. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

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    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  19. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

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    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)

    1999-06-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 99m}Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  20. HIV-2 neutralization by intact V3-specific Fab fragments

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    Sourial Samer

    2008-08-01

    Full Text Available Abstract The V3 region of both HIV-1 gp120 and HIV-2 gp125 surface glycoprotein has been described as a target for neutralizing antibodies. In this study a conformation-sensitive (3C4 and a linear site-specific (7C8 anti-HIV-2 V3 monoclonal antibody (mAb were characterized. The neutralization capacity of the purified mAbs and their respective papain-generated Fab fragments was analyzed. The Fabs were further characterized by sequence analysis. Our results demonstrate that neither purified mAbs were capable of neutralizing HIV-2, while intact Fab fragments from both mAbs blocked in vitro infection of HIV-2 isolates. Moreover, the conformation sensitive 3C4 Fab neutralized both subtype A and B HIV-2 isolates and SIVsm. Sequence analysis of the hypervariable regions of 3C4 Fab and 7C8 Fab revealed that the third CDR of the heavy chain (CDRH3 of the antibodies was not as long as many of the previously characterized neutralizing antibodies. Our findings suggest that whole 7C8 and 3C4 mAbs are sterically hindered from neutralizing HIV-2, whereas the smaller size of Fab fragments enables access to the V3 region on the virion surface.

  1. Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

    Science.gov (United States)

    Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

    2015-01-01

    In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

  2. GTP-specific fab fragment-based GTPase activity assay.

    Science.gov (United States)

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  3. Preparation of F(ab')2 fragments of immunoglobulin G.

    Science.gov (United States)

    Killion, J J; Holtgrewe, E M

    1983-11-01

    We describe a simple protocol for the preparation of F(ab')2 fragments of immunoglobulin G, based upon the known Fc- binding properties of protein A-Sepharose. The fragment preparations of xenogeneic and allogeneic anti-IgG were noncytotoxic to intact target cells, and were able to block the cytotoxicity of intact antibody. This method should therefore be useful for functional studies not requiring biochemical homogeneity.

  4. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that...

  5. Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

    Science.gov (United States)

    Crottet, P; Corthésy, B

    1998-11-15

    Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

  6. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  7. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

    Science.gov (United States)

    Schlapschy, Martin; Fogarasi, Marton; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Aguib, Yasmine; Skerra, Arne

    2009-03-01

    An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

  8. Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

    Science.gov (United States)

    Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

    2016-01-01

    Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

  9. Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of bio-therapeutic Fab fragments.

    Science.gov (United States)

    Cresswell, Chrissie; Newcombe, Anthony R; Davies, Susannah; Macpherson, Ian; Nelson, Paul; O'Donovan, Kieran; Francis, Richard

    2005-10-01

    In the present paper, we describe a rapid method for the determination of optimum conditions for papain digestion of polyclonal ovine IgG (purified by Na(2)SO(4) precipitation) for the production of bio-therapeutic Fabs (antigen-binding fragments). To determine the optimum conditions for digestion, a factorial approach to the design of experiments was undertaken. The resulting experimental data were used to construct the mathematical models using Design Expert 6.06(R) (Stat-Ease, Minneapolis, MN, U.S.A.) to predict the optimum conditions for a robust IgG digestion step. Optimum conditions were evaluated experimentally, and the applicability of the conditions for large-scale manufacture of bio-therapeutic Fab fragments was assessed. The results and methods described in the present paper suggest that, provided the time and temperature are maintained at the high settings evaluated (24 h, 40 degrees C), the modelled data predict IgG digestion close to 100% for all the papain concentrations used. Provided papain is used at >2.5% (w/w), either time and/or temperature may be reduced. The results and methods described in the present paper may also be applicable to the generation of therapeutic Fab fragments from other immunoglobulins, including monoclonal antibodies purified from mammalian cell culture.

  10. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

    Science.gov (United States)

    Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; Montaño, Sherwin P.; Kurosawa, Kohei; Zheng, Yupeng; Akin, Louesa R.; Świst-Rosowska, Kalina M.; Grzybowski, Adrian T.; Koide, Akiko; Krajewski, Krzysztof; Strahl, Brian D.; Kelleher, Neil L.; Ruthenburg, Alexander J.; Koide, Shohei

    2016-01-01

    Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies. PMID:26862167

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  12. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    Science.gov (United States)

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  13. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    Science.gov (United States)

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  14. Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen.

    Science.gov (United States)

    Cierniewski, C S; Janiak, A; Wyroba, E

    1986-01-01

    Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.

  15. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  16. Immunoglobulin fragments, F(ab')2, that are cytotoxic to enzyme-treated cells.

    Science.gov (United States)

    Holtgrewe, E M; Killion, J J

    1984-07-01

    Bivalent immunoglobulin fragments of IgG, F(ab')2, prepared from normal murine sera were found to be cytotoxic to neuraminidase-treated cells. The fragments were cytotoxic to both allogenic and syngeneic targets (with respect to the source of the sera), suggesting that the antigen bound by the F(ab')2 is not related to the major histocompatibility locus of mice (H-2).

  17. Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, V. V., E-mail: vvo@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Lapuk, V. A. [Russian Academy of Sciences, Zelinskii Institute of Organic Chemistry (Russian Federation); Shtykova, E. V.; Stepina, N. D.; Dembo, K. A.; Sokolova, A. V.; Amarantov, S. V. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Timofeev, V. P. [Russian Academy of Sciences, Engelhardt Institute of Molecular Biology (Russian Federation); Ziganshin, R. Kh. [Russian Academy of Sciences, Shemyakin Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Varlamova, E. Yu. [Russian Academy of Medical Sciences, Hematology Research Center (Russian Federation)

    2008-05-15

    The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that for Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.

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

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

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

  19. Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

    Science.gov (United States)

    Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

    2016-07-01

    Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

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

    Science.gov (United States)

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

    2015-11-01

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

  1. The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F-ab and F-c fragments

    NARCIS (Netherlands)

    Vermeer, AWP; Norde, W; van Amerongen, A

    2000-01-01

    The unfolding and further denaturation of IgG and its F-ab and F-c fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F-ab and F-c units was ret

  2. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    Science.gov (United States)

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

  3. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  4. Crystal Structure of the Fab Fragment of an Anti-factor IX Antibody 10C12

    Institute of Scientific and Technical Information of China (English)

    SHI Xiao-Li; ZENG Tu; HUANG Ming-Dong

    2008-01-01

    10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C 12.

  5. [Digitoxin poisoning: reversing ventricular fibrillation with Fab fragments of anti-digoxin antibody].

    Science.gov (United States)

    Domart, Y; Bismuth, C; Schermann, J M; Abuaf, N; Pontal, P G; Baud, F; Bolo, A; Gailliot, M; Fournier, P E

    1982-12-25

    Purified Fab fragments of ovine anti-digoxin antibodies (Wellcome Foundation) were used to treat a patient who attempted suicide by absorbing 10 mg of digitoxin (serum concentration 265 micrograms/l). The poor prognosis, as assessed clinically and from serum potassium levels (7.5 mEq/l), seemed to warrant such a treatment. The weak (6.85%) cross-reactivity elicited in vitro between the anti-digoxin antibodies and digitoxin was compensated by increasing the doses, but improvement was observed with 3.6 g, i.e. about half the effective dosage initially considered. The criteria of effectiveness were clinical, electrocardiographic (reversal of the ventricular fibrillation), biochemical (simultaneous and opposite changes in extra- and intracellular potassium levels, suggesting that ATPase inhibition by digitalis is a reversible process) and toxicological: there was an increase in digitoxin serum levels suggesting displacement of the drug from tissue sites to plasma and other extracellular compartments where the Fab fragments are distributed, and Fab-bound digitoxin appeared fairly rapidly in the urine, which suggested shunting of the normal hepatic metabolic pathway.

  6. Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.

    Science.gov (United States)

    Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

    2015-03-01

    The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.

  7. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    Science.gov (United States)

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies.

  8. Cholestatic liver disease after rituximab and adalimumab and the possible role of cross-reacting antibodies to Fab 2 fragments.

    Directory of Open Access Journals (Sweden)

    Joerg Latus

    Full Text Available BACKGROUND: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA-associated granulomatosis with polyangiitis (GPA who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. METHODS: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. RESULTS: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients' sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. CONCLUSION: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects

  9. Construction, Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    Feng WANG; Ping LEI; Ping HU; Lijuan ZHU; Huifeng ZHU; Yue ZHANG; Jing YANG; Guanxin SHEN

    2008-01-01

    Summary: The purpose of this study was to construct expression vectors of idiotype (Id) Smlg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id,and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by 1PTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ, in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. Coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.

  10. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    Science.gov (United States)

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  11. Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

    Science.gov (United States)

    Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

    2013-10-01

    A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

  12. 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.

    2015-01-01

    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 sub

  13. 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.

    2015-01-01

    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 su

  14. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

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

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su

    2015-12-01

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

  16. 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.

    Directory of Open Access Journals (Sweden)

    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.

  17. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  18. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    Science.gov (United States)

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  19. Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.

    Science.gov (United States)

    Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2016-01-01

    The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

  20. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

    Science.gov (United States)

    Graille, M; Stura, E A; Corper, A L; Sutton, B J; Taussig, M J; Charbonnier, J B; Silverman, G J

    2000-05-09

    Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

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

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-15

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

  3. Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

    Science.gov (United States)

    Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

    2017-07-12

    Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

  4. Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

    Science.gov (United States)

    Song, Hyung-Nam; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2015-01-01

    Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

  5. An improved single-chain Fab platform for efficient display and recombinant expression.

    Science.gov (United States)

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

  6. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    Science.gov (United States)

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.

  7. Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

    Energy Technology Data Exchange (ETDEWEB)

    Niemi, Merja, E-mail: merja.niemi@joensuu.fi; Jänis, Janne [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland); Jylhä, Sirpa [VTT Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT (Finland); Kallio, Johanna M.; Hakulinen, Nina [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland); Laukkanen, Marja-Leena; Takkinen, Kristiina [VTT Technical Research Centre of Finland, PO Box 1000, FIN-02044 VTT (Finland); Rouvinen, Juha [Department of Chemistry, University of Joensuu, PO Box 111, FIN-80101 Joensuu (Finland)

    2008-01-01

    The high-resolution mass-spectrometric characterization, crystallization and X-ray diffraction studies of a recombinant IgE Fab fragment in complex with bovine β-lactoglobulin are reported. A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT–ICR mass spectrometry and crystallized with bovine β-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å. The three-dimensional structure of the D1 Fab fragment–BLG complex will provide the first insight into IgE antibody–allergen interactions at the molecular level.

  8. Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain.

    Science.gov (United States)

    Kanje, Sara; von Witting, Emma; Chiang, Samuel C C; Bryceson, Yenan T; Hober, Sophia

    2016-09-21

    Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

  9. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

  10. Structural and functional insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite.

    Science.gov (United States)

    Gutiérrez, Lucas Joel; Andujar, Sebastián Antonio; Enriz, Ricardo Daniel; Baldoni, Héctor Armando

    2014-01-01

    A molecular modeling study giving structural, functional, and mutagenesis insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite is reported. Our results allow extending experimental data resulting from X-ray diffraction experiments in order to examine unknown aspects for the Fab-BACE1 recognition and its binding mode. Thus, the study performed here allows extending the inherently static nature of crystallographic structures in order to gain a deeper understanding of the structural and dynamical basis at the atomic level. The characteristics and strength of the interatomic interactions involved in the immune complex formation are exhaustively analyzed. The results might explain how the anti-BACE1 Fab fragment and other BACE1 exosite binders are capable to produce an allosteric modulation of the BACE1 activity. Our site-directed mutagenesis study indicated that the functional anti-BACE1 paratope, residues Tyr32 (H1), Trp50 (H2), Arg98 (H3), Phe101 (H3), Trp104 (H3) and Tyr94 (L3), strongly dominates the binding energetics with the BACE1 exosite. The mutational studies described in this work might accelerate the development of new BACE1 exosite binders with interesting pharmacological activity.

  11. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    Science.gov (United States)

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  12. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    Science.gov (United States)

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    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 subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  13. LABELING McAb 3H11 AND ITS Fab FRAGMENT WITH 211At AND THEIR IMMUNOREACTIVITIES AND INJURY EFFECTS ON HUMAN GASTRIC CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    刘宁; 罗德元; 等

    1995-01-01

    An antigastric cancer monoclonal antibody,3H11,and its Fab fragment,were labeled using p-(211At)-astatobenzoic acid(pAtBA) intermediate,in the yields of more than 30%.The results of in vitro experiments show that 211 At-3H11 and 211At-3H11 Fab are stable,and have specific immunoreactivities and cytotoxic effects to human gastric cancer cell M85,The cytotoxic effects are dependent on the concentration of 211At-3H11 or 211 At-3H11 Fab and obviously stronger than that of Na211At.

  14. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    Science.gov (United States)

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  15. Screening of Human Antibody Fab Fragment against HBsAg and the Construction of its dsFv Form

    Directory of Open Access Journals (Sweden)

    Leili Jia, Jiyun Yu, Hongbin Song, Xuelin Liu, Weina Ma, Yuanyong Xu, Chuanfu Zhang, Shicun Dong, Qiao Li

    2008-01-01

    Full Text Available The objective of this study was to pursue the techniques involving the screening of the human antibody Fab fragment against hepatitis B virus surface antigen (HBsAg and the construction of its disulfide-stabilized Fv fragment (dsFv. The phage antibody Fab fragments against HBsAg were screened from the human combinatorial immunoglobulin library. Sequence analysis revealed that its heavy chain gene was complete, but the light chain gene was lost. To improve the affinity of the antibody by chain shuffling, a human antibody light chain gene repertoire was generated by reverse transcriptase-polymerase chain reaction (RT-PCR from the human peripheral blood lymphocytes. A phage antibody sub-library was then constructed by inserting the light chain gene repertoire into the phagmid that contained the Fd gene. Five clones with appreciably higher absorbance than that of the original clone were obtained, which indicated that the affinity of the light chain-shuffled phage antibodies was improved. Then, the mutated genes of dsFv against HBsAg were constructed by using PCR-based point mutagenesis method. Purified VH and VL proteins were folded into a 25-kDa protein, designated as anti-HBsAg dsFv. ELISA and competition ELISA revealed that the dsFv maintained the specificity of the Fab by binding to HBsAg, even through with a lower binding activity. These results have facilitated the undertaking of further functional analyses of the constructed dsFv, and may therefore provide an improved technique for the production and application of dsFvs against HBsAg.

  16. Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

    Science.gov (United States)

    Shatz, Whitney; Hass, Philip E; Mathieu, Mary; Kim, Hok Seon; Leach, Kim; Zhou, Michelle; Crawford, Yongping; Shen, Amy; Wang, Kathryn; Chang, Debby P; Maia, Mauricio; Crowell, Susan R; Dickmann, Leslie; Scheer, Justin M; Kelley, Robert F

    2016-09-01

    We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

  17. A kit to prepare (111)In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer.

    Science.gov (United States)

    Scollard, Deborah A; Chan, Conrad; Holloway, Claire M B; Reilly, Raymond M

    2011-01-01

    The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for (111)In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ((111)In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of (111)In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37°C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (≥ 85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of (111)In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K(a) = 0.6-9.6 × 10(7) L/mol; B(max) = 0.6-10.4 × 10(6) sites/cell). (111)In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of (111)InCl(3) to a single kit vial and incubating for 30 min at room temperature. (111)In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility. Pure and

  18. A kit to prepare {sup 111}In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Scollard, Deborah A.; Chan, Conrad [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Holloway, Claire M.B. [Department of Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 1H1 (Canada); Reilly, Raymond M., E-mail: raymond.reilly@utoronto.c [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3E2 (Canada); Toronto General Research Institute, University Health Network, Toronto, ON, M5G 2M9 (Canada)

    2011-01-15

    Introduction: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for {sup 111}In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ({sup 111}In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of {sup 111}In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. Methods: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37{sup o}C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency ({>=}85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of {sup 111}In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K{sub a}=0.6-9.6x10{sup 7} L/mol; B{sub max}=0.6-10.4x10{sup 6} sites/cell). {sup 111}In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of {sup 111}InCl{sub 3} to a single kit vial and incubating for 30 min at room temperature. {sup 111}In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for p

  19. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    Science.gov (United States)

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  20. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Science.gov (United States)

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  1. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Directory of Open Access Journals (Sweden)

    Tong Li

    2015-07-01

    Full Text Available The effects of somatic mutations that transform polyspecific germline (GL antibodies to affinity mature (AM antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM. We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab, and subsequently, the DCM was combined with molecular dynamics (MD simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  2. High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

    Science.gov (United States)

    Papalia, Giuseppe A; Baer, Mark; Luehrsen, Kenneth; Nordin, Helena; Flynn, Peter; Myszka, David G

    2006-12-01

    A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

  3. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    Science.gov (United States)

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  4. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    Science.gov (United States)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  5. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

    Science.gov (United States)

    Blaise, Lydia; Wehnert, Anita; Steukers, Mieke P G; van den Beucken, Twan; Hoogenboom, Hennie R; Hufton, Simon E

    2004-11-24

    Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

  6. Intracellular routing in breast cancer cells of streptavidin-conjugated trastuzumab Fab fragments linked to biotinylated doxorubicin-functionalized metal chelating polymers.

    Science.gov (United States)

    Liu, Peng; Cai, Zhongli; Kang, Jae W; Boyle, Amanda J; Adams, Jarret; Lu, Yijie; Ngo Ndjock Mbong, Ghislaine; Sidhu, Sachdev; Reilly, Raymond M; Winnik, Mitchell A

    2014-03-10

    We describe the synthesis of a heterotelechelic metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree of polymerization DPn = 50 (PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic acid (DTPA) pendant groups as the binding sites for metal ions. We compared its behavior in cell-uptake experiments with a similar polymer (Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab (tmFab) fragments covalently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2 ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with (111)In (an Auger electron emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In(3+) and visualized their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with (111)In. Both radiolabeled complexes showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in human breast cancer cells

  7. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells.

    Science.gov (United States)

    Röhm, Martina; Handl, Alina; König, Maria; Mavoungou, Chrystelle; Handrick, René; Schindowski, Katharina

    2016-09-01

    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab')2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I) and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand.

  8. A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages.

    Science.gov (United States)

    Wang, Maorong; Zheng, Wenkai; Zhu, Xuhui; Xu, Jing; Cai, Binggang; Zhang, Yiqing; Zheng, Feng; Zhou, Linfu; Yang, Zhiguo; Zhang, Xin; Wang, Changjun; Nie, Shinan; Zhu, Jin

    2016-01-01

    The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis.

  9. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    Science.gov (United States)

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

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

    Science.gov (United States)

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

    2014-01-01

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

  11. In vitro Fab display: a cell-free system for IgG discovery.

    Science.gov (United States)

    Stafford, Ryan L; Matsumoto, Marissa L; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R; Baliga, Ramesh; Murray, Christopher J; Thanos, Christopher D; Hallam, Trevor J; Sato, Aaron K

    2014-04-01

    Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

  12. A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

    Science.gov (United States)

    Portolano, S; Seto, P; Chazenbalk, G D; Nagayama, Y; McLachlan, S M; Rapoport, B

    1991-08-30

    A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

  13. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  14. Uptake of /sup 99m/Tc labelled (Fab')/sub 2/ fragments of monoclonal antibody 225. 28S by a benign ocular naevus

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-06-01

    Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, /sup 99m/Tc radiolabelled (Fab')/sub 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.

  15. Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

    Science.gov (United States)

    Ding, Haiming; Carlton, Michelle M; Povoski, Stephen P; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H; Colcher, David; Brody, Rich; Davis, Paul D; Pokora, Alex; Phelps, Mitchell; Martin, Edward W; Tweedle, Michael F

    2013-11-20

    The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

  16. A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

    Science.gov (United States)

    Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

    2014-06-15

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

  17. Preparation and activity of conjugate of monoclonal antibody HAbl8 against hepatoma F(ab')2 fragment and staphylococcal enterotoxin A

    Institute of Scientific and Technical Information of China (English)

    Lian Jun Yang; Yan Fang Sui; Zhi Nan Chen

    2001-01-01

    AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION

  18. Decisive diagnosis of infected mandibular osteoradionecrosis with a Tc-99m-labelled anti-granulocyte Fab'-fragment

    Energy Technology Data Exchange (ETDEWEB)

    Kampen, W.U.; Brenner, W.; Henze, E. [Kiel Univ. (Germany). Klinik fuer Nuklearmedizin; Terheyden, H. [Kiel Univ. (DE). Clinic of Oral and Maxillofacial Surgery (Germany); Bohuslavizki, K.H. [Universitaetskrankenhaus Eppendorf, Hamburg (Germany). Abt. fuer Nuklearmedizin

    1999-07-01

    The accepted golden standard for detection of inflammatory bone disease is conventional three-phase bone scanning. Hyperperfusion, a high blood-pool activity and elevated bone metabolism are typical signs for an acute osteomyelitis. However, in case of subacute, chronic inflammation, neither elevated blood flow nor high blood-pool activity may be seen. This may cause difficulties in differentiating such cases from neoplastic or postoperative changes. This case report verifies the possible advantage of immunoscintigraphy with Tc-99m-labelled antigranulocyte Fab'-fragments (LeukoScan {sup trademark}) in a patient with infected mandibular osteoradionecrosis, who had equivocal clinical symptomes and questionable radiographic results. LeukoScan {sup trademark} is shown to be more sensitive in case of subacute bone inflammation compared with three-phase bone scanning. However, acquisition of delayed images after 24 hours including SPECT is inevitable in case of negative scans during the first hours of investigation. (orig.) [German] Die konventionelle Drei-Phasen-Skelettszintigraphie ist der Goldstandard in der Diagnostik entzuendlicher Knochenprozesse. Hyperperfusion, hohe Blutpool-Aktivitaet und ein erhoehter Knochenstoffwechsel sind typisch fuer eine akute Osteomyelitis. Bei chronisch-subakut verlaufenden Osteomyelitiden koennen Hyperperfusion und Blutpool-Aktivitaet jedoch fehlen, was zu Schwierigkeiten bei der differentialdiagnostischen Abgrenzung von neoplastischen oder postoperativen Veraenderungen fuehrt. Die vorgestellte Kasuistik belegt den potentiellen Vorteil der Entzuendungs-Szintigraphie mit Tc-99m-markierten Antigranulozyten-Antikoerperfragmenten (LeukoScan {sup trademark}) bei einem Patienten mit infizierter Osteoradionekrose der Mandibula bei untypischer klinischer Symptomatik und unklarer Bildgebung. LeukoScan {sup trademark} erwies sich als sensitiver bei der Diagnose der subakuten ossaeren Entzuendung im Vergleich zur Drei

  19. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells

    Directory of Open Access Journals (Sweden)

    Martina Röhm

    2016-09-01

    Full Text Available This data article focuses on the production of monoclonal antibodies (mAb and their fragments Fab and F(ab′2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand.

  20. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin.

    Science.gov (United States)

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

  1. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells

    OpenAIRE

    Martina Röhm; Alina Handl; Maria König; Chrystelle Mavoungou; René Handrick; Katharina Schindowski

    2016-01-01

    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab′)2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like...

  2. Matching the decay half-life with the biological half-life: ImmunoPET imaging with (44)Sc-labeled cetuximab Fab fragment.

    Science.gov (United States)

    Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F; Hernandez, Reinier; Hong, Hao; Nickles, Robert J; Cai, Weibo

    2014-12-17

    Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with (44)Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after (44)Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of (44)Sc-CHX-A″-DTPA-Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using (44)Sc-CHX-A″-DTPA-Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.

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

    Science.gov (United States)

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

    2012-12-28

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

  4. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G. (Purdue); (WU-MED); (Crucell)

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  5. Biodistribution and planar gamma camera imaging of {sup 123}I- and {sup 131}I-labeled F(ab'){sub 2} and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor

    Energy Technology Data Exchange (ETDEWEB)

    Burvenich, Ingrid J.G. [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)]. E-mail: ingrid.burvenich@ugent.be; Schoonooghe, Steve [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Blanckaert, Peter [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Bacher, Klaus [Department of Medical Physics and Radiation Protection, Ghent University, B-9000 Ghent (Belgium); Vervoort, Liesbet [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Coene, Elisabeth [N. Goormaghtigh Institute of Pathology, Ghent University, B-9000 Ghent (Belgium); Mertens, Nico [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Vos, Filip de [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Slegers, Guido [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)

    2007-04-15

    Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab'){sub 2} and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. Methods: F(ab'){sub 2} and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of {sup 125}I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of {sup 131}I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these {sup 123}I-labeled 14C5 fragments for radioimmunodetection (RID). Results: Saturation binding experiments showed highest affinity for {sup 125}I-labeled F(ab'){sub 2} fragments (K {sub d}=0.37{+-}0.10 nmol/L) and lowest affinity for {sup 125}I-labeled Fab fragments (K {sub d}=2.25{+-}0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t1/2{alpha}) value for Fab, F(ab'){sub 2} and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t1/2{beta} value for Fab, F(ab'){sub 2} and mAb 14C5 was 439, 627 and 4067 min, respectively. {sup 131}I-Fab fragments showed highest tumor uptake 3 h after injection (2.4{+-}0.8 %ID/g), {sup 131}I-labeled F(ab'){sub 2} showed highest tumor uptake 6 h after injection (4.7{+-}0.7 %ID/g) and for {sup 131}I-labeled mAb highest tumor uptake was observed at 24 h (10.7{+-}2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than {sup 123}I-mAb 14C5. Conclusion: Fab and F(ab'){sub 2} fragments derived from intact mAb 14C5 have

  6. Function of individual 30S subunit proteins of Escherichia coli. Effect of specific immunoglobulin fragments (Fab) on activities of ribosomal decoding sites.

    Science.gov (United States)

    Lelong, J C; Gros, D; Gros, F; Bollen, A; Maschler, R; Stöffler, G

    1974-02-01

    Specific anti-30S protein immunoglobulin G fragments (Fab) were used to determine the contribution of each of the 30S ribosomal proteins to: (1) polyphenylalanine synthesis, (2) initiation factor-dependent binding of fMet-tRNA, (3) T-factor-dependent binding of phenylalanyl-tRNA, and (4) fixation of radioactive dihydrostreptomycin. Twenty of the 21 possible antibodies (antibody against S17 excepted) were used. In conditions where all the 30S proteins were accessible to Fabs, all of these monovalent antibodies strongly inhibited polyphenylalanine synthesis in vitro. Antibodies against S4, S6, S7, S12, S15, and S16, however, showed a weaker effect.30S proteins can be classified into four categories by their contributions to the function of sites "A" and "P": class I appears nonessential for tRNA positioning at either site (S4, S7, S15, and S16); class II includes proteins whose role in initiation is critical (S2, S5, S6, S12, and S13); class III (S8, S9, S11, and S18) corresponds to proteins whose blockade prevents internal (elongation factor Tudependent) positioning; and class IV includes entities that are essential for activities of both "A" and "P" sites (S1, S3, S10, S14, S19, S20, and S21). Dihydrostreptomycin fixation to the 30S or 70S ribosomes was inhibited by antibodies against S1, S10, S11, S18, S19, S20, and S21, but only weakly by the anti-S12 (Str A protein) Fab. The significance of these results is discussed in relation to 30S protein function, heterogeneity, and topography.

  7. ANTITUMOR EFFECTS OF PINGYANGMYCIN CONJUGATED WITH Fab' FRAGMENT OF MONOCLONAL ANTIBODY%平阳霉素与单克隆抗体Fab'片段偶联物的抗肿瘤作用

    Institute of Scientific and Technical Information of China (English)

    刘小云; 刘秀均; 李毅; 王维刚; 甄永苏

    2000-01-01

    目的研制一种以单抗Fab'片段为基础的抗肿瘤导向药物.方法制备单抗3A5 Fab'片段及其与平阳霉素(PYM)偶联物Fab'-PYM后,测定Fab'-PYM与肿瘤细胞的免疫反应性、偶联物中PYM的抑菌活性、对肿瘤细胞的杀伤作用和体内抑瘤作用.结果 Fab'及Fab'-PYM保持了与靶细胞C26的免疫反应性;偶联物中PYM的抑菌活性为游离PYM的15%;Fab'-PYM对C26细胞的杀伤作用强于PYM;对非靶细胞KB的杀伤作用与PYM相似;ip和iv给药,Fab'-PYM对小鼠皮下接种的肠癌26生长抑制作用均强于3A5-PYM和PYM.结论 Fab'-PYM具有比PYM及3A5-PYM更强的体内外抗肿瘤作用.

  8. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

    Science.gov (United States)

    Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

    2014-11-01

    The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.

  9. Chemically optimized antimyosin Fab conjugates with chelating polymers: importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization.

    Science.gov (United States)

    Trubetskoy, V S; Narula, J; Khaw, B A; Torchilin, V P

    1993-01-01

    Murine antimyosin Fab fragment was conjugated with 111In-labeled N-terminal-modified DTPA-polylysine using three bifunctional reagents: N-hydroxysuccinimide esters of 3-(2-pyridyldithio)propionic acid (SPDP conjugate), 4-(maleimidomethyl)cyclohexanecarboxylic acid (SMCC conjugate) and bromoacetic acid (BrAc conjugate) for potential localization of experimental myocardial infarction. Using various antibody preparations and a rabbit acute myocardial infarction model the following parameters were observed: (1) an in vitro antigen binding activity of SPDP conjugate = SMCC conjugate > BrAc conjugate, (2) a blood clearance rate of SPDP conjugate > BrAc conjugate > SMCC conjugate, (3) a liver and splenic accumulation of SPDP conjugate > BrAc conjugate > SMCC conjugate, and (4) the infarcted tissue activity showed an accumulation of SMCC conjugate > SPDP conjugate > BrAc conjugate. This study exemplifies the importance of rational chemical design of antimyosin Fab-chelating polymer conjugate for improved target tissue localization in vivo.

  10. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    Science.gov (United States)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  11. Imaging of deep venous thrombosis in patients using a radiolabelled anti-D-dimer Fab' fragment ({sup 99m}Tc-DI-DD3B6/22-80B3): results of a phase I trial

    Energy Technology Data Exchange (ETDEWEB)

    Macfarlane, David [University of Queensland, School of Medicine, Brisbane (Australia); Socrates, Angelides; Larcos, George [University of Sydney, Department of Medicine, Sydney (Australia)]|[Westmead Hospital, Department of Nuclear Medicine and Ultrasound, Westmead (Australia)]|[Westmead Hospital, Centre for Biomedical Imaging and Research, Westmead (Australia); Eisenberg, Paul [Amgen Inc, Thousand Oaks, CA (United States); Roach, Paul [University of Sydney, Department of Medicine, Sydney (Australia)]|[Royal North Shore Hospital, Nuclear Medicine, St. Leonards (Australia); Gerometta, Michael [Agen Biomedical Pty Ltd, Brisbane (Australia); Smart, Richard; Tsui, Wendy [St. George Hospital, Nuclear Medicine Department, Sydney (Australia)]|[University of New South Wales, Department of Medicine, Sydney (Australia); Scott, Andrew M. [Austin Hospital, Centre for PET, Melbourne (Australia)]|[Ludwig Institute, Melbourne (Australia)

    2009-02-15

    {sup 99m}Tc-DI-DD3B6/22-80B3 (ThromboView registered, hereafter abbreviated to {sup 99m}Tc-DI-80B3 Fab') is a radiolabelled humanised monoclonal Fab' fragment with affinity and specificity for D-dimer domains of cross-linked fibrin. Detection of thromboembolic events has been demonstrated in canine models. The study objectives were evaluation of safety and characterisation of biodistribution, immunogenicity and pharmacokinetic profile of increasing doses of {sup 99m}Tc-DI-80B3 Fab' in subjects with acute lower-limb DVT. Twenty-six patients with acute lower limb DVT were enrolled. Of these, 21 received a single intravenous dose of 0.5 mg (n = 6), 1.0 mg (n = 9) or 2 mg (n = 6) {sup 99m}Tc-DI-80B3 Fab'. Blood and urine samples and gamma camera images were collected to 24 h after administration for pharmacokinetic and dosimetry analysis. Vital signs, electrocardiography, hematological and biochemical data and human anti-human antibody (HAHA) levels were monitored for up to 30 days following administration. Patients were assigned to either planar or single photon emission computed tomographic (SPECT) imaging of the thorax at 4 h following injection. Thirty-five adverse events were reported in 15 of the 21 subjects. Those deemed possibly related to administration of {sup 99m}Tc-DI-80B3 Fab' included mild hypertension, mild elevation of LD (lactate dehydrogenase) and moderate elevation of ALT (alanine transaminase). HAHA assays remained negative. Pharmacokinetics and organ dosimetry were comparable to prior normal volunteer data. Localisation of Thromboview registered to sites of known thrombus was evident as early as 30 min post-injection. In subjects with acute DVT, {sup 99m}Tc-DI-80B3 Fab' was well tolerated with favourable characteristics for the detection of acute venous thrombosis. (orig.)

  12. Imaging of deep venous thrombosis in patients using a radiolabelled anti-D-dimer Fab' fragment (99mTc-DI-DD3B6/22-80B3): results of a phase I trial.

    Science.gov (United States)

    Macfarlane, David; Socrates, Angelides; Eisenberg, Paul; Larcos, George; Roach, Paul; Gerometta, Michael; Smart, Richard; Tsui, Wendy; Scott, Andrew M

    2009-02-01

    (99m)Tc-DI-DD3B6/22-80B3 (ThromboView, hereafter abbreviated to (99m)Tc-DI-80B3 Fab') is a radiolabelled humanised monoclonal Fab' fragment with affinity and specificity for D-dimer domains of cross-linked fibrin. Detection of thromboembolic events has been demonstrated in canine models. The study objectives were evaluation of safety and characterisation of biodistribution, immunogenicity and pharmacokinetic profile of increasing doses of (99m)Tc-DI-80B3 Fab' in subjects with acute lower-limb DVT. Twenty-six patients with acute lower limb DVT were enrolled. Of these, 21 received a single intravenous dose of 0.5 mg (n = 6), 1.0 mg (n = 9) or 2 mg (n = 6) (99m)Tc-DI-80B3 Fab'. Blood and urine samples and gamma camera images were collected to 24 h after administration for pharmacokinetic and dosimetry analysis. Vital signs, electrocardiography, hematological and biochemical data and human anti-human antibody (HAHA) levels were monitored for up to 30 days following administration. Patients were assigned to either planar or single photon emission computed tomographic (SPECT) imaging of the thorax at 4 h following injection. Thirty-five adverse events were reported in 15 of the 21 subjects. Those deemed possibly related to administration of (99m)Tc-DI-80B3 Fab' included mild hypertension, mild elevation of LD (lactate dehydrogenase) and moderate elevation of ALT (alanine transaminase). HAHA assays remained negative. Pharmacokinetics and organ dosimetry were comparable to prior normal volunteer data. Localisation of Thromboview to sites of known thrombus was evident as early as 30 min post-injection. In subjects with acute DVT, (99m)Tc-DI-80B3 Fab' was well tolerated with favourable characteristics for the detection of acute venous thrombosis.

  13. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.

  14. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    Science.gov (United States)

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  15. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M;

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-01

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

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

    Science.gov (United States)

    McKinstry, William J; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T; Martin, Thomas J; Parker, Michael W

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A 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.

  18. Phase I trial of intraoperative detection of tumor margins in patients with HER2-positive carcinoma of the breast following administration of 111In-DTPA-trastuzumab Fab fragments.

    Science.gov (United States)

    Holloway, Claire M B; Scollard, Deborah A; Caldwell, Curtis B; Ehrlich, Lisa; Kahn, Harriette J; Reilly, Raymond M

    2013-07-01

    Our aim was to conduct a Phase I clinical trial to determine the feasibility of intraoperative detection of tumor margins in HER2 positive breast carcinoma using a hand-held γ-probe following administration of (111)In-DTPA-trastuzumab Fab fragments. Accurate delineation of tumor margins is important for preventing local recurrence. Six patients with HER2-positive in situ or invasive ductal carcinoma were administered 74MBq (0.5mg) of (111)In-DTPA-trastuzumab Fab fragments and counts in the tumor, surgical cavity wall and en face margins were measured intraoperatively at 72h post-injection using the Navigator or C-Trak γ-probes. Margins were evaluated histologically. Quantitative whole body planar imaging was performed to estimate radiation absorbed doses using OLINDA/EXM software. SPECT imaging of the thorax was performed to evaluate tumor uptake. The pharmacokinetics of elimination from the blood and plasma were determined over 72h. There were no acute adverse reactions from (111)In-DTPA-trastuzumab Fab fragments and no changes in hematological or biochemical indices were found over a 3month period. (111)In-DTPA-trastuzumab Fab fragments exhibited a biphasic elimination from the blood and plasma with t1/2α=11.9h and 7.5h, respectively, and t1/2β=26.6 and 20.7h, respectively. The radiopharmaceutical accumulated in the liver, spleen and kidneys. SPECT imaging did not reveal tumor in any patient. The mean effective dose was 0.146mSv/MBq (10.8mSv for 74MBq). Counts in excised tumors were low but were higher than in margins. Margins in two patients harboured tumor but this was not correlated with counts obtained using the γ-probes. Surgical cavity counts were high and likely due to detection of γ-photons outside the surgical field. We conclude that it was not feasible, at least at the administered amount of radioactivity used in this study, to reliably detect the margins of disease in patients with in situ or invasive ductal carcinoma intraoperatively using a

  19. Choice of labeling and cell line influences interactions between the Fab fragment AbD15179 and its target antigen CD44v6.

    Science.gov (United States)

    Stenberg, Jonas; Spiegelberg, Diana; Karlsson, Hampus; Nestor, Marika

    2014-02-01

    Medical imaging by use of immunotargeting generally relies on a labeled molecule binding to a specific target on the cell surface. It is important to utilize both cell-based and time-resolved binding assays in order to understand the properties of such molecular interactions in a relevant setting. In this report we describe the detailed characterization of the interaction properties for AbD15179, a promising CD44v6-targeting antibody fragment for radio-immunotargeting. Influence of labeling and cell-line model on the protein interaction kinetics was assessed using three different labeling approaches ((111)In, (125)I and FITC) on three different squamous carcinoma cell lines. Interactions were measured using time-resolved assays on living cells, and further analyzed with Interaction Map®. Results demonstrated a general biphasic appearance of a high- and a low-affinity binding event in all cases. The relative contribution from these two interactions differed between conjugates. For (125)I-Fab, the population of low-affinity binders could be significantly increased by extending the chloramine T exposure during labeling, whereas the (111)In-labeling predominantly resulted in a high-affinity interaction. Interactions were also shown to be cell line dependent, with e.g. SCC-25 cells generally mediating a faster dissociation of conjugates compared to the other cell lines. In conclusion, we report both cell line dependent and labeling associated variations in interaction kinetics for AbD15179 binding to CD44v6. This has implications for cell-based kinetic assays and applications based on labeled conjugates in general, as well as in a clinical setting, where each individual tumor may create different kinetic profiles for the same conjugate. © 2014.

  20. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage.

    Science.gov (United States)

    Cai, Binggang; Wang, Maorong; Zhu, Xuhui; Xu, Jing; Zheng, Wenkai; Zhang, Yiqing; Zheng, Feng; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  1. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4 Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage

    Directory of Open Access Journals (Sweden)

    Binggang Cai

    2015-10-01

    Full Text Available Lipopolysaccharides (LPS can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4 signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2 complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  2. Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using {sup 111}In-trastuzumab (Herceptin) Fab fragments

    Energy Technology Data Exchange (ETDEWEB)

    Tang Ying [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada); Wang, Judy [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Scollard, Deborah A. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Mondal, Hridya [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Holloway, Claire [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Kahn, Harriette J. [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, Ontario, M5S 3E2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

    2005-01-01

    Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with {sup 111}In. The dissociation constant (K{sub d}) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (K{sub d}=47 nM). {sup 111}In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8{+-}0.7% injected dose (ID)/g and tumor/blood ratio of 25.2{+-}1.6 at 72 h postinjection compared with 2.7{+-}0.7% ID/g and 7.0{+-}0.9 for {sup 111}In-HuM195 anti-CD33 Fab (significantly different, P<.001). Small (3-5 mm in diameter) BT-474 tumors were imaged with {sup 111}In-trastuzumab Fab as early as 24 h postinjection.

  3. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    Science.gov (United States)

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  4. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J. (SVIMR-A); (HeidelbergU); (WEHI); (Melbourne)

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  5. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Directory of Open Access Journals (Sweden)

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  6. Beyond Fab Four

    Science.gov (United States)

    Babichev, E.; Charmousis, C.; Langlois, D.; Saito, R.

    2015-12-01

    We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the 'John' and 'Paul' terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four.

  7. Beyond Fab Four

    CERN Document Server

    Babichev, E; Langlois, D; Saito, R

    2015-01-01

    We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the "John" and "Paul" terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four.

  8. 双功能螯合剂BAT与鼠IgG1 Fab'片段的点特异性交联方法的研究%Site-Specific Conjugation of Bifunctional Chelator BAT to Mouse IgG1 Fab' Fragment

    Institute of Scientific and Technical Information of China (English)

    李君; 王学浩; 成峰; 王晓明; 陈兆雷; 沈喜平

    2005-01-01

    目的报道一种用双功能螯合剂BAT与鼠单克隆抗体Fab'片段行点特异性交联的新方法.方法通过位于Fab'铰链区、远离抗原结合位点的活性巯基(-SH)直接与BAT相交联.用简单的两步法制备B43单抗的Fab'片段.首先单抗用胃蛋白酶处理,产生稳定的F(ab')2 片段,然后 F(ab')2经半胱氨酸还原后产生Fab'片段,最后Fab'片段直接与BAT交联.结果每个Fab'分子平均含1.8个-SH基团,74%的Fab'片段可与BAT生成交联物,平均每个Fab'分子携带1.28个BAT.经检测,F(ab')2、Fab' 及 Fab'-BAT 均保持着良好的免疫活性.结论该交联法简单、高效,为双功能螯合剂BAT与鼠单抗Fab'片段的交联提供了一个新途径.

  9. Stirred batch crystallization of a therapeutic antibody fragment.

    Science.gov (United States)

    Hebel, Dirk; Huber, Sabine; Stanislawski, Bernd; Hekmat, Dariusch

    2013-07-20

    Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

  10. Imaging of low-grade bone infection with a technetium-99m labelled monoclonal anti-NCA-90 Fab' fragment in patients with previous joint surgery

    Energy Technology Data Exchange (ETDEWEB)

    Ivaneeviae, V.; Sandrock, D.; Munz, D.L. [Clinic for Nuclear Medicine, University Hospital Charite, Humboldt University of Berlin (Germany); Perka, C.; Hasart, O. [Orthopaedic Clinic, University Hospital Charite, Humboldt University of Berlin (Germany)

    2002-04-01

    We analysed 38 scans in 30 consecutive patients (age range, 30-85 years; median age, 62 years) referred for suspected low-grade bone infection. There were 17 patients (21 scans) with total hip arthroplasty (THA), six with total knee arthroplasty (TKA), three who had undergone hip or knee surgery for trauma and five (seven scans) with resected hips and no endoprostheses (Girdlestone situations); There were no patients with rheumatoid arthritis as the underlying disease. Results were verified by means of bacteriological cultures, histopathological findings and/or follow-up and compared with the respective Zimmerli scores, which were used for clinical assessment of inflammatory activity. In one patient, the final diagnosis could not be established. One, 5 and 24 h after intravenous injection of up to 1.1 GBq of MN3 Fab', whole-body and planar scans were performed using a dual-head gamma camera. Scans were analysed visually and semiquantitatively adopting an arbitrary score ranging from 0 to 3. There were 13 true positive, 14 true negative and 10 false positive outcomes, yielding an overall sensitivity of 100%, an overall specificity of 58%, an accuracy of 73% and positive and negative predictive values of 57% and 100%, respectively. In patients with THA or TKA, accuracy was 81% and 80%, respectively, while it dropped to 43% in patients with Girdlestone situations owing to a high proportion of false positive findings (4/7) in this subgroup. Scintigraphic score was 1 in all of the false positive and in 11/13 true positive findings. The two remaining true positive findings displayed scintigraphic scores of 2 and 3, respectively. Scintigraphic and Zimmerli scores were loosely correlated (Spearman {rho}=0.38, P<0.05). Infection was excluded in 22/24 investigations with Zimmerli scores of <6. In this group, there were 13 scintigraphically true negative, nine false positive outcomes, and just two true positive outcomes. In 11/12 investigations with Zimmerli scores of 6

  11. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

    Science.gov (United States)

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute

    2011-07-01

    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

  12. A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

    DEFF Research Database (Denmark)

    Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik

    2016-01-01

    molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel...

  13. Purification and Analysis of Structure of Fab Fragment of Recombinant Human Anti-HBs%重组人抗HBs-Fab抗体的纯化及结构分析

    Institute of Scientific and Technical Information of China (English)

    饶桂荣; 陈文吟; 粟宽源; 任向荣; 郑业华; 余宙耀

    2006-01-01

    目的研究重组人抗HBs-Fab抗体的纯化工艺,并对其结构等特性进行分析.方法采用离子交换-分子筛层析法分离纯化由酵母工程菌(GS115/Fab)发酵的重组人抗HBsAg Fab抗体,并经ELISA检测其抗体活性,等电聚焦电泳法检测其等电点(pI),基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)检测其相对分子质量和肽质量图谱.结果纯化的重组Fab抗体的纯度可达90%以上,经Sephacryl-100进一步层析后,纯度达99%以上,总收率可达80%以上.Fab抗体具有较好的抗体活性,其pI值为7.6,为一碱性蛋白,相对分子质量为50 494,比其理论值约多2 579.35,经Endoglycosidase H内切酶消化后的相对分子质量为49 609,证明Fab的一级结构中有糖基化现象,且分布于Fab抗体的H链和L链上.胰酶酶解肽段中有21个与理论肽段相符,另检测到1对二硫键正确.结论已建立了重组人抗HBs-Fab抗体的稳定的纯化工艺,且Fab抗体的一级结构正确.

  14. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

    Energy Technology Data Exchange (ETDEWEB)

    Obmolova, Galina, E-mail: gobmolov@its.jnj.com; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L., E-mail: gobmolov@its.jnj.com [Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477 (United States)

    2014-07-23

    The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

  15. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    Science.gov (United States)

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  16. Yeast mating for combinatorial Fab library generation and surface display

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Jane M.; Lou, Jianlong; Coleman, James R.; Siegel, Robert W.; Marks, James D.; Feldhaus, Michael

    2004-04-23

    Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv (scFv) antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

  17. Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab' fragment of monoclonal antibody directed against type IV collagenase

    Institute of Scientific and Technical Information of China (English)

    WANG; Fengqiang; SHANG; Boyang; ZHEN; Yongsu

    2004-01-01

    Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab′-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab′ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab′-LDM conjugate was 65 kD with a 1:1 molecular ratio of Fab′ and LDM. The Fab′-LDM conjugate maintained most part of the immunoreactivity of Fab′ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab′-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab′-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab′-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab′-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab′-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.

  18. FabIO

    DEFF Research Database (Denmark)

    Bergbäck Knudsen, Erik; Sørensen, Henning O.; Wright, Jonathan P.

    2013-01-01

    FabIO is a Python module written for easy and transparent reading of raw two-dimensional data from various X-ray detectors. The module provides a function for reading any image and returning a fabioimage object which contains both metadata (header information) and the raw data. All fabioimage...

  19. Fab Four Neutron Stars

    CERN Document Server

    Maselli, Andrea; Minamitsuji, Masato; Berti, Emanuele

    2016-01-01

    Horndeski's theory of gravity is the most general scalar-tensor theory with a single scalar whose equations of motion contain at most second-order derivatives. A subsector of Horndeski's theory known as "Fab Four" gravity allows for dynamical self-tuning of the quantum vacuum energy, and therefore it has received particular attention in cosmology as a possible alternative to the $\\Lambda$CDM model. Here we study compact stars in Fab Four gravity, which includes as special cases general relativity ("George"), Einstein-dilaton-Gauss-Bonnet gravity ("Ringo"), theories with a nonminimal coupling with the Einstein tensor ("John") and theories involving the double-dual of the Riemann tensor ("Paul"). We generalize and extend previous results in theories of the John class and we show that there are no viable compact star solutions in theories of the Paul class.

  20. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    Science.gov (United States)

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  1. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    Science.gov (United States)

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  2. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    Science.gov (United States)

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  3. 131I-肝癌单抗片段HAb18F(ab')2注射液药盒的制备%Preparation of 131I Labeled Hepatoma Monoclonal Antibody Fragment HAb18F(ab')2 Kit

    Institute of Scientific and Technical Information of China (English)

    冯强; 米力; 边惠洁; 余晓玲; 陈志南

    2002-01-01

    选择Mather高效碘标法(NBS法)制备应用于肝癌治疗的131I-肝癌单抗片段HAb18F(ab')2注射液药盒,确定并优化了冷药盒各组分的剂型、标记条件和纯化方法.制备出的药盒抗体标记物的放化纯度大于95%,整个标记过程可在10 min内完成,10 ℃以下干燥处可保存两年以上,适于临床应用.

  4. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.

    Science.gov (United States)

    Rauchenberger, Robert; Borges, Eric; Thomassen-Wolf, Elisabeth; Rom, Eran; Adar, Rivka; Yaniv, Yael; Malka, Michael; Chumakov, Irina; Kotzer, Sarit; Resnitzky, Dalia; Knappik, Achim; Reiffert, Silke; Prassler, Josef; Jury, Karin; Waldherr, Dirk; Bauer, Susanne; Kretzschmar, Titus; Yayon, Avner; Rothe, Christine

    2003-10-03

    The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

  5. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

  6. The ceramide transporter and the Goodpasture antigen binding protein: one protein--one function?

    Science.gov (United States)

    Mencarelli, Chiara; Losen, Mario; Hammels, Caroline; De Vry, Jochen; Hesselink, Matthijs K C; Steinbusch, Harry W M; De Baets, Marc H; Martínez-Martínez, Pilar

    2010-06-01

    The Goodpasture antigen-binding protein (GPBP) and its splice variant the ceramide transporter (CERT) are multifunctional proteins that have been found to play important roles in brain development and biology. However, the function of GPBP and CERT is controversial because of their involvement in two apparently unrelated research fields: GPBP was initially isolated as a protein associated with collagen IV in patients with the autoimmune disease Goodpasture syndrome. Subsequently, a splice variant lacking a serine-rich domain of 26 amino acids (GPBPDelta26) was found to mediate the cytosolic transport of ceramide and was therefore (re)named CERT. The two splice forms likely carry out different functions in specific sub-cellular localizations. Selective GPBP knockdown induces extensive apoptosis and tissue loss in the brain of zebrafish. GPBP/GPBPDelta26 knock-out mice die as a result of structural and functional defects in endoplasmic reticulum and mitochondria. Because both mitochondria and ceramide play an important role in many biological events that regulate neuronal differentiation, cellular senescence, proliferation and cell death, we propose that GPBP and CERT are pivotal in neurodegenerative processes. In this review, we discuss the current state of knowledge on GPBP and CERT, including the molecular and biochemical characterization of GPBP in the field of autoimmunity as well as the fundamental research on CERT in ceramide transport, biosynthesis, localization, metabolism and cell homeostasis.

  7. The expression of the Goodpasture antigen-binding protein (ceramide transporter) in adult rat brain.

    Science.gov (United States)

    Mencarelli, Chiara; Hammels, Caroline; Van Den Broeck, Joost; Losen, Mario; Steinbusch, Hellen; Revert, Francisco; Saus, Juan; Hopkins, David A; De Baets, Marc H; Steinbusch, Harry W; Martinez-Martinez, Pilar

    2009-10-01

    The Goodpasture antigen-binding protein (GPBP) plays a critical role in brain development. Knockdown of GPBP leads to loss of myelinated tracts in the central nervous system and to extensive apoptosis in the brain during early embryogenesis. GPBP was initially identified as a protein associated with the autoantigen in Goodpasture autoimmune syndrome, where it was shown to be a kinase that regulates type IV collagen organization. GPBP isoforms bind and transport ceramide from the endoplasmic reticulum to the Golgi apparatus and are therefore also known as ceramide transporters (CERT). Ceramide dysregulation is involved in autoimmunity and neurodegenerative disorders. In order to analyze the possible role of GPBP in neuroinflammation and neurodegeneration we studied the basal GPBP expression in normal rat brain. High levels of immunoreactivity were detected in neurons of the cerebral cortex, hippocampal formation, the basal ganglia, the olfactory bulb and nuclei of the thalamus, the hypothalamus and the septal area. Lower expression levels of GPBP were observed widely throughout the brain, suggesting that GPBP plays an important role in central nervous system neuron function.

  8. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  9. "Fab 13": The Learning Factory.

    Science.gov (United States)

    Crooks, Steven M.; Eucker, Tom R.

    2001-01-01

    Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

  10. "Fab 13": The Learning Factory.

    Science.gov (United States)

    Crooks, Steven M.; Eucker, Tom R.

    2001-01-01

    Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

  11. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    Science.gov (United States)

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  12. Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

    Science.gov (United States)

    Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

    2016-11-01

    Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM.

  13. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

    Science.gov (United States)

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  14. Generation and selection of immunized Fab phage display library against human B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Yongmei Shen; Xiaochun Yang; Ningzheng Dong; Xiaofang Xie; Xia Bai; Yizhen Shi

    2007-01-01

    The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the K light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×107. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

  15. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    Science.gov (United States)

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. Published by Elsevier Ltd.

  16. Inhibition of histo-blood group antigen binding as a novel strategy to block norovirus infections.

    Directory of Open Access Journals (Sweden)

    Xu-Fu Zhang

    Full Text Available Noroviruses (NoVs are the most important viral pathogens that cause epidemic acute gastroenteritis. NoVs recognize human histo-blood group antigens (HBGAs as receptors or attachment factors. The elucidation of crystal structures of the HBGA-binding interfaces of a number of human NoVs representing different HBGA binding patterns opens a new strategy for the development of antiviral compounds against NoVs through rational drug design and computer-aided virtual screening methods. In this study, docking simulations and virtual screening were used to identify hit compounds targeting the A and B antigens binding sites on the surface of the capsid P protein of a GII.4 NoV (VA387. Following validation by re-docking of the A and B ligands, these structural models and AutoDock suite of programs were used to screen a large drug-like compound library (derived from ZINC library for inhibitors blocking GII.4 binding to HBGAs. After screening >2 million compounds using multistage protocol, 160 hit compounds with best predicted binding affinities and representing a number of distinct chemical classes have been selected for subsequent experimental validation. Twenty of the 160 compounds were found to be able to block the VA387 P dimers binding to the A and/or B HBGAs at an IC50<40.0 µM, with top 5 compounds blocking the HBGA binding at an IC50<10.0 µM in both oligosaccharide- and saliva-based blocking assays. Interestingly, 4 of the top-5 compounds shared the basic structure of cyclopenta [a] dimethyl phenanthren, indicating a promising structural template for further improvement by rational design.

  17. Uncommon Structural Motifs Dominate the Antigen Binding Site in Human Autoantibodies Reactive with Basement Membrane Collagen

    Science.gov (United States)

    Foster, Mary H.; Buckley, Elizabeth S.; Chen, Benny J.; Hwang, Kwan-Ki; Clark, Amy G.

    2016-01-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients’ IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20–36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  18. Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity

    Science.gov (United States)

    Miralem, Tihomir; Gibbs, Peter E. M.; Revert, Fernando; Saus, Juan; Maines, Mahin D.

    2010-01-01

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis. PMID:20177069

  19. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J. (SAIC); (NCI)

    2012-10-16

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

  20. 重组人抗HBsAg Fab抗体的纯化方法比较研究%Study on the purification of recombinant human anti-HBsAg Fab fragment

    Institute of Scientific and Technical Information of China (English)

    饶桂荣; 粟宽源; 陈文吟; 余宙耀

    2005-01-01

    目的研究重组人抗HBsAg Fab抗体的纯化条件.方法采用羊抗人Fab抗体亲和层析柱、14F7单克隆抗体亲和层析柱、离子交换-分子筛层析柱,分别纯化由酵母工程菌(GS115/Fab)发酵的重组人抗HBsAg Fab抗体,并对3种纯化方法所得Fab抗体的纯度、收率、与HBsAg的结合活性进行比较.结果 3种纯化方法中,14F7单克隆抗体柱纯化的Fab抗体的纯度达98%左右,Fab抗体柱纯化的Fab抗体的纯度为95%,但这两种亲和柱的目的蛋白收率都不高,分别为35%、55%.而离子交换柱纯化的Fab抗体的纯度为93.8%,经分子筛柱进一步纯化后,可达98%以上,Fab抗体蛋白收率可达80%以上.经ELISA分析,3种方法纯化的Fab抗体均具有较高的HBsAg抗原结合力和特异性.结论通过对3种纯化方法的比较得出,离子交换-分子筛层析法是重组人抗HBsAg Fab抗体的最佳纯化方法,这为抗HBsAg Fab抗体的产业化生产和临床研究打下良好基础.

  1. Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

    Directory of Open Access Journals (Sweden)

    Sebastian Fenn

    Full Text Available Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

  2. Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

    Science.gov (United States)

    Caballero, A; Foletti, D; Bierdeman, M; Tang, A; Arana, A; Hasa-Moreno, A; Sangalang, E; O'Callaghan, R J

    2014-06-01

    Abstract Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

  3. Disulfide cross-linked Fab-aggregates: preparation and biodistribution.

    Science.gov (United States)

    Dalkara, S; Petrov, A; Trubetskoy, V S; Khaw, B A; Torchilin, V P

    1998-01-01

    The high-molecular-weight soluble aggregates of Fab fragments of murine antibodies against cardiac myosin were prepared as a potential long-circulating and low immunogenic pharmaceutical carriers by conjugation of thiolated Fab and Fab modified with succinimidyl 3-(2-pyridyldithio)propionate. The clearance time and biodistribution of 111In-radiolabeled aggregates were studied in normal and nude-mice bearing human breast tumor implant and in rabbits with experimental myocardial infarction. The aggregates had a prolonged circulation time (half clearance time ca. 3-5 h) and ability to concentrate in the tumor and in the necrotic area of infarcted myocardium. Similar tumor-to-normal and infarct-to-normal accumulation ratios (ca. 3 h in both cases) suggest that combination of long circulation with impaired filtration in necrotic tissues is responsible for this accumulation rather than a specific interaction. The aggregates prepared may serve as long-circulating drug carriers able to deliver pharmaceuticals into areas with affected and leaky vasculature.

  4. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  5. Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand.

    Science.gov (United States)

    Parreira, P; Shi, Q; Magalhaes, A; Reis, C A; Bugaytsova, J; Borén, T; Leckband, D; Martins, M C L

    2014-12-01

    The strength of binding between the Helicobacter pylori blood group antigen-binding adhesin (BabA) and its cognate glycan receptor, the Lewis b blood group antigen (Le(b)), was measured by means of atomic force microscopy. High-resolution measurements of rupture forces between single receptor-ligand pairs were performed between the purified BabA and immobilized Le(b) structures on self-assembled monolayers. Dynamic force spectroscopy revealed two similar but statistically different bond populations. These findings suggest that the BabA may form different adhesive attachments to the gastric mucosa in ways that enhance the efficiency and stability of bacterial adhesion.

  6. Crystal structure determination of anti-DNA Fab A52.

    Science.gov (United States)

    Stanfield, Robyn L; Eilat, Dan

    2014-08-01

    A52 is a murine monoclonal antibody isolated from autoimmune New Zealand Black/New Zealand White F1 mice that recognizes single and double stranded DNA. This mouse strain spontaneously develops systemic lupus erythematosus-like symptoms and has served as a model for that disease for many years. The 1.62 Å crystal structure of the A52 Fab fragment reveals an H3 complementarity determining region with four closely spaced arginine residues, creating a positively charged surface to accommodate bound DNA.

  7. Study of antibody/antigen binding kinetics by total internal reflection ellipsometry.

    Science.gov (United States)

    Baleviciute, Ieva; Balevicius, Zigmas; Makaraviciute, Asta; Ramanaviciene, Almira; Ramanavicius, Arunas

    2013-01-15

    Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.

  8. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  9. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    Energy Technology Data Exchange (ETDEWEB)

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.; (Stanford-MED); (CH-Boston)

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  10. Structure of the omalizumab Fab.

    Science.gov (United States)

    Jensen, Rasmus K; Plum, Melanie; Tjerrild, Luna; Jakob, Thilo; Spillner, Edzard; Andersen, Gregers Rom

    2015-04-01

    Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

  11. Display and selection of chicken IgA Fab fragments

    NARCIS (Netherlands)

    Wieland, W.H.; Orzaéz, D.; Lammers, A.; Parmentier, H.K.; Schots, A.

    2006-01-01

    Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicke

  12. Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques

    NARCIS (Netherlands)

    Mencarelli, Chiara; Bode, Gerard H.; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C.; Veerhuis, Robert; Steinbusch, Harry W. M.; De Baets, Marc H.; Nicolaes, Gerry A. F.; Martinez-Martinez, Pilar

    2012-01-01

    Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  14. A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties.

    Directory of Open Access Journals (Sweden)

    Felix Unverdorben

    Full Text Available Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD from streptococcal protein G (SpGC3 as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv and bispecific single-chain diabody (scDb. Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

  15. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

    Directory of Open Access Journals (Sweden)

    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  16. FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

    Science.gov (United States)

    Pagano, John

    2015-01-01

    This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

  17. Rapportage LTO FAB II 2008 : Functionele Agro Biodiversiteit

    NARCIS (Netherlands)

    Scheele, J.; Gurp, van H.; Alebeek, van F.A.N.; Belder, den E.; Elderson, J.

    2009-01-01

    Doel van het LTO FAB II project is een gebruiksklaar FAB concept te ontwikkelen voor een aantal ziekten en plagen in een aantal gewassen die op eenvoudige wijze door telers benut kan worden en voor de toepasser kostenneutraal zijn. Uitgangspunt in het FAB project is een evenwichtige balans tussen de

  18. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    OpenAIRE

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.; Rock, Charles O.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were...

  19. Antibacterial FabH Inhibitors with Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance Mutation Mapping.

    Science.gov (United States)

    McKinney, David C; Eyermann, Charles J; Gu, Rong-Fang; Hu, Jun; Kazmirski, Steven L; Lahiri, Sushmita D; McKenzie, Andrew R; Shapiro, Adam B; Breault, Gloria

    2016-07-01

    Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (β-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery.

  20. Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

    Directory of Open Access Journals (Sweden)

    Olga Kyrchanova

    2016-07-01

    Full Text Available Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

  1. Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

    Science.gov (United States)

    Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

    2016-07-01

    Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

  2. PEGylation of antibody fragments for half-life extension.

    Science.gov (United States)

    Jevševar, Simona; Kusterle, Mateja; Kenig, Maja

    2012-01-01

    Antibody fragments (Fab's) represent important structure for creating new therapeutics. Compared to full antibodies Fab' fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crystallizable (Fc) region-mediated functions such as antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The main drawback for the use of Fab's in clinical applications is associated with their short half-life in vivo, which is a consequence of no longer having the Fc region. To exert meaningful clinical effects, the half-life of Fab's need to be extended, which has been achieved by postproduction chemical attachment of polyethylene glycol (PEG) chain to protein using PEGylation technology. The most suitable approach employs PEG-maleimide attachment to cysteines, either to the free hinge cysteine or to C-terminal cysteines involved in interchain disulfide linkage of the heavy and light chain. Hence, protocols for mono-PEGylation of Fab via free cysteine in the hinge region and di-PEGylation of Fab via interchain disulfide bridge are provided in this chapter.

  3. Monitoring strategy to match the advanced fabs

    Science.gov (United States)

    Ackmann, Paul W.

    2004-06-01

    The reduction in feature size below the exposure wavelength, the requirement for high yields, the expectation for consistent cycletime and shipment to mix, all mean that the reticle industry must be like advanced wafer fabrication centers. Due to the lower output of write tools versus steppers, and the fact that a reticle is a lot of one instead of 25 or 50 wafers as well as the need to match ship data to Fab ramp, the reticle line monitoring strategy must be optimized for small sample size. The use of tool time and alternative inspection strategies can lead to the early detection of problems. Because every reticle is a customer specific design, the monitoring strategy takes on a new look compared to the Fab. We have organized the AMTC to resemble a wafer fab. We have a dedicated Integration group that works with the customers and technologists, to monitor the needs of the customers and then drive the development programs that improve reticle capability. We have dedicated yield team to identify and classify the yield loss mechanisms and define probable causes. The teams then work with the Process owners to fix the source of yield loss and track the corrective actions. All sources of variations must be modeled and then sources of errors reduced to levels below the tool specification. The manufacturing organization has all the process and tool experts to focus on Pilot Line and Development tasks to meet the advance needs of our customers. With the organization in place we can then develop the methods based on Reticle and Fab manufacturing to best control the line and provide development with manufacturing cycle times.

  4. Mask qualification strategies in a wafer fab

    Science.gov (United States)

    Jaehnert, Carmen; Kunowski, Angela

    2007-02-01

    Having consistent high quality photo masks is one of the key factors in lithography in the wafer fab. Combined with stable exposure- and resist processes, it ensures yield increases in production and fast learning cycles for technology development and design evaluation. Preventive controlling of incoming masks and quality monitoring while using the mask in production is essential for the fab to avoid yield loss or technical problems caused by mask issues, which eventually result in delivery problems to the customer. In this paper an overview of the procedures used for mask qualification and production release, for both logic and DRAM, at Infineon Dresden is presented. Incoming qualification procedures, such as specification checks, incoming inspection, and inline litho process window evaluation, are described here. Pinching and electrical tests, including compatibility tests for mask copies for high volume products on optimized litho processes, are also explained. To avoid mask degradation over lifetime, re-inspection checks are done for re-qualification while using the mask in production. The necessity of mask incoming inspection and re-qualification, due to the repeater printing from either the processing defects of the original mask or degrading defects of being used in the fab (i.e. haze, ESD, and moving particles, etc.), is demonstrated. The need and impact of tight mask specifications, such as CD uniformity signatures and corresponding electrical results, are shown with examples of mask-wafer CD correlation.

  5. Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

    OpenAIRE

    Hoang, T.T.; Schweizer, H P

    1997-01-01

    The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemop...

  6. Identification and Function Reasearch of fabA and fabB of Sinorhizobium meliloti%苜蓿中华根瘤菌fabA和fabB基因功能的鉴定

    Institute of Scientific and Technical Information of China (English)

    胡喆; 马金成; 蒋晶晶; 王海洪

    2013-01-01

    在大肠杆菌(Escherichia coli)脂肪酸合成酶体系中,fabA基因编码有双功能的3-羟基脂酰ACP脱水异构酶,其异构产物能被fabB基因编码的3-酮基脂酰ACP合成酶Ⅰ延伸,合成不饱和脂肪酸,该FabA-FabB途径被认为是缺氧条件下不饱和脂肪酸合成的经典途径.生物信息学分析发现,苜蓿中华根瘤菌(Sinorhizobium meliloti)的SmFabA与EcFabA相似性达到60.6%,具有相同的保守活性位点和两个保守的α螺旋结构;SmFabB与EcFabB相似性达到61.1%,具有相同的Cys-His-His活性中心.用携带SmfabA和SmfabB的质粒载体遗传互补大肠杆菌湿度敏感突变株CY57和CY242,在添加三氯森(TCL)抑制烯脂酰ACP还原酶活性的条件下,转化子能在42℃恢复生长,且放射性薄层层析能检测到转化子中不饱和脂肪酸棕榈油酸(A9C16:1)和十八碳烯酸(△11C18:1)的合成.体外重建脂肪酸合成反应表明,SmFabA能催化羟脂酰ACP的脱水反应且能够使反-2-癸烯酰ACP异构化,SmFabB能催化不同链长的脂酰ACP和丙二酸单酰ACP的聚合反应.另外,未得到SmFabA和SmFabB的突变株,表明SmFabA和SmFabB可能是苜蓿中华根瘤菌脂肪酸合成酶系中必不可少的关键蛋白.上述结果证实了苜蓿中华根瘤菌fabA和fabB两个基因在不饱和脂肪酸合成中的功能.

  7. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  8. The cosmology of the Fab-Four

    CERN Document Server

    Copeland, Edmund J; Saffin, Paul M

    2012-01-01

    We have recently proposed a novel self tuning mechanism to alleviate the famous cosmological constant problem, based on the general scalar tensor theory proposed by Horndeski. The self-tuning model ends up consisting of four geometric terms in the action, with each term containing a free potential function of the scalar field; the four together being labeled as the Fab-Four. In this paper we begin the important task of deriving the cosmology associated with the Fab-Four Lagrangian. Performing a phase plane analysis of the system we are able to obtain a number of fixed points for the system, with some remarkable new solutions emerging from the trade-off between the various potentials. As well as obtaining inflationary solutions we also find conventional radiation/matter-like solutions, but in regimes where the energy density is dominated by a cosmological constant, and where we do not have any explicit forms of radiation or matter. Stability conditions for matter solutions are obtained and we show how it is po...

  9. Een inventarisatie naar de bekendheid van Functionele Agrobiodiversiteit (FAB) en de mogelijkheden om FAB met andere agroranden te combineren

    NARCIS (Netherlands)

    Geerts, R.H.E.M.; Meerburg, B.G.

    2011-01-01

    Op dit moment zijn er regio's waar het FAB concept al breed wordt toegepast en goed is ingebed (West-Brabant/Zeeland), waar het in opmars is (Flevoland en Zuid-Holland) en enkele regio's waar het FAB gedachtegoed slechts matig aanwezig is (Groningen / Drenthe).

  10. COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

    Institute of Scientific and Technical Information of China (English)

    邹颖; 卞美璐; 杨子义; 连利娟; 刘文淑; 许秀英

    1995-01-01

    In the present study,four different proteases (pepsin,papain,bromelain and ficin) were screened with a murine monoclonal antibody OC859,in order to verify whether different digestion procedures could improve yield and stability of the F(ab')2 or Fab fragments.The yields of F(ab')2 or Fab fragments from digestion with pepsin,papain,bromelain and ficin were respectively 20.3+/-2.0%,50.5%+/-5.0%,74.4+/-2.7% and 82.8+/-10.2% of the theoretical maximum.Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%,36+/-5%,60+/-6% and 75+/-6% of the intact OC859 IgG.These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

  11. Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

    Science.gov (United States)

    Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

    2015-12-01

    Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

  12. Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

    Science.gov (United States)

    Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

    2016-04-01

    The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

  13. Improved renal clearance and tumor targeting of {sup 99m}Tc-labeled anti-Tac monoclonal antibody Fab by chemical modifications

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Meyoung-kon; Jeong, Hyeh-Jean; Kao, Chih-Hao K.; Yao, Zhengsheng; Paik, David S.; Pie, Jae Eun; Kobayashi, Hisataka; Waldmann, Thomas A.; Carrasquillo, Jorge A.; Paik, Chang H. E-mail: cpaik@mail.cc.nih.gov

    2002-02-01

    This study was undertaken to improve the renal clearance and tumor targeting properties of {sup 99m}Tc-labeled humanized anti-Tac (HuTac) monoclonal antibody Fab fragments using two chemical approaches: 1) labeling with a renal secretion agent {sup 99m}Tc-mercaptoacetyltriglycine (MAG3) and 2) lowering its isoelectric point (pI) by acylation. HuTac Fab (3.3 mg/mL) was reacted with a trifluorophenyl ester (TFP) of {sup 99m}Tc-MAG3 alone or was additionally reacted with TFP-glycolate to reduce the pI. In Balb/c mice, {sup 99m}Tc-MAG3-Fab (pI>9.3) rapidly accumulated in the kidneys (177% injected dose [ID]/g at 15 min) and then gradually cleared out of the kidneys. In contrast, the glycolation (pI 4.6{approx}6.6) drastically reduced the renal uptake (31% ID/g) and also the whole-body retention (82% ID vs 101% for the nonglycolated) at 15 min, indicating that the glycolated {sup 99m}Tc-MAG3-Fab (pI 4.6{approx}6.6) was rapidly excreted. The glycolated remained in the blood longer than the nonglycolated (1.2% vs 0.3% ID/g at 360 min), but this effect was less drastic than the effect shown on the renal uptake. In nude mice bearing receptor-positive (ATAC4) tumors, the glycolated {sup 99m}Tc-MAG3-Fab increased the peak tumor uptake to 14.8% ID/g from 8.3% ID/g for {sup 99m}Tc-MAG3-Fab, whereas the glycolation resulted in a drastic reduction of the renal uptake at 15 min. We demonstrated that the renal clearance and the tumor targeting of Fab could be optimized by chemical modifications.

  14. Semiconductor industry wafer fab exhaust management

    CERN Document Server

    Sherer, Michael J

    2005-01-01

    Given the myriad exhaust compounds and the corresponding problems that they can pose in an exhaust management system, the proper choice of such systems is a complex task. Presenting the fundamentals, technical details, and general solutions to real-world problems, Semiconductor Industry: Wafer Fab Exhaust Management offers practical guidance on selecting an appropriate system for a given application. Using examples that provide a clear understanding of the concepts discussed, Sherer covers facility layout, support facilities operations, and semiconductor process equipment, followed by exhaust types and challenges. He reviews exhaust point-of-use devices and exhaust line requirements needed between process equipment and the centralized exhaust system. The book includes information on wet scrubbers for a centralized acid exhaust system and a centralized ammonia exhaust system and on centralized equipment to control volatile organic compounds. It concludes with a chapter devoted to emergency releases and a separ...

  15. How Fabulous Is Fab 5 Cosmology?

    CERN Document Server

    Linder, Eric V

    2013-01-01

    Extended gravity origins for cosmic acceleration can solve some fine tuning issues and have useful characteristics, but generally have little to say regarding the cosmological constant problem. Fab 5 gravity can be ghost free and stable, have attractor solutions in the past and future, and possess self tuning that solves the original cosmological constant problem. Here we show however it does not possess all these qualities at the same time. We also demonstrate that the self tuning is so powerful that it not only cancels the cosmological constant but also all other energy density, and we derive the scalings of its approach to a renormalized de Sitter cosmology. While this strong cancellation is bad for the late universe, it greatly eases early universe inflation.

  16. The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors.

    Science.gov (United States)

    Houimel, Mehdi

    2014-08-01

    A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74-92% and 90-96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3-IGKV1, IGHV3-IGVL1, and IGHV3-IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.

  17. A Case Study of a High School Fab Lab

    Science.gov (United States)

    Lacy, Jennifer E.

    This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

  18. Synthesis of CCK-8 Tetrapeptide Fragment by Enzymatic Method

    Institute of Scientific and Technical Information of China (English)

    XIANG Guangya (项光亚); Heiner Eckstein

    2003-01-01

    The enzymatic synthesis of a tetrapeptide Phac-Met-Gly-Trp-Met-OEt, a fragment of thecholecystokinin C-terminal octapeptide CCK-8, was reported. This fragment was synthesized bycoupling Phac-Met-OEt with Gly-OMe, Trp-OMe and Met-OEt successively. These three stepswere catalyzed by α-chymotrpsin, Papain and α-chymotrpsin respectively. The results of FAB-MSshowed that all the products had the correct molecular mass.

  19. Increased Fab thermoresistance via VH-targeted directed evolution.

    Science.gov (United States)

    Entzminger, Kevin C; Johnson, Jennifer L; Hyun, Jeongmin; Lieberman, Raquel L; Maynard, Jennifer A

    2015-10-01

    Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.

  20. Implementation of high-resolution reticle inspection in wafer fabs

    Science.gov (United States)

    Dayal, Aditya; Bergmann, Nathan M.; Sanchez, Peter

    2003-05-01

    Many advanced wafer fabs are currently fabricating devices with 130nm or smaller design rules. To meet the challenges at these sub-wavelength technology nodes, fabs are using a variety of resolution enhancement techniques (RETs) in lithography and exploring new methods of processing, inspecting and requalifying photomasks. The acceleration of the lithography roadmap imposes more stringent requirements on mask qualification and requalification to ensure that device yields are not compromised: mask inspection tools of today need to find smaller defects on reticles against considerably more complicated patterns or tighter critical dimensions (CDs). In this paper we describe the early stages of implementation and proliferation of advanced reticle inspection tools at high volume manufacturing wafer fabs. The fabs run incoming multi-surface contamination inspections on masks sent from the mask shop (Intel Mask Operations, IMO), and follow them up with periodic inspections/review to make sure any new contaminant or damage does not go undetected. When necessary, images of defects are electronically presented to engineers at IMO for review. Reticle requalification with these inspection tools reduces or eliminates the need for print test verification. We describe the tools and procedure used to streamline reticle requalification at the fabs and improve the feedback loop between the fabs and the mask shop.

  1. Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of 227 antibody-antigen complexes.

    Science.gov (United States)

    Robin, Gautier; Sato, Yoshiteru; Desplancq, Dominique; Rochel, Natacha; Weiss, Etienne; Martineau, Pierre

    2014-11-11

    Antibody molecules are able to recognize any antigen with high affinity and specificity. To get insight into the molecular diversity at the source of this functional diversity, we compiled and analyzed a non-redundant aligned collection of 227 structures of antibody-antigen complexes. Free energy of binding of all the residue side chains was quantified by computational alanine scanning, allowing the first large-scale quantitative description of antibody paratopes. This demonstrated that as few as 8 residues among 30 key positions are sufficient to explain 80% of the binding free energy in most complexes. At these positions, the residue distribution is not only different from that of other surface residues but also dependent on the role played by the side chain in the interaction, residues participating in the binding energy being mainly aromatic residues, and Gly or Ser otherwise. To question the generality of these binding characteristics, we isolated an antibody fragment by phage display using a biased synthetic repertoire with only two diversified complementarity-determining regions and solved its structure in complex with its antigen. Despite this restricted diversity, the structure demonstrated that all complementarity-determining regions were involved in the interaction with the antigen and that the rules derived from the natural antibody repertoire apply to this synthetic binder, thus demonstrating the robustness and universality of our results. Copyright © 2014. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2015-12-24

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

  3. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon, E-mail: choe@salk.edu [Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (United States)

    2006-09-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K{sup +} channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

  4. Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2%人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    张为家; 刘孝荣; 李官成; 贺智敏

    2011-01-01

    .The humanized Fab phage antibody library against HEF2 ECD was constructed by infection of helper phage VCSM13.The libraries were enrich after panned three cycles by purification protein of recombinant HER2 ECD.Then random clones were tested by ELISA to select the positive ones, which were furher identified their antigen binding acticities by Western blot, and the strongest binding to HER2 ECD clone was sequenced.RESULTS: The Fab phage antibody library with 2.5 × 107 volume was constructed and four positive clones which specifically recognized the HER2 ECD were isolated and further demonstrated by Western blot.Sequence analysis of the positivest clone showed that the variable heavy domains(VH) and variable light domains(VL) were highly homologous with the human embryonal Ig heavy chain V region sequences and kappa light chain sequences, respectively.CONCLUSION: A fully humanized Fab phage antibody library is successfully constructed and specific antibodies against HER2 ECD are obtained, which provides an experimental foundation for new humanized anti-HER2 ECD monoclonal antibodies.

  5. 抗内毒素Fab'的制备%Production of Fab' of the chicken egg yolk antibody against lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    马思远; 张雅萍

    2007-01-01

    目的 研究抗内毒素卵黄免疫球蛋白(IgY)的活性片断Fab',探讨防治内毒素血症的新途径.方法 用内毒素(LPS)作为抗原免疫25周龄德国罗曼鸡,改良水溶法提取抗内毒素IgY,胃蛋白酶切后提取Fab'片断,光密度法测抗内毒素Fab'的浓度和含量、ELISA检测抗内毒素Fab'效价、SDS-聚丙烯酰胺凝胶电泳检测其分子量及纯度. 结果 抗内毒素Fab'含量为4.2 mg/mL蛋黄液,效价为1∶51 200,纯度为92%,相对分子质量为44 000. 结论 抗内毒素Fab'产量大、效价高、特异性强.

  6. Human milk sIgA molecules contain various combinations of different antigen-binding sites resulting in a multiple binding specificity of antibodies and enzymatic activities of abzymes.

    Directory of Open Access Journals (Sweden)

    Sergey E Sedykh

    Full Text Available In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11-20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S and the join chain (J can combine molecules of dimeric H(2L(2 λ-IgAs and κ-IgAs against different antigens forming many different variants of H(4L(4SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning

  7. Self-tuning and the derivation of the Fab Four

    CERN Document Server

    Charmousis, Christos; Padilla, Antonio; Saffin, Paul M

    2011-01-01

    We have recently proposed a special class of scalar tensor theories known as the Fab Four. These arose from attempts to analyse the cosmological constant problem within the context of Horndeski's most general scalar tensor theory. The Fab Four together give rise to a model of self-tuning, with the relevant solutions evading Weinberg's no-go theorem by relaxing the condition of Poincare invariance in the scalar sector. The Fab Four are made up of four geometric terms in the action with each term containing a free potential function of the scalar field. In this paper we rigorously derive this model from the general model of Horndeski, proving that the Fab Four represents the only classical scalar tensor theory of this type that has any hope of tackling the cosmological constant problem. We present the full equations of motion for this theory, and give an heuristic argument to suggest that one might be able to keep radiative corrections under control. We also give the Fab Four in terms of the potentials presente...

  8. Independent evolution of Fc- and Fab-mediated HIV-1-specific antiviral antibody activity following acute infection

    Science.gov (United States)

    Dugast, Anne-Sophie; Stamatatos, Leonidas; Tonelli, Andrew; Suscovich, Todd J.; Licht, Anna F.; Mikell, Iliyana; Ackerman, Margaret E.; Streeck, Hendrik; Klasse, P.J.; Moore, John P.; Alter, Galit

    2014-01-01

    Fc-related antibody activities, such as antibody-dependent cellular cytotoxicity (ADCC), or more broadly, antibody-mediated cellular viral inhibition (ADCVI), play a role in curbing early SIV viral replication, are enriched in human long-term infected non-progressors, and could potentially contribute to protection from infection. However, little is known about the mechanism by which such humoral immune responses are naturally induced following infection. Here we focused on the early evolution of the functional antibody response, largely driven by the Fc portion of the antibody, in the context of the evolving binding and neutralizing antibody response, which is driven mainly by the antibody binding fragment (Fab). We show that ADCVI/ADCC-inducing responses in humans are rapidly generated following acute HIV-1 infection, peak at approximately 6 months post-infection, but decay rapidly in the setting of persistent immune activation, as Fab-related activities persistently increase. Moreover, the loss of Fc activity occurred in synchrony with a loss of HIV-specific IgG3 responses. Our data strongly suggest that Fc- and Fab-related antibody functions are modulated in a distinct manner following acute HIV infection. Vaccination strategies intended to optimally induce both sets of antiviral antibody activities may, therefore, require a fine-tuning of the inflammatory response. PMID:25043633

  9. Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody.

    Science.gov (United States)

    Cignetto, Simona; Modica, Chiara; Chiriaco, Cristina; Fontani, Lara; Milla, Paola; Michieli, Paolo; Comoglio, Paolo M; Vigna, Elisa

    2016-06-01

    The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors.

  10. Enterococcus faecalis endocarditis severity in rabbits is reduced by IgG Fabs interfering with aggregation substance.

    Directory of Open Access Journals (Sweden)

    Patrick M Schlievert

    Full Text Available BACKGROUND: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue. Our previous research determined that enterococcal aggregation substance (AS is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS+ E. faecalis endocarditis. METHODOLOGY/PRINCIPAL FINDINGS: When challenged with AS+ E. faecalis, 10 rabbits actively immunized against AS+ E. faecalis developed more significant vegetations than 9 animals immunized against AS⁻E. faecalis, and 9/10 succumbed compared to 2/9 (p<0.005, suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group immediately before challenge with AS+E. faecalis, reduced total vegetation (endocarditis lesion microbial counts (7.9 x 10⁶ versus 2.0 x 10⁵, p = 0.02 and size (40 mg versus 10, p = 0.05. In vitro, the Fabs prevented enterococcal aggregation. CONCLUSIONS/SIGNIFICANCE: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS+E. faecalis illness.

  11. Generation, characterization, and docking studies of DNA-hydrolyzing recombinant F(ab) antibodies.

    Science.gov (United States)

    Zein, Haggag S; El-Sehemy, Ahmed A; Fares, Mohamed O; ElHefnawi, Mahmoud; da Silva, Jaime A Teixeira; Miyatake, Kazutaka

    2011-01-01

    Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs.

  12. 20 CFR 30.316 - How does the FAB issue a final decision on a claim?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final...

  13. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  14. Thermodynamic stability and flexibility characteristics of antibody fragment complexes.

    Science.gov (United States)

    Li, Tong; Verma, Deeptak; Tracka, Malgorzata B; Casas-Finet, Jose; Livesay, Dennis R; Jacobs, Donald J

    2014-01-01

    Free energy landscapes, backbone flexibility and residue-residue couplings for being co-rigid or co-flexible are calculated from the minimal Distance Constraint Model (mDCM) on an exploratory dataset consisting of VL, scFv and Fab antibody fragments. Experimental heat capacity curves are reproduced markedly well, and an analysis of quantitative stability/flexibility relationships (QSFR) is applied to a representative VL domain and several complexes in the scFv and Fab forms. Global flexibility in the denatured ensemble typically decreases in the larger complexes due to domain-domain interfaces. Slight decreases in global flexibility also occur in the native state of the larger fragments, but with a concurrent large increase in correlated flexibility. Typically, a VL fragment has more co-rigid residue pairs when isolated compared to the scFv and Fab forms, where correlated flexibility appears upon complex formation. This context dependence on residue- residue couplings in the VL domain across length scales of a complex is consistent with the evolutionary hypothesis of antibody maturation. In comparing two scFv mutants with similar thermodynamic stability, local and long-ranged changes in backbone flexibility are observed. In the case of anti-p24 HIV-1 Fab, a variety of QSFR metrics were found to be atypical, which includes comparatively greater co-flexibility in the VH domain and less co-flexibility in the VL domain. Interestingly, this fragment is the only example of a polyspecific antibody in our dataset. Finally, the mDCM method is extended to cases where thermodynamic data is incomplete, enabling high throughput QSFR studies on large numbers of antibody fragments and their complexes.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial...... 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...

  16. Pollution prevention opportunity assessment for MicroFab and SiFab facilities at Sandia National Laboratories.

    Energy Technology Data Exchange (ETDEWEB)

    Gerard, Morgan Evan

    2011-12-01

    This Pollution Prevention Opportunity Assessment (PPOA) was conducted for the MicroFab and SiFab facilities at Sandia National Laboratories/New Mexico in Fiscal Year 2011. The primary purpose of this PPOA is to provide recommendations to assist organizations in reducing the generation of waste and improving the efficiency of their processes and procedures. This report contains a summary of the information collected, the analyses performed, and recommended options for implementation. The Sandia National Laboratories Environmental Management System (EMS) and Pollution Prevention (P2) staff will continue to work with the organizations to implement the recommendations.

  17. Molecular Dynamics Simulation of the Crystallizable Fragment of IgG1—Insights for the Design of Fcabs

    Directory of Open Access Journals (Sweden)

    Balder Lai

    2014-01-01

    Full Text Available An interesting format in the development of therapeutic monoclonal antibodies uses the crystallizable fragment of IgG1 as starting scaffold. Engineering of its structural loops allows generation of an antigen binding site. However, this might impair the molecule’s conformational stability, which can be overcome by introducing stabilizing point mutations in the CH3 domains. These point mutations often affect the stability and unfolding behavior of both the CH2 and CH3 domains. In order to understand this cross-talk, molecular dynamics simulations of the domains of the Fc fragment of human IgG1 are reported. The structure of human IgG1-Fc obtained from X-ray crystallography is used as a starting point for simulations of the wild-type protein at two different pH values. The stabilizing effect of a single point mutation in the CH3 domain as well as the impact of the hinge region and the glycan tree structure connected to the CH2 domains is investigated. Regions of high local flexibility were identified as potential sites for engineering antigen binding sites. Obtained data are discussed with respect to the available X-ray structure of IgG1-Fc, directed evolution approaches that screen for stability and use of the scaffold IgG1-Fc in the design of antigen binding Fc proteins.

  18. Human milk IgGs contain various combinations of different antigen-binding sites resulting in multiple variants of their bispecificity.

    Directory of Open Access Journals (Sweden)

    Sergey E Sedykh

    Full Text Available In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies (Abs recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, human milk IgGs to different antigens undergo extensive half-molecule exchange. In the IgGs pool, only 33 ± 5% and 13 ± 5% of Abs contained light chains exclusively of kappa- or lambda-type, respectively, while 54 ± 10% of the IgGs contained both kappa- and lambda- light chains. All Ab preparations contained different amounts of IgGs of all four subclasses. Interestingly, lambda-IgGs contained an increased amount of IgG2 (87% and only 3-6% of each of IgG1, IgG3, and IgG4, while kappa-IgGs consisted of comparable (17-32% amounts of all IgG subtypes. Chimeric kappa-lambda-IgGs consisted of ~74% IgG1, ~16% IgG2, ~5% IgG3 and ~5% IgG4. As the result of the exchange, all IgG fractions eluted from several specific affinity sorbents under the conditions destroying strong immunocomplexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, oligosaccharides, phosphorylation of proteins, lipids, and oligosaccharides. In vitro, an addition of reduced glutathione and milk plasma to two IgG fractions with different affinity for DNA-cellulose led to a transition of 25-60% of Ab of one fraction to the other fraction. Our data are indicative of the possibility of half-molecule exchange between milk IgGs of various subclasses, raised against different antigens (including abzymes, which explains the polyspecificity and cross-reactivity of these IgGs.

  19. Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression.

    Science.gov (United States)

    Miralem, Tihomir; Gibbs, Peter E M; Revert, Fernando; Saus, Juan; Maines, Mahin D

    2010-04-23

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-kappaB show the hBVR role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the (281)CX(10)C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC(281), corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

  20. Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis.

    Science.gov (United States)

    Li, Meng; Meng, Qiu; Fu, Huihui; Luo, Qixia; Gao, Haichun

    2016-11-15

    As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis In fabB(+) or desA(+) (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1 There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.

  1. 20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

    Science.gov (United States)

    2010-04-01

    ... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of...

  2. Quantum fragmentation

    CERN Document Server

    Peschanski, R

    1993-01-01

    Phenomenological and theoretical aspects of fragmentation for elementary particles (resp. nuclei) are discussed. It is shown that some concepts of classical fragmentation remain relevant in a microscopic framework, exhibiting non-trivial properties of quantum relativistic field theory (resp. lattice percolation). Email contact: pesch@amoco.saclay.cea.fr

  3. Safety, pharmacokinetic and dosimetry evaluation of the proposed thrombus imaging agent {sup 99m}Tc-DI-DD-3B6/22-80B3 Fab'

    Energy Technology Data Exchange (ETDEWEB)

    Macfarlane, David J. [Royal Brisbane and Women' s Hospital, Department of Nuclear Medicine, Brisbane (Australia); Smart, Richard C. [St George Hospital, Department of Nuclear Medicine, Sydney (Australia); Tsui, Wendy W. [St George Hospital, Department of Nuclear Medicine, Sydney (Australia); University of New South Wales, School of Medicine, Sydney (Australia); Gerometta, Michael [AGEN Biomedical Limited, Research and Development, Brisbane (Australia); Eisenberg, Paul R. [Eli Lilly Company, Lilly Research Laboratories, Indianapolis (United States); Scott, Andrew M. [Austin Health, Centre for PET, Melbourne (Australia); Ludwig Institute for Cancer Research, Melbourne (Australia)

    2006-06-15

    {sup 99m}Tc-DI-DD-3B6/22-80B3 (Thromboview, hereafter abbreviated to {sup 99m}Tc-DI-80B3 Fab') is a humanised, radiolabelled monoclonal antibody Fab' fragment with high affinity and specificity for the D-dimer domain of cross-linked fibrin. The purpose of this study was to evaluate the safety, pharmacokinetics and dosimetry of four increasing doses of {sup 99m}Tc-DI-80B3 Fab' in healthy volunteers. Thirty-two healthy volunteers (18-70 years; 16 male, 16 female) received a single intravenous injection of 0.5, 1.0, 2.0 or 4.0 mg of {sup 99m}Tc-DI-80B3 Fab'. Safety outcomes (vital signs, electrocardiography, haematology, biochemistry, adverse events and development of human anti-human antibodies) were assessed up to 30 days post injection. Blood and urine samples were collected up to 48 h post injection. Gamma camera images were acquired at 0.5, 1, 2, 4, 6 and 24 h post injection. Dosimetry was performed using standard MIRD methodology. No adverse events considered to be drug related were observed. Human anti-human antibody was not detectable in any subject during the follow-up period. {sup 99m}Tc-DI-80B3 Fab' had a rapid initial plasma clearance (t{sub 1/2}{alpha}=1 h). The pharmacokinetic profile of the Fab' fragment was generally linear across the four dose cohorts. By 24 h, 30-35% of the administered radioactivity appeared in the urine. There was marked renal accumulation with time, but no specific uptake was identified within other normal tissues. The effective dose was 9 mSv/750 MBq. (orig.)

  4. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    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...... and the Party-state, I suggest the notion of integrated fragmentation....

  5. Characterization of molecular mechanisms controlling fabAB transcription in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Herbert P Schweizer

    Full Text Available BACKGROUND: The FabAB pathway is one of the unsaturated fatty acid (UFA synthesis pathways for Pseudomonas aeruginosa. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt fabA upstream sequence, which is conserved in P. aeruginosa, P. putida, and P. syringae, led to a significant decrease in fabA transcription, suggesting positive regulation by an unknown positive regulatory mechanism. METHODS/PRINCIPAL FINDINGS: Here, genetic and biochemical approaches were employed to identify a potential fabAB activator. Deletion of candidate genes such as PA1611 or PA1627 was performed to determine if any of these gene products act as a fabAB activator. However, none of these genes were involved in the regulation of fabAB transcription. Use of mariner-based random mutagenesis to screen for fabA activator(s showed that several genes encoding unknown functions, rpoN and DesA may be involved in fabA regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of fabAB operon. CONCLUSION/SIGNIFICANCE: The data suggest that fabA expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism.

  6. Multidisciplinary Analysis of Cyclophilin A Function in Human Breast Cancer

    Science.gov (United States)

    2010-03-01

    diagram of synthetic antibody production, which involves target protein immobilization, phase display library screening , and structural and functional...proteins, phase display library screening , and functional analysis of selected antigen-binding fragments (FABs) (Figure 4A). In brief, recombinant his...display library screening results in the generation of total 18 CypA- specific FABs including CsA sensitive (#1-11) and non CsA-sensitive (#12-18). A free

  7. Spherical Cows in the Sky with Fab Four

    CERN Document Server

    Kaloper, Nemanja

    2013-01-01

    We explore spherically symmetric static solutions in a subclass of unitary scalar-tensor theories of gravity, called the `Fab Four' models. The weak field large distance solutions may be phenomenologically viable, but only if the Gauss-Bonnet term is negligible. Only in this limit will the Vainshtein mechanism work consistently. Further, classical constraints and unitarity bounds constrain the models quite tightly. Nevertheless, in the limits where the range of individual terms at large scales is respectively Kinetic Braiding, Horndeski, and Gauss-Bonnet, the horizon scale effects may occur while the theory satisfies Solar system constraints and, marginally, unitarity bounds. On the other hand, to bring the cutoff down to below a millimeter constrains all the couplings scales such that `Fab Fours' can't be heard outside of the Solar system.

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

    Science.gov (United States)

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

    2016-12-01

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

  9. Strategy optimization for mask rule check in wafer fab

    Science.gov (United States)

    Yang, Chuen Huei; Lin, Shaina; Lin, Roger; Wang, Alice; Lee, Rachel; Deng, Erwin

    2015-07-01

    Photolithography process is getting more and more sophisticated for wafer production following Moore's law. Therefore, for wafer fab, consolidated and close cooperation with mask house is a key to achieve silicon wafer success. However, generally speaking, it is not easy to preserve such partnership because many engineering efforts and frequent communication are indispensable. The inattentive connection is obvious in mask rule check (MRC). Mask houses will do their own MRC at job deck stage, but the checking is only for identification of mask process limitation including writing, etching, inspection, metrology, etc. No further checking in terms of wafer process concerned mask data errors will be implemented after data files of whole mask are composed in mask house. There are still many potential data errors even post-OPC verification has been done for main circuits. What mentioned here are the kinds of errors which will only occur as main circuits combined with frame and dummy patterns to form whole reticle. Therefore, strategy optimization is on-going in UMC to evaluate MRC especially for wafer fab concerned errors. The prerequisite is that no impact on mask delivery cycle time even adding this extra checking. A full-mask checking based on job deck in gds or oasis format is necessary in order to secure acceptable run time. Form of the summarized error report generated by this checking is also crucial because user friendly interface will shorten engineers' judgment time to release mask for writing. This paper will survey the key factors of MRC in wafer fab.

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Magma Fragmentation

    Science.gov (United States)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmatic volatiles, decompression of low-viscosity magma leads to rapid expansion. The magma is torn into fragments, as it is stretched into hydrodynamically unstable sheets and filaments. If the magma is highly viscous, resistance to bubble growth will instead lead to excess gas pressure and the magma will deform viscoelastically by fracturing like a glassy solid, resulting in the formation of a violently expanding gas-pyroclast mixture. In either case, fragmentation represents the conversion of potential energy into the surface energy of the newly created fragments and the kinetic energy of the expanding gas-pyroclast mixture. If magma comes into contact with external water, the conversion of thermal energy will vaporize water and quench magma at the melt-water interface, thus creating dynamic stresses that cause fragmentation and the release of kinetic energy. Lastly, shear deformation of highly viscous magma may cause brittle fractures and release seismic energy.

  12. Framing Fragmentation

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte

    2009-01-01

    , contain distinctive architectural traits, not only based on rational repetition, but also supporting composition and montage as dynamic concepts. Prefab architecture is an architecture of fragmentation, individualization and changeability, and this sets up new challenges for the architect. This paper...... into separate parts or systems: skeleton, skin, services, internal cladding, etc. Each building part/system is being conceived, produced, delivered and maintained by different construction companies. Basically the building is being fragmented into separate parts living their separate lives. The architect has...... 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...

  13. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  14. The role of indium-111 antimyosin (Fab) imaging as a noninvasive surveillance method of human heart transplant rejection

    Energy Technology Data Exchange (ETDEWEB)

    De Nardo, D.; Scibilia, G.; Macchiarelli, A.G.; Cassisi, A.; Tonelli, E.; Papalia, U.; Gallo, P.; Antolini, M.; Pitucco, G.; Reale, A. (Universita degli Studi di Roma I La Sapienza Policlinico Umberto I (Italy))

    1989-09-01

    The identification of rejection after heart transplantation in patients receiving cyclosporine immunosuppressive therapy requires the endomyocardial biopsy, an invasive method associated with a finite morbidity. To evaluate the role of indium-111 antimyosin (Fab) scintigraphy as a noninvasive surveillance method of heart transplant rejection, the Fab fragment of murine monoclonal antimyosin antibodies labeled with indium-111 was administered intravenously in 30 scintigraphic studies to 10 consecutive heart transplant recipients. Endomyocardial biopsy specimens were obtained 72 hours after each scintigraphic study. Nineteen scintigraphic studies had negative findings; no false negative finding was obtained. Eleven antimyosin scintigraphic studies had positive findings, and in these studies endomyocardial biopsy revealed mild rejection in two cases, moderate acute rejection with myocyte necrosis in two cases, myocyte necrosis as a consequence of ischemic injury in six cases, and possibly cytotoxic damage in one case. Antimyosin scintigraphy may represent a reliable screening method for the surveillance of heart transplant patients. In the presence of a negative finding from antimyosin scintigraphy, it may be possible to avoid endomyocardial biopsy. Conversely, in patients who have a positive finding from antimyosin scintigraphy, the endomyocardial biopsy is mandatory to establish the definitive diagnosis by histologic examination of the myocardium.

  15. Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097.

    Science.gov (United States)

    Boschert, Verena; Muth, Eva Maria; Knappik, Achim; Frisch, Christian; Mueller, Thomas D

    2015-04-01

    The secreted cystine-knot protein sclerostin was first identified from genetic screening of patients suffering from the rare bone-overgrowth diseases sclerosteosis and van Buchem disease. Sclerostin acts a negative regulator of bone growth through inhibiting the canonical Wnt signalling cascade by binding to and blocking the Wnt co-receptor LRP5/6. Its function in blocking osteoblastogenesis makes it an important target for osteoanabolic therapy approaches to treat osteoporosis, which is characterized by a progressive decrease in bone mass and density. In this work, the production, crystallization and preliminary X-ray diffraction data analysis of a sclerostin-neutralizing human Fab antibody fragment, AbD09097, obtained from a naive antibody library are reported. Crystals of the Fab AbD09097 belonged to space group P21, with unit-cell parameters a = 45.19, b = 78.49, c = 59.20 Å, β = 95.71° and diffracted X-rays to a resolution of 1.8 Å.

  16. Apoptosis function on tumor cell by SEA-Fab' coupled protein%SWA-Fab'对肿瘤细胞的凋亡作用

    Institute of Scientific and Technical Information of China (English)

    舒晓刚; 王国斌

    2005-01-01

    目的研究SEA-Fab'对肿瘤细胞的凋亡作用及单克隆抗体于肿瘤定向治疗作用.方法实验分为三组:SEA-Fab'、SEA及对照组,三组肿瘤细胞分别用SEA-Fab'、SEA及PBMC+Walker-256细胞处理,在24~72 h内观察肿瘤细胞的凋亡情况.结果SEA-Fab'组、SEA组的肿瘤细胞指数分别为(34.6%~68.9%)和(15.5%~31.9%)高于对照组(5.5%~12.5%),差异存在显著性,SEA-Fab'组高于SEA组差异显著.结论SEA-Fab'在激活T细胞和杀死肿瘤细胞效果好于SEA,将来有可能利用单克隆抗体来作定向靶位治疗.%[Objective] To Study the anti-tumor effect of the SEA-Fab' coupled protein and the possibility of themonoclonal antibody Fab's targeted in immunotherapy of human tumor. [Methods] The experiment group was treat-ed by SEA-Fab' and SEA, the contrast group was treated by PBMC+Walker-256 cell. The apoptotic index was ob-served in 24~72 h. [Results] The poptotic index was significantly higher in the SEA-Fab's (34.6~68.9%) and SEAgroups (15.5~31.9%) compared with the PBMC+Wallker-256 cell group (5.5~12.8%). There was significant differ-ence between SEA-Fab group and SEA group (P<0.01). [Conclusion] SEA-Fab' is more effective to activate Tcells and kill tumor cell than SEA, there is ossibility of The monoclonal antibody targeted in immunotherapy of hu-man tumor.

  17. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    OpenAIRE

    Jeffrey D. Nanson; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II f...

  18. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Haustraete Jurgen

    2009-08-01

    Full Text Available Abstract Background Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. Results Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. Conclusion Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression

  19. Specific polyclonal F(ab')2 neutralize a large panel of highly pathogenic avian influenza A viruses (H5N1) and control infection in mice.

    Science.gov (United States)

    Herbreteau, Cécile Hélène; Jacquot, Frédéric; Rith, Sareth; Vacher, Laurent; Nguyen, Ludovic; Carbonnelle, Caroline; Lotteau, Vincent; Jolivet, Michel; Raoul, Hervé; Buchy, Philippe; Saluzzo, Jean-François

    2014-01-01

    There is still no specific therapy for infection with the highly pathogenic avian influenza A virus (HPAI) H5N1, which caused 39 human cases with a 64% fatality rate in 2013. We prepared highly purified specific equine polyclonal immunoglobulin fragments (F(ab')2) against H5N1 and tested them for efficacy in vitro and with different administration schedules in H5N1-challenged BALB/c mice. in vitro, F(ab')2 neutralized 21 different H5N1 strains from different areas, representative of 11 different clades and sub-clades and 9 years of evolution of the virus. In vivo mouse experiments identified that the most efficient administration protocol consists of five consecutive daily injections after infection; 10 mg/kg giving a 60% increase in survival. These data demonstrate the ability of anti-H5N1 F(ab')2 to markedly reduce the mortality and morbidity associated with infection of mice with HPAI H5N1 virus, and their potential for human therapy.

  20. Functionally Approached Body (FAB) Strategies for Young Children Who Have Behavioral and Sensory Processing Challenges

    Science.gov (United States)

    Pagano, John

    2005-01-01

    Functionally Approached Body (FAB) Strategies offer a clinical approach to help parents of young children with behavioral and sensory processing strategies. This article introduces the FAB Strategies, clinical strategies developed by the author for understanding and addressing young children's behavioral and sensory processing challenges. The FAB…

  1. Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antimyosin Fab

    Energy Technology Data Exchange (ETDEWEB)

    Addonizio, L.J.; Michler, R.E.; Marboe, C.; Esser, P.E.; Johnson, L.L.; Seldin, D.W.; Gersony, W.M.; Alderson, P.O.; Rose, E.A.; Cannon, P.J.

    1987-03-01

    The acute rejection of cardiac allografts is currently diagnosed by the presence of myocyte necrosis on endomyocardial biopsy. We evaluated the efficacy of noninvasive scintigraphic imaging with indium-111-labeled anticardiac myosin Fab fragments (indium-111 antimyosin) to detect and quantify cardiac allograft rejection. Six dogs that had intrathoracic heterotopic cardiac allograft transplantation were injected with indium-111 antimyosin and planar and single photon emission computed tomographic (SPECT) images were obtained in various stages of acute and subacute rejection. Four dogs had an allograft older than 8 months and had been on long-term immunosuppressive therapy; two dogs had an allograft less than 2 weeks old and were not on immunosuppressive therapy. Count ratios comparing heterotopic with native hearts were calculated from both SPECT images and in vitro scans of excised and sectioned hearts and were compared with the degree of rejection scored by an independent histopathologic review. Indium-111 antimyosin uptake was not visible in planar or SPECT images of native hearts. Faint diffuse uptake was apparent in cardiac allografts during long-term immunosuppression and intense radioactivity was present in hearts with electrocardiographic evidence of rejection. The heterotopic to native heart count ratios in SPECT images correlated significantly with the count ratios in the excised hearts (r = 0.93) and with the histopathologic rejection score (r = 0.97). The distribution of indium-111 antimyosin activity in right and left ventricles corresponded to areas of histopathologic abnormalities.

  2. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  3. The Emerging Importance of IgG Fab Glycosylation in Immunity.

    Science.gov (United States)

    van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

    2016-02-15

    Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

  4. Preparation and development of equine hyperimmune globulin F(ab')2 against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    Jia-hai LU; Bing L WONG; Nan-shan ZHONG; Zhong-min GUO; Wen-yu HAN; Guo-ling WANG; Ding-mei ZHANG; Yi-fei WANG; Sheng-yun SUN; Qin-he YANG; Huan-ying ZHENG

    2005-01-01

    Aim: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood,and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. Methods: SARS coronavirus (CoV)F69 (AY313906)and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab')2 fragments. Results: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab')2 was obtained from 1 litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. Conclusion: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.

  5. 75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

    Science.gov (United States)

    2010-04-23

    ... Employment and Training Administration Intel Corporation, Fab 20 Division, Including On-Site Leased Workers... for Worker Adjustment Assistance on March 10, 2010, applicable to workers of Intel Corporation, Fab 20... the Hillsboro, Oregon location of Intel Corporation, Fab 20 Division. The Department has...

  6. 20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316,...

  7. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    Science.gov (United States)

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  8. Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

    Science.gov (United States)

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R K; Burger, Christa; Goodman, Simon L; Arnaout, M Amin

    2014-05-16

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

  9. Bespoke Fragments

    DEFF Research Database (Denmark)

    Kruse Aagaard, Anders

    2016-01-01

    The Ph.D. -project Bespoke Fragments seeks to explore and utilise the space emerging between the potentials of digital drawing and fabrication and the field of materials and their properties and capacities. Within this span, the project is situated in a shuttling between the virtual and the actual......, the emergence of virtual space is no longer limited to the computer's digital world, but extends into the materials' world. Creation and uncertainty are allowed as virtual parameters in both the digital and reality. Based on this notion the project suggests utilising that exact potential to develop...

  10. sup 111 In-antimyosin Fab scintigraphy in cardiovascular diseases; Multicenter clinical trial

    Energy Technology Data Exchange (ETDEWEB)

    Kawai, Chuichi; Matsumori, Akira; Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine); Nishimura, Tsunehiko

    1990-12-01

    In a multicenter study, a total of 380 patients with myocardial infarction, myocarditis and cardiomyopathy underwent {sup 111}In-antimyosin Fab myocardial imaging. {sup 111}In-antimyosin Fab was administered intravenously and myocardial images were obtained 48 hours later. Only 3 patients developed mild adverse effects. Human antimouse antibodies were detected in 7 patients. Positive scans in patients with myocardial infarction were seen in 92/119 (77%) within 2 weeks after the onset of myocardial infarction, in 58/71 (82%) at 3-4 weeks, in 20/22 (91%) at 4-8 weeks and 17/31 (55%) thereafter. The location of myocardial damage delineated by {sup 111}In-antimyosin Fab imaging was concordant with the infarct location by ECG and coronary angiography. In patients with myocarditis, {sup 111}In-antimyosin Fab uptake was positive in 7/12 (58%) within 8 weeks and 6/17 (35%) thereafter. Positive {sup 111}In-antimyosin Fab scans were seen in 12/36 (33%) in dilated cardiomyopathy and in 17/19 (89%) in hypertrophic cardiomyopathy. Although the mechanism of persistently positive {sup 111}In-antimyosin Fab images in the subacute to chronic stage of myocardial infarction and myocarditis remains to be clarified, {sup 111}In-antimyosin Fab may be useful for the detection of the disease and in evaluating the prognosis of patients with cardiomyopathy. (author).

  11. The community FabLab platform: applications and implications in biomedical engineering.

    Science.gov (United States)

    Stephenson, Makeda K; Dow, Douglas E

    2014-01-01

    Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

  12. Hepatic targeting and hypocholesterolemic effect of lactosaminated Fab against low density lipoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Bernini, F.; Bocan, T.M.A.; Via, D.P.; Gotto, A.M. Jr.; Smith, L.C.

    1986-03-01

    Lactosaminated Fab (lac-Fab) specific for human LDL induces plasma clearance and uptake of circulating (/sup 125/-I)-iodo-LDL in rat, a process mediated by galactose receptors of the liver. This study demonstrates that lac-Fab is a specific carrier of LDL to the liver parenchymal cells and exhibits hypocholesterolemic activity in vivo. Rats were injected with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-LDL (diI-LDL) or 6 mg of LDL plus tracer amounts of (/sup 125/I)-iodo-LDL. After 10-20 min, the animals received 3-10 mg of lac-Fab. Histologic examination of the liver sections showed the uptake of diI-LDL in the parenchymal cells, as compared to diI-acetyl-LDL which was localized in sinusoidal cells. More than 85% of human LDL disappeared within 2.5 hr after lac-Fab injection, reducing plasma cholesterol from 133.0 +/- 12.6 mg/dl to 66.4 +/- 8.0 mg/dl, the basal value in the rat. In control rats, only about 20% of radioactivity and cholesterol disappeared at 2.5 hr. HDL levels were unaffected. The authors conclude that lac-Fab is a specific carrier of LDL to hepatocytes and can lower plasma LDL-cholesterol in vivo. Lac-Fab specific for other antigens may act as specific carriers of molecule or macromolecules to hepatocytes.

  13. 20 CFR 30.318 - Can the FAB consider objections to HHS's reconstruction of a radiation dose or to the guidelines...

    Science.gov (United States)

    2010-04-01

    ... at 42 CFR part 82, is binding on the FAB. The FAB reviewer may determine, however, that objections... CFR part 81, is also binding on the FAB (see § 30.213). However, since OWCP applies this methodology... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB consider objections to...

  14. Meleagrin, a new FabI inhibitor from Penicillium chryosogenum with at least one additional mode of action.

    Directory of Open Access Journals (Sweden)

    Chang Ji Zheng

    Full Text Available Bacterial enoyl-acyl carrier protein reductase (FabI is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.

  15. Direct-write scanning probe lithography: towards a desktop fab

    Science.gov (United States)

    Giam, Louise R.; Senesi, Andrew J.; Liao, Xing; Wong, Lu Shin; Chai, Jinan; Eichelsdoerfer, Daniel J.; Shim, Wooyoung; Rasin, Boris; He, Shu; Mirkin, Chad A.

    2011-06-01

    Massively parallel scanning-probe based methods have been used to address the challenges of nanometer to millimeter scale printing for a variety of materials and mark a step towards the realization of a "desktop fab." Such tools enable simple, flexible, high-throughput, and low-cost nano- and microscale patterning, which allow researchers to rapidly synthesize and study systems ranging from nanoparticle synthesis to biological processes. We have developed a novel scanning probe-based cantilever-free printing method termed polymer pen lithography (PPL), which uses an array of elastomeric tips to transfer materials (e.g. alkanethiols, proteins, polymers) in a direct-write manner onto a variety of surfaces. This technique takes the best attributes of dip-pen nanolithography (DPN) and eliminates many of the disadvantages of contact printing. Various related techniques such as beam pen lithography (BPL), scanning probe block copolymer lithography (SPBCL), and hard-tip, soft spring lithography (HSL) are also discussed.

  16. Integrated fab process for metal oxide EUV photoresist

    Science.gov (United States)

    Grenville, Andrew; Anderson, Jeremy T.; Clark, Benjamin L.; De Schepper, Peter; Edson, Joseph; Greer, Michael; Jiang, Kai; Kocsis, Michael; Meyers, Stephen T.; Stowers, Jason K.; Telecky, Alan J.; De Simone, Danilo; Vandenberghe, Geert

    2015-03-01

    Inpria is developing directly patternable, metal oxide hardmasks as robust, high-resolution photoresists for EUV lithography. Targeted formulations have achieved 13nm half-pitch at 35 mJ/cm2 on an ASML's NXE:3300B scanner. Inpria's second-generation materials have an absorbance of 20/μm, thereby enabling an equivalent photon shot noise compared to conventional resists at a dose lower by a factor of 4X. These photoresists have ~40:1 etch selectivity into a typical carbon underlayer, so ultrathin 20nm films are possible, mitigating pattern collapse. In addition to lithographic performance, we review progress in parallel advances required to enable the transition from lab to fab for such a metal oxide photoresist. This includes considerations and data related to: solvent compatibility, metals cross-contamination, coat uniformity, stability, outgassing, and rework.

  17. 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.; Graaf, W.T. van der; Oyen, W.J.G.; Boerman, O.C.

    2012-01-01

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

  18. Measurement of infarct size and percentage myocardium infarcted in a dog preparation with single photon-emission computed tomography, thallium-201, and indium 111-monoclonal antimyosin Fab

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, L.L.; Lerrick, K.S.; Coromilas, J.; Seldin, D.W.; Esser, P.D.; Zimmerman, J.M.; Keller, A.M.; Alderson, P.O.; Bigger, J.T. Jr.; Cannon, P.J.

    1987-07-01

    Single photon-emission tomography (SPECT) and indium 111-labeled monoclonal antimyosin Fab fragments were used to measure myocardial infarct size in 12 dogs, six subjected to balloon catheter-induced coronary artery occlusion for 6 hr (late reperfusion) and six subjected to occlusion with reperfusion at 2 hr (early reperfusion). Tomographic imaging was performed 24 hr after the intravenous injection of labeled Fab fragments with the use of a dual-head SPECT camera with medium-energy collimators. Immediately after the first tomographic scan, thallium-201 was injected into nine of 12 dogs and imaging was repeated. Estimated infarct size in grams was calculated from transaxially reconstructed, normalized, and background-corrected indium SPECT images with the use of a threshold technique for edge detection. Estimated noninfarcted myocardium in grams was calculated from obliquely reconstructed thallium SPECT images by a similar method. The animals were killed and infarct size in grams and true infarct size as a percentage of total left ventricular myocardial volume were measured by triphenyl tetrazolium chloride staining. Estimated infarct size from indium SPECT images showed an excellent correlation with true infarct size (r = .95, SEE = 4.1 g). Estimated percentage myocardium infarcted was calculated by dividing estimated infarct size from indium images by the sum of estimated infarct size plus estimated noninfarcted myocardium obtained from thallium images. Correlation between the estimated percentage of myocardium infarcted and true percentage of myocardium infarcted was excellent.

  19. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

    Science.gov (United States)

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J

    2006-01-01

    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  20. Establishment of bacteria display technology for Fab antibody library screening%筛选Fab抗体库的细菌展示技术的建立

    Institute of Scientific and Technical Information of China (English)

    徐黎明; 尹成凯; 任桂萍; 田辉; 王雪苯; 丁良君; 李德山

    2011-01-01

    fragments and the light chains of the anti-human IL-1β (hIL-1β) antibody were inserted downstream of the NlpA leader and pelB leader respectively to construct the pBFD-Fab for Fab antibody display. Then pBFD-Fab transformed E. Coli DH5a was induced by IPTG to express the Fab antibodies, as detected by flow cytometry ( FCM), and positive populations were sorted. Instead of PCR, plasmids were extracted for rescue purpose. The rescue plasmids were retransformed to E. Coli DH5a and FCM was performed again. RESULTS: The pBFD-Fab-transformed bacteria were incubated with antigen and antigen specific FITC-antibody, and showed strong fluorescence as detected by FCM in a dose-dependent manner. The rescued pBFD-Fab displayed similar fluorescence intensity, indicating the reliability of this technology. CONCLU-SION: The Fab expressed by the bacterial display system folds efficiently and binds to hIL-ip specifically. The plasmid rescue works well and it can avoid mutation and mis-pairing chains. This bacterial display technology has the stability of antibody expression. This study has used the technology to screen anti-hlL-ip Fab antibody Library successfully.

  1. SpiderFab: Architecture for On-Orbit Construction of Kilometer-Scale Apertures Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The SpiderFab effort has investigated the value proposition and technical feasibility of radically changing the way we build and deploy spacecraft in order to escape...

  2. Preparation and Purification of IgY and Fab' Against Human Rotavirus%抗轮状病毒IgY和Fab'的分离与纯化

    Institute of Scientific and Technical Information of China (English)

    孙淑清; 段春燕; 胡彦涛; 孟岩

    2006-01-01

    抗轮状病毒IgY可用两步盐析结合凝胶过滤从蛋黄中分离出来,用SDS-PAGE检测其纯度可达到95%以上.纯的IgY经胃蛋白酶分解得到的抗体片断(Fab'),经SDS-PAGE和MALDI质谱法测定,其纯度达到99%以上.结果表明,所设计的分离抗轮状病毒IgY和Fab'的方法简单、有效.经ELISA法检测,Fab'的活性仍保持在IgY原始活性的70%以上.

  3. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans

    2008-08-01

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

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

    Science.gov (United States)

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

    2007-12-01

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

  5. Structural Basis for Enhanced HIV-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12

    Directory of Open Access Journals (Sweden)

    Yunji Wu

    2013-12-01

    Full Text Available The human immunoglobulin G (IgG 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs are intramolecularly domain exchanged, resulting in a rigid (Fab2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.

  6. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

    OpenAIRE

    Bondt, Albert; Wuhrer, Manfred; Kuijper, Martijn; Hazes, Mieke; Dolhain, Radboud

    2016-01-01

    Background Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from i...

  7. Anti-tumor Effect and Mechanism of SEA-Fab' Coupled Protein on Gastric Tumor

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The anti-tumor effect and mechanism of SEA-Fab' coupled protein on gastric tumor was studied. The target cell Walker-256 was treated with SEA-Fab' synthesized chemically or SEA respectively for 24 h, 36 h or 72 h. PBMC+Walke-256 cells served as controls. The apoptotic index of SEA-Fab' against effector cells was detected. In the mouse gastric cancer models (n=60), SEAFab', SEA and normal saline was injected in experimental group, SEA group and control group respectively. The occurrence and weight of tumor was observed. The results showed that the apoptotic index was significantly higher in the SEA-Fab' (34.6 %-68.9 %) and SEA group (15.5 %-31.9 %) than in PBMC+Walker-256 group (5.5 %-12.8 %) with the difference being significant (P<0.01). And there was significant difference between SEA-Fab' group and SEA group (P <0. 01). The tumor weight in SEA-Fab', SEA and control groups was 3. 64±0. 53 g, 0. 78±0.26 g and 0.49 ±0.17 g respectively with the difference being statistically significant between the SEAFab' group, SEA group and the control group (P<0.01). In the SEA-Fab's and SEA groups,there were CD4+ T and CD8+ T cell infiltrates, but in the cotnrol group, no or few T lymphocytes were seen in the mouse tumor tissue. It was concluded that SEA-Fab' was more effective to activate T lymphocytes to kill the tumor cells than SEA used alone. It was feasibility by using the monoclonal antibody as carrier to perform the targeted immunotherapy of gatric tumor.

  8. Functional characterization of triclosan-resistant enoyl-acyl-carrier protein reductase (FabV in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Yong-Heng Huang

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR, FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholera fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acylhomoserine lactones (AHLs production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore to the fabV mutant strain the ability to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity.

  9. Functional Characterization of Triclosan-Resistant Enoyl-acyl-carrier Protein Reductase (FabV) in Pseudomonas aeruginosa

    Science.gov (United States)

    Huang, Yong-Heng; Lin, Jin-Shui; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR), FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholerae fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acyl-homoserine lactones (AHLs) production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore the fabV mutant strain to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity. PMID:27965638

  10. Anticancer effects of combination of anti-EBV-LMP1 humanized antibody Fab and mitomycin C on nasopharyngeal carcinoma xenografts in nude mice%人源抗EB病毒潜伏膜蛋白1抗体Fab联合丝裂霉素C对鼻咽癌裸鼠移植瘤生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    毛圆; 张大为; 陈仁杰; 朱进; 冯振卿

    2011-01-01

    目的:观察联合应用人源抗EB病毒鼻咽癌潜伏膜蛋白1(latent membrane protein 1,LMP1)抗体Fab片段与丝裂霉素C(mitomycin C,MMC)对LMP1阳性鼻咽癌裸鼠移植瘤生长的抑制作用.方法:建立LMP1阳性鼻咽癌裸鼠移植瘤模型,20只成瘤裸鼠随机分成4组,即Fab组、MMC组、Fab+MMC组和对照组.腹腔注射药物,每3天1次,共5次.观察肿瘤生长、测量肿瘤体积,计算抑瘤率.免疫组化法检测肿瘤组织血管内皮生长因子(vascular endothelial growth factor,VEGF)表达、流式细胞术检测肿瘤细胞凋亡情况.结果:Fab组、MMC组和Fab+MMC组抑瘤率分别为18.90%、28.95%、49.12%.Fab组、Fab+MMC组VEGF表达量明显低于对照组(P0.05).MMC组、Fab+MMC组肿瘤细胞凋亡率分别为(11.84±1.50)%、(17.55±3.21)%,与对照组凋亡率(3.90±0.55)%相比,有显著差异(P0.05).结论:Fab及MMC均有抑瘤作用,联合使用效果优于单独使用,二者抑制肿瘤的作用途径不完全相同.Fab可能与抑制肿瘤血管生成有关,MMC可能与诱导肿瘤细胞凋亡增加有关.%Objective: To investigate the inhibitory activity of anti-latent membrane protein 1 (LMPl)antibody fragment (Fab) combined with mitomycin C (MMC) on nasopharyngeal carcinoma NPC-LMPl xenografts in nude mice. Methods:Nasopharyngeal carcinoma LMPl cells (5×lO6) were subcutaneously transplanted in to 20 nude mice, which were randomly divided into Fab group,MMC group, Fab+MMC group and control group. The mice were injected once every 3 days. After corresponding treatments for 5 times,the tumor inhibition rate was evaluated, vascular endothelial growth factor (VEGF) was measured with immunohistochemistry, and the apoptotic rates were analyzed by flow cytometry. Results:The tumor growth inhibition rates in Fab group, MMC group, Fab+MMC group were 18.90%,28.95% and 49.12%,respectively. VEGF in Fab group and Fab+MMC group significantly reduced and showed lower expression compared with control group (P

  11. Small animal PET imaging of TAG-72 expressing tumor using {sup 68}Ga-NOTA-3E8 Fab radioimmunoconjugate

    Energy Technology Data Exchange (ETDEWEB)

    Jung, J. H.; Lee, T. S.; Woo, S. K.; Woo, K. S.; Chung, W. S.; Kang, J. H.; Cheon, G. J.; Choi, C. W.; Lim, S. M. [KIRAMS, Seoul (Korea, Republic of); Hong, H. J. [KRIBB, Daejeon (Korea, Republic of)

    2009-05-15

    The tumor-associated glycoprotein TAG-72 is express ed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancer. 3E8 is anti-TAG-72 humanized antibody. Antibody fragments have some advantages such as improved pharmacokinetics and reduced immunogenicity compared to whole IgG. {sup 68}Ga is a short-lived positron emitter (t{sub 1/2} {sup 68} min; {beta}+, 88%) that is produced, independent from a cyclotron, by a {sup 68}Ge/{sup 68}Ga generator. The parent nuclide {sup 68}Ge has a long half-life (270.8 day), allowing its use as a generator for more than 1 year. A {sup 68}Ga is labeled with antibodies through bifunctional chelators, which allows possible kit formulation and which wide availability of the nuclear imaging agents. In this study, Fab fragment of anti-TAG-72 humanized Ab (3E8) was conjugated with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4, 7-triacetic acid (p-SCN-Bn-NOTA) and radiolabeled with {sup 68}Ga and acquire small animal PET image.

  12. Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes

    Science.gov (United States)

    Soares da Costa, Tatiana P.; Nanson, Jeffrey D.; Forwood, Jade K.

    2017-01-01

    Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis. PMID:28045020

  13. Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

    Science.gov (United States)

    Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

    2015-11-01

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

  14. Structural requirements of the major protective antibody to Haemophilus influenzae type b

    DEFF Research Database (Denmark)

    Hougs, L; Juul, L; Svejgaard, A

    1999-01-01

    expressed as antigen-binding fragments (Fabs) in Escherichia coli, define amino acids involved in antigen binding and idiotype expression, and propose a three-dimensional structure for the variable domains. We found that canonical Fabs, unlike a noncanonical Fab, bound effectively to HibCP in the absence......Protective antibodies to the important childhood pathogen Haemophilus influenzae type b (Hib) are directed against the capsular polysaccharide (HibCP). Most of the antibody is encoded by a well-defined set of ("canonical") immunoglobulin genes, including the Vkappa A2 gene, and expresses...... an idiotypic marker (HibId-1). In comparison to noncanonical antibodies, the canonical antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats. Using site-directed mutagenesis, we here characterize canonical HibCP antibodies...

  15. Fragmentation and Hadronization

    OpenAIRE

    Webber, B. R.

    1999-01-01

    Experimental data, theoretical ideas and models concerning jet fragmentation and the hadronization process are reviewed, concentrating on the following topics: factorization and small-x resummation of fragmentation functions, hadronization models, single-particle yields and spectra in Z decay, comparisons between quark and gluon jets, current and target fragmentation in deep inelastic scattering, heavy quark fragmentation, Bose-Einstein correlations and WW fragmentation.

  16. Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

    Science.gov (United States)

    Torán, J L; Sánchez-Pulido, L; Kremer, L; del Real, G; Valencia, A; Martínez-A, C

    2001-01-01

    We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

  17. Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

    Science.gov (United States)

    Benvenuto, Pasquale; Neves, Miguel A D; Blaszykowski, Christophe; Romaschin, Alexander; Chung, Timothy; Kim, Sa Rang; Thompson, Michael

    2015-05-19

    This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

  18. Fab Four: When John and George play gravitation and cosmology

    CERN Document Server

    Bruneton, Jean-Philippe; Kanfon, Antonin; Hees, Aurélien; Schlögel, Sandrine; Füzfa, André

    2012-01-01

    Scalar-tensor theories of gravitation have recently regained a great interest after the discovery of the Chameleon mechanism and of the Galileon models. The former allows, in principle, to reconcile the presence of cosmological scalar fields with the constraints from experiments at the Solar System scale. The latter open up the possibility of building inflationary models that, among other things, do not need ad hoc potentials. Further generalizations have finally led to the most general tensor-scalar theory, recently dubbed the "Fab Four", with only first and second order derivatives of the fields in the equations of motion and that self-tune to a vanishing cosmological constant. This model has a very rich phenomenology that needs to be explored and confronted with experimental data in order to constrain a very large parameter space. In this paper, we present some results regarding a subset of the theory named "John", which corresponds to a non-minimal derivative coupling between the scalar field and the Eins...

  19. Fab Four: When John and George Play Gravitation and Cosmology

    Directory of Open Access Journals (Sweden)

    J.-P. Bruneton

    2012-01-01

    Full Text Available Scalar-tensor theories of gravitation attract again a great interest since the discovery of the Chameleon mechanism and of the Galileon models. The former allows reconciling the presence of a scalar field with the constraints from Solar System experiments. The latter leads to inflationary models that do not need ad hoc potentials. Further generalizations lead to a tensor-scalar theory, dubbed the “Fab Four,” with only first and second order derivatives of the fields in the equations of motion that self-tune to a vanishing cosmological constant. This model needs to be confronted with experimental data in order to constrain its large parameter space. We present some results regarding a subset of this theory named “John,” which corresponds to a nonminimal derivative coupling between the scalar field and the Einstein tensor in the action. We show that this coupling gives rise to an inflationary model with very unnatural initial conditions. Thus, we include the term named “George,” namely, a nonminimal, but nonderivative, coupling between the scalar field and Ricci scalar. We find a more natural inflationary model, and, by performing a post-Newtonian analysis, we derive the set of equations that constrain the parameter space with data from experiments in the Solar System.

  20. Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV.

    Science.gov (United States)

    Khan, Raees; Lee, Myung Hwan; Joo, Hae-Jin; Jung, Yong-Hoon; Ahmad, Shabir; Choi, Jin-Hee; Lee, Seon-Woo

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

  1. RetroFab: A Design Tool for Retrofitting Physical Interfaces using Actuators, Sensors and 3D Printing

    OpenAIRE

    2016-01-01

    We present RetroFab, an end-to-end design and fabrication environment that allows non-experts to retrofit physical interfaces. Our approach allows for changing the layout and behavior of physical interfaces. Unlike customizing software interfaces, physical interfaces are often challenging to adapt because of their rigidity. With RetroFab, a new physical interface is designed that serves as a proxy interface for the legacy controls that are now operated by actuators. RetroFab makes this concep...

  2. 3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

    Science.gov (United States)

    2013-07-01

    FabI, but share low sequence identity and are poorly inhibited by triclosan.25,26 S. pneumoniae and P. aeruginosa contain FabK,24 and Vibrio cholerae ,27...Massengo-Tiasse, R. P.; Cronan, J. E. Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J. Biol. Chem. 2008, 283, 1308...of enoyl- (acyl-carrier protein) reductase, FabV, from Vibrio fischeri. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 78−80. (27

  3. Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

    Science.gov (United States)

    Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

    2016-01-01

    Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

  4. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA.

    Science.gov (United States)

    Moynié, Lucile; Hope, Anthony G; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M; Schneider, Gunter; Burkart, Michael D; Smith, Andrew D; Gray, David W; Naismith, James H

    2016-01-16

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.

  5. Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments.

    Science.gov (United States)

    Liu, Yuan; Lin, Manman; Zhang, Xifeng; Hu, Xiaodan; Lin, Jieru; Hao, Jia; He, Dan; Zhang, Xiao; Xu, Chongxin; Zhong, Jianfeng; Xie, Yajing; Zhang, Cunzheng; Liu, Xianjin

    2017-05-15

    Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10(-8) M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 μg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

  6. Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

    Science.gov (United States)

    Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad

    2015-12-01

    A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.

  7. Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

    Energy Technology Data Exchange (ETDEWEB)

    Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C., E-mail: jcwilliams@coh.org [Beckman Research Institute of City of Hope, 1710 Flower Street, Duarte, CA 91010 (United States)

    2016-05-23

    An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.

  8. Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

    Directory of Open Access Journals (Sweden)

    Justin M Scheer

    Full Text Available The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab'(2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

  9. Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

    Science.gov (United States)

    Nanson, Jeffrey D; Forwood, Jade K

    2015-01-01

    Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

  10. Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Nanson

    Full Text Available Ketoacyl-acyl carrier protein reductases (FabG are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP linked thioesters within the bacterial type II fatty acid synthesis (FASII pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG, the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

  11. Treatment of digitalis intoxication with emphasis on the clinical use of digoxin immune Fab.

    Science.gov (United States)

    Allen, N M; Dunham, G D

    1990-10-01

    Many studies and cases of digitalis intoxication have been reported since the time of William Withering's first publication in 1785. Recognition and management of digitalis toxicity is challenging. Before digoxin immune Fab was commercially available, treatment consisted of managing the signs and symptoms of toxicity until the digitalis was eliminated. Digoxin immune Fab offers a safe, effective, and specific method of quickly reversing digitalis toxicity. Factors that must be considered with the clinical use of this agent include the dosage calculation, administration technique, postdose monitoring, pharmacokinetics, mechanism of action, interference with commercially available digoxin assays, partial neutralizing dosing, rebound of free digoxin, and indications for use. For severe, life-threatening toxicity, digoxin immune Fab is the treatment of choice.

  12. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    Science.gov (United States)

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  13. 3D printing in social education: Eki-Fab and student PBL

    Science.gov (United States)

    Makino, Masato; Saito, Azusa; Kodama, Mai; Takamatsu, Kyuuichiro; Tamate, Hideaki; Sakai, Kazuyuki; Wada, Masato; Khosla, Ajit; Kawakami, Masaru; Furukawa, Hidemitsu

    2017-04-01

    Additive manufacturing or 3D printer is one of the most innovative material processing methods. We are considering that human resources for 3D printing would be needed in the future. To educate the abilities of the digital fabrication, we have the public digital fabrication space "Eki-Fab" for junior and high school students and Project Based Learning (PBL) class for undergraduate students. Eki-Fab is held on every Saturday at the Yonezawa train station. In the "Eki-Fab", anybody can study the utilizing of 3D printer and modeling technics under the instruction of staff in Yamagata University. In the PBL class, we have the class every Thursday. The students get the techniques of the digital fabrication through the PBL.

  14. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  15. 20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the...

  16. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

    Science.gov (United States)

    Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

    2016-12-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma

    Institute of Scientific and Technical Information of China (English)

    Jin-Liang Xing; Xiang-Min Yang; Xi-Ying Yao; Fei Song; Zhi-Nan Chen

    2004-01-01

    AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody.METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then,the competent E. colicells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting,ELISA and HPLC.RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg/mL. The renatured cFab could specifically bind to related antigen with high affinity.CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

  18. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    Science.gov (United States)

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property. PMID:23383298

  19. Research advances on Fabs antibodies%Fab类抗体的研究进展

    Institute of Scientific and Technical Information of China (English)

    刘美君; 高向东; 徐晨

    2014-01-01

    In genetic engineering antibodies,Fabs antibodies have many advantages,such as lower relative molecular mass, specific tissue distribution and lower immunogenicity, which have made Fabs antibodies the research hotspot for the past few decades. Besides the widely used enzymatic generation, multiple expression systems which include E. coli. and insects are used in the generation of Fabs antibodies. E. coli. expression system is most thoroughly explored and has been used in the preparation of clinical Fabs drugs. There are six Fab drugs that have been approved by FDA, such as certolizumabpegol and many Fabs are still in clinical research. This review discusses the recent research progress of Fabs drugs, including Fabs′ structure and characteristics, the generation and expression of Fabs, strategies to increase production and clinical applications, with the emphasis on generation, expression and clinical applications.%在基因工程抗体中,Fab类(Fabs)抗体有许多优势,如相对分子质量小、组织分布特异性强和免疫原性低等,使Fab类抗体成为近几十年来的研究热点。除了应用较广泛的体外酶消化法外,多种表达系统(如大肠杆菌和昆虫等)皆用于Fab类抗体的获得,其中大肠杆菌表达的系统研究最为彻底,且已经应用于制备Fab类临床药物。目前已经有赛妥珠单抗等6种Fab类抗体药物通过了美国FDA申请并上市,并有多种Fab类药物正处于临床试验阶段。本文分别对Fab类抗体的基本结构及特点、Fab类抗体的生成、表达和提高产量的策略、临床应用的研究进展进行介绍。

  20. International Foot and Ankle Biomechanics Community (i-FAB: past, present and beyond

    Directory of Open Access Journals (Sweden)

    Rosenbaum Dieter

    2009-06-01

    Full Text Available Abstract The International Foot and Ankle Biomechanics Community (i-FAB is an international collaborative activity which will have an important impact on the foot and ankle biomechanics community. It was launched on July 2nd 2007 at the foot and ankle session of the International Society of Biomechanics (ISB meeting in Taipei, Taiwan. i-FAB is driven by the desire to improve our understanding of foot and ankle biomechanics as it applies to health, disease, and the design, development and evaluation of foot and ankle surgery, and interventions such as footwear, insoles and surfaces.

  1. Anti-fouling properties of Fab’ fragments immobilized on silane-based adlayers

    Energy Technology Data Exchange (ETDEWEB)

    Crivianu-Gaita, Victor [Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6 (Canada); Romaschin, Alexander [Clinical Biochemistry, St. Michael' s Hospital, Toronto, ON M5B 1W8 (Canada); Thompson, Michael, E-mail: mikethom@chem.utoronto.ca [Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6 (Canada)

    2015-12-30

    Highlights: • Simple and mixed adlayers formed with Fab’ linker and/or spacers. • Binding of Fab’ fragments through TUBTS linker resulted in oriented immobilization. • Immobilized Fab’ fragments have inherent anti-fouling character. • Up to 80% fouling reduction when Fab’ fragments introduced to surfaces. • Used the minimally fouling surfaces to detect a cancer biomarker (PTHrP) in serum. - Graphical abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab’) in biosensors. One Fab’ linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab’ fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab’ fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab’-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection. - Abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a

  2. High quality mask storage in an advanced Logic-Fab

    Science.gov (United States)

    Jähnert, Carmen; Fritsche, Silvio

    2012-02-01

    High efficient mask logistics as well as safe and high quality mask storage are essential requirements within an advanced lithography area of a modern logic waferfab. Fast operational availability of the required masks at the exposure tool with excellent mask condition requires a safe mask handling, safeguarding of high mask quality over the whole mask usage time without any quality degradation and an intelligent mask logistics. One big challenge is the prevention of haze on high advanced phase shift masks used in a high volume production line for some thousands of 248nm or 193nm exposures. In 2008 Infineon Dresden qualified a customer specific developed semi-bare mask storage system from DMSDynamic Micro Systems in combination with a high advanced mask handling and an interconnected complex logistic system. This high-capacity mask storage system DMS M1900.22 for more than 3000 masks with fully automated mask and box handling as well as full-blown XCDA purge has been developed and adapted to the Infineon Lithotoollandscape using Nikon and SMIF reticle cases. Advanced features for ESD safety and mask security, mask tracking via RFID and interactions with the exposure tools were developed and implemented. The stocker is remote controlled by the iCADA-RSM system, ordering of the requested mask directly from the affected exposure tool allows fast access. This paper discusses the advantages and challenges for this approach as well as the practical experience gained during the implementation of the new system which improves the fab performance with respect to mask quality, security and throughput. Especially the realization of an extremely low and stable humidity level in addition with a well controlled air flow at each mask surface, preventing masks from haze degradation and particle contamination, turns out to be a notable technical achievement. The longterm stability of haze critical masks has been improved significantly. Relevant environmental parameters like

  3. AGILE integration into APC for high mix logic fab

    Science.gov (United States)

    Gatefait, M.; Lam, A.; Le Gratiet, B.; Mikolajczak, M.; Morin, V.; Chojnowski, N.; Kocsis, Z.; Smith, I.; Decaunes, J.; Ostrovsky, A.; Monget, C.

    2015-09-01

    mix logic Fab) in term of product and technology portfolio AGILE corrects for up to 120nm of product topography error on process layer with less than 50nm depth of focus Based on tool functionalities delivered by ASML and on high volume manufacturing requirement, AGILE integration is a real challenge. Regarding ST requirements "Automatic AGILE" functionality developed by ASML was not a turnkey solution and a dedicated functionality was needed. A "ST homemade AGILE integration" has been fully developed and implemented within ASML and ST constraints. This paper describes this integration in our Advanced Process Control platform (APC).

  4. 20 CFR 30.317 - Can the FAB request a further response from the claimant or return a claim to the district office?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB request a further response from....317 Can the FAB request a further response from the claimant or return a claim to the district office? At any time before the issuance of its final decision, the FAB may request that the claimant...

  5. 20 CFR 30.312 - What will the FAB do if the claimant objects to the recommended decision but does not request a...

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false What will the FAB do if the claimant objects....312 What will the FAB do if the claimant objects to the recommended decision but does not request a... period of time allotted in § 30.310 but does not request a hearing, the FAB will consider any...

  6. Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen

    Directory of Open Access Journals (Sweden)

    Fabio Selis

    2016-04-01

    Full Text Available PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

  7. Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen

    Science.gov (United States)

    Selis, Fabio; Focà, Giuseppina; Sandomenico, Annamaria; Marra, Carla; Di Mauro, Concetta; Saccani Jotti, Gloria; Scaramuzza, Silvia; Politano, Annalisa; Sanna, Riccardo; Ruvo, Menotti; Tonon, Giancarlo

    2016-01-01

    PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments. PMID:27043557

  8. Progress and improvement of the manufacturing process of snake antivenom

    Directory of Open Access Journals (Sweden)

    Zolfagharian H.

    2013-05-01

    Full Text Available Antivenoms have been used successfully for more than a century and up to now constitute the only effectivetreatment for snakebites .The production of antivenin started long time ago when the calmette was preparedthe antivenom in 1894.The method currently used to prepare antivenom by most of the manufacturers areoriginated from the method of Pope which was develop in 1938. Several new approaches in the production ofantivenom have been proposed to produce IgG, F(ab2, F(ab antivenin to improve their quality .Theseimprovement include complete or partial modification in the antivenom production regarding animal,immunization protocols , new adjuvants in hyperimmunization of animals , purification processes ( caprylicacid ,chromatography , diafiltration and ulterafiltration ,enzymatic digestion of IgG (pepsin, papain andfractionation of venom .When the IgG is digested enzymatically, different fragments are obtained depending on the enzyme used, that is, if papain is used, three fragments are obtained, the crystallizing fragment (Fc and two antigen-binding fragments F(ab and, if pepsin is used, one F(ab'2 fragment is obtained, while thecrystallizing fragment is digested. Fab and F(ab2 fragments conserve their capacity to specifically bind to the antigen that gave rise to them.

  9. Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan: kinetic analysis of mutant FabIs.

    Science.gov (United States)

    Sivaraman, Sharada; Zwahlen, Jacque; Bell, Alasdair F; Hedstrom, Lizbeth; Tonge, Peter J

    2003-04-22

    Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.

  10. Quench-condensing superconducting thin films using the Fab on a Chip approach

    Science.gov (United States)

    Han, Han; Imboden, Matthias; Del Corro, Pablo; Stark, Thomas; Lally, Richard; Pardo, Flavio; Bolle, Cristian; Bishop, David

    Micro-electromechanical systems (MEMS) being manufactured in a macroscopic fab inspires the idea of getting the process further down to fabricate even smaller structures, namely nano-structures, using MEMS. The Fab on a Chip concept was proposed based on such ideas. By implementing the final-step, additive fabrication approach, manufacturing, characterization and experiments of nano-structures are integrated in-situ. Due to the miniature size of MEMS, the thickness precision is significantly improved while the power consumption is significantly depressed, making the quench-condensation of very thin films well controlled and easily achievable. Among various types of nano-structures, quench-condensed superconducting thin films are of great interest for physicists. Here we present such experiments done on superconducting thin films quench-condensed using the Fab on a Chip. We show that we are able to fabricate very thin films with its thickness precisely controlled, and the base temperature kept under ~3K during the process. The resistivity data demonstrates the high purity and uniformity of the film, as well as the annealing effect when cycling to higher temperatures. Based on the tremendous results obtained from the superconducting thin films, more complex nano-circuits can be fabricated and investigated using the Fab on a Chip, enabling a new approach for novel condensed matter physics experiments. This research is funded by the NSF through their CMMI division. This research is funded by the NSF through their CMMI division.

  11. Integrated MEMS mass sensor and atom source for a ``Fab on a Chip''

    Science.gov (United States)

    Han, Han; Imboden, Matthias; Stark, Thomas; Bishop, David

    2014-03-01

    ``Fab on a Chip'' is a new concept suggesting that the semiconductor fabrication facility can be integrated into a single silicon chip for nano-manufacturing. Such a chip contains various MEMS devices which can work together, operating in a similar way as a conventional fab does, to fabricate nano-structures. Here we present two crucial ``Fab on a chip'' components: the MEMS mass sensor and atomic evaporation source. The mass sensor is essentially a parallel plate capacitor with one suspended plate. When incident atoms deposit on the suspended plate, the mass change of the plate can be measured by detecting the resonant frequency shift. Using the mass sensor, a mass resolution of 3 fg is achieved. The MEMS evaporation source consists of a polysilicon plate suspended by two electrical leads with constrictions. By resistively heating the plate, this device works as a tunable atom flux source. By arranging many of these devices into an array, one can build a multi-element atom evaporator. The mass sensor and atom source are integrated so that the mass sensor is used to monitor and characterize the atomic flux. A material source and a sensor to monitor the fabrication are two integral components for our ``Fab on a Chip.''

  12. 75 FR 9438 - Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation...

    Science.gov (United States)

    2010-03-02

    ... Employment and Training Administration Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung..., applicable to workers of Samsung Austin Semiconductor, LLC, a subsidiary of Samsung Electronics Corporation... Systems, Inc. were employed on-site at the Austin, Texas location of Samsung Austin Semiconductor, LLC,...

  13. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

    NARCIS (Netherlands)

    A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

    2016-01-01

    textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

  14. Part I, FAB evaluation & application trials AFUE measurements: Part II, Integrated heating system (IHS) development

    Energy Technology Data Exchange (ETDEWEB)

    Leigh, R.W. [Brookhaven National Lab., Upton, NY (United States); Fisher, L. [BNL Consultant, Colrain, MA (United States)

    1996-07-01

    An oil burner/boiler efficiency test stand has been set up in the BNL oil heat laboratory which can measure the Annual Fuel Utilization Efficiency (AFUE) of burner/boiler combinations in accordance with ASHRAE and DOE standards. Measurements include both steady state efficiencies and heat-up and cool-down characteristics so that cycling effects can be included in an estimate of seasonal average performance. In addition to AFUE measurements, the direct conversion of fuel energy content to enthalpy increase in the boiler water is monitored. The system is largely automated, with most control functions under computer control and data taken electronically and permanently recorded on disks for future reference. To date, a retention-head burner and a fan atomized burner (FAB) have been tested in a steel boiler, the latter operating at two different fuel flow rates. The results are presented below, and verify that the very tight construction of the FAB`s fan results in a significant decrease in off-cycle sensible heat losses. Tests were also performed on a center-flue water heater fired with a conventional retention-head burner and with an FAB. The tests conformed to DOE standard procedures for hot water heaters, and the results are discussed below.

  15. Novel Schiff-base-derived FabH inhibitors with dioxygenated rings as antibiotic agents.

    Science.gov (United States)

    Zhou, Yang; Du, Qian-Ru; Sun, Jian; Li, Jing-Ran; Fang, Fei; Li, Dong-Dong; Qian, Yong; Gong, Hai-Bin; Zhao, Jing; Zhu, Hai-Liang

    2013-03-01

    Fatty acid biosynthesis plays a vital role in bacterial survival and several key enzymes involved in this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. Of these promising targets, β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target that could trigger the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Designing small molecules with FabH inhibitory activity displays great significance for developing antibiotic agents, which should be highly selective, nontoxic and broad-spectrum. In this manuscript, a series of novel Schiff base compounds were designed and synthesized, and their biological activities were evaluated as potential inhibitors. Among these 21 new compounds, (E)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)hexadecan-1-amine (10) showed the most potent antibacterial activity with a MIC value of 3.89-7.81 μM(-1) against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with an IC(50) value of 1.6 μM. Docking simulation was performed to position compound 10 into the E. coli FabH active site to determine the probable binding conformation.

  16. Cyclization strategies of meditopes: affinity and diffraction studies of meditope-Fab complexes.

    Science.gov (United States)

    Bzymek, Krzysztof P; Ma, Yuelong; Avery, Kendra A; Horne, David A; Williams, John C

    2016-06-01

    Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic `pocket' and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope-Fab complex.

  17. Orienterende Fast Atom Bombardment (FAB) experimenten met de VG-70-SQ massaspectrometer

    NARCIS (Netherlands)

    Hove GJ ten; Boer AC den; Burgers PC; Jong APJM de

    1988-01-01

    Eerste orienterende metingen met fast atom bombardment (FAB) ionisatietechniek zijn uitgevoerd. De techniek werd toegepast bij de analyse van korte-keten polypeptiden (n=2-5), cyclosporine, NADP en microperoxidase. Onderzocht werd de invloed van de aard van de matrix (glycerol, thioglycerol) op

  18. Impact of Fab Lab Tulsa on Student Self-Efficacy toward STEM Education

    Science.gov (United States)

    Dubriwny, Nicholas; Pritchett, Nathan; Hardesty, Michelle; Hellman, Chan M.

    2016-01-01

    Student self-confidence is important to any attempt to increase interest and achievement in Science, Technology, Engineering, and Math (STEM) education. This study presents a longitudinal examination of Fab Lab Tulsa's impact on attitude and self-efficacy toward STEM education among middle-school aged students. Paired samples t-test showed a…

  19. Various Energy-Saving Approaches to a TFT-LCD Panel Fab

    Directory of Open Access Journals (Sweden)

    Cheng-Kuang Chang

    2016-09-01

    Full Text Available This study employs the developed simulation software for the energy use of the high-tech fabrication plant (hereafter referred as a fab to examine six energy-saving approaches for the make-up air unit (MAU of a TFT-LCD (thin-film transistor liquid-crystal display fab. The studied approaches include: (1 Approach 1: adjust the set point of dry bulb temperature and relative humidity in the cleanroom; (2 Approach 2: lower the flow rate of supply air volume in the MAU; (3 Approach 3: use a draw-through type instead of push through type MAU; (4 Approach 4: combine the two stage cooling coils in MAU to a single stage coil; (5 Approach 5: reduce the original MAU exit temperature from 16.5 °C to 14.5 °C; and (6 Approach 6: avoid an excessive increase in pressure drop over the filter by replacing the HEPA filter more frequently. The simulated results are further compared to the measured data of the studied TFT-LCD fab in Taiwan. The simulated results showed that Approach 1 exhibits more significant influence on annual power consumption than the other approaches. The advantage/disadvantage of each approach is elaborated. The impact of the six approaches on the annual power consumption of the fab is also discussed.

  20. The tumor-inhibitory effectiveness of a novel anti-Trop2 Fab conjugate in pancreatic cancer.

    Science.gov (United States)

    Mao, Yuan; Wang, Xiaoying; Zheng, Feng; Wang, Changjun; Tang, Qi; Tang, Xiaojun; Xu, Ning; Zhang, Huiling; Zhang, Dawei; Xiong, Lin; Liang, Jie; Zhu, Jin

    2016-04-26

    Human trophoblastic cell surface antigen 2 (Trop2) has been reported to act oncogenically. In this study, one-step quantitative real-time polymerase chain reaction (qPCR) test and immunohistochemistry (IHC) analysis with were employed to evaluate the relationship between Trop2 expression and the clinicopathological features of patients with PC. Then a novel anti-Trop2 Fab antibody was conjugated with Doxorubicin (DOX) to form Trop2Fab-DOX, an antibody-drug conjugate. This Trop2Fab-DOX conjugate was characterized by cell ELISA and immunofluorescence assay. MTT and wound healing analyses were used to evaluate the inhibitory effect of Trop2Fab-DOX on PC cell growth in vitro, while xenograft nude mice model was established to examine the tumor-inhibitory effects of PC in vivo. High Trop2 expression was observed in PC tissues and Trop2 expression was associated with several malignant attributes of PC patients, including overall survival. Trop2Fab-DOX can bind to the Trop2-expressing PC cells and provide an improved releasing type of DOX. In addition, Trop2Fab-DOX inhibited the proliferation and suppressed the migration of PC cells in a dose-dependent manner in vitro, while inhibited the growth of PC xenografts in vivo. Trop2 is a specific marker for PC, and a novel Trop2Fab-DOX ADC has a potent antitumor activity.

  1. Docking and molecular dynamics studies on triclosan derivatives binding to FabI.

    Science.gov (United States)

    Yang, Xuyun; Lu, Junrui; Ying, Ming; Mu, Jiangbei; Li, Peichun; Liu, Yue

    2017-01-01

    FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2'-position and 4'-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3'-position and 5'-position of B ring destroy the π-π stacking interaction of A ring and NAD(+) which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.

  2. FabSim: facilitating computational research through automation on large-scale and distributed e-infrastructures

    CERN Document Server

    Groen, Derek; Suter, James; Hetherington, James; Zasada, Stefan; Coveney, Peter

    2015-01-01

    We present FabSim, a toolkit developed to simplify a range of computational tasks for researchers in diverse disciplines. FabSim is flexible, adaptable, and allows users to perform a wide range of tasks with ease. It also provides a systematic way to automate the use of resourcess, including HPC and distributed resources, and to make tasks easier to repeat by recording contextual information. To demonstrate this, we present three use cases where FabSim has enhanced our research productivity. These include simulating cerebrovascular bloodflow, modelling clay-polymer nanocomposites across multiple scales, and calculating ligand-protein binding affinities.

  3. Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

    Science.gov (United States)

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

    2016-07-01

    Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

  4. Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.

    Science.gov (United States)

    Billah, Md Morsaline; Hodges, Christopher S; Hays, Henry C W; Millner, P A

    2010-11-01

    The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10(-13)M to 10(-6)M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

  5. IgG Fc Fragment as a Scaffold for Development of Targeted Therapeutics.

    Science.gov (United States)

    Wozniak-Knopp, Gordana; Stadlmayr, Gerhard; Rueker, Florian

    2016-11-14

    Monoclonal antibodies and Fc fusion proteins have been successful therapeutics in the field of cancer and immune disease. As their pharmacological activity is dependent on the Fc fragment governing their effector functions and long in vivo half-life, the extensive engineering of the Fc for altered binding of its natural ligands that enable these properties has delivered molecules optimized for their therapeutic effect. Recently, the IgG1 Fc region itself and its subunits monomeric Fc fragment, CH2 and monomeric CH3 domain, have been engineered into scaffolds with favorable biophysical properties and a high potential for de novo antigen recognition. A dimeric Fc fragment with an antigen binding site has proven suitable for evaluation in animal models and will soon be entering human trials. Such modified constant domains can easily be incorporated into an antibody or fused with antibody domains of a second specificity. The small size of the Fc and its subunits that enhances their tissue penetration, as well as the unique topology of their binding sites that allows novel modes of contact with the antigen, are attractive features that prompt their development into promising candidate therapeutics.

  6. Mechanisms in Impact Fragmentation

    OpenAIRE

    Wittel, Falk K.; Carmona, Humberto A.; Kun, Ferenc; Herrmann, Hans J.

    2015-01-01

    The brittle fragmentation of spheres is studied numerically by a 3D Discrete Element Model. Large scale computer simulations are performed with models that consist of agglomerates of many spherical particles, interconnected by beam-truss elements. We focus on a detailed description of the fragmentation process and study several fragmentation mechanisms involved. The evolution of meridional cracks is studied in detail. These cracks are found to initiate in the inside of the specimen with quasi...

  7. 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.

  8. Determining Specificity of Fab Antibody against Mouse Serologically Detected Male Antigen%血清学检测的雄性特异性抗原Fab抗体的特异性鉴定

    Institute of Scientific and Technical Information of China (English)

    王乃东; 袁安文; 邓治邦; 屠迪; 许道军; 夏南松; 杨大为; 薛立群

    2011-01-01

    旨在研究血清学检测的雄性特异性抗原噬菌体Fab抗体的特异性.以从噬菌体Fab抗体库中筛选到具有较高雄性特异性结合活性的重组噬菌体克隆为基础,构建Fab抗体的可溶性表达噬质粒,并诱导和纯化该抗体片段,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性.结果表明,该抗体在约49 ku处有条带出现.在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌、雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清.石蜡切片免疫荧光定量分析显示,Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.002 3<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.019 2<0.05).以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示,Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清.结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体在具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作,以获得高亲和力、高雄性特异性的可溶性Fab抗体分子.%To study specificity of Fab antibody against mouse serologically detected male antigen, based on recombinant phage clone with male specific activity screened from phage Fab antibody library, expression plasmid of soluble Fab antibody was constructed, antibody fragment was induced and purified, the antibody specificity was determined by immunofluorescence staining with FITC/DAPI and ELISA. The results showed that an about 49 ku band appeared in the SDS-PAGE, comparison of cell immunofluorescence staining of Fab antibody and H-Y antiserum showed that male specificity and binding activity of Fab antibody was higher than that of antiserum, based on number of positive cells and mean

  9. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  10. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    Energy Technology Data Exchange (ETDEWEB)

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong; (Houston)

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  11. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    Science.gov (United States)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  12. Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-Specific Monoclonal Antibody and Recombinant F(ab

    Directory of Open Access Journals (Sweden)

    Lawrence K. Silbart

    2013-03-01

    Full Text Available Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs such as 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

  13. Activités insecticides de Striga hermonthica (Del.) Benth (Scrophulariaceae) sur Callosobrichus maculatus (Fab.) (Coleoptera : Bruchidae)

    OpenAIRE

    Nacoulma OG.; Ouedraogo AP.; Kiendrebeogo M.

    2006-01-01

    Insecticidal activities of Striga hermonthica (Del.) Benth (Scrophulariacecae) on Callobruchus maculatus (Fab.) (Coleptera Bruchidae). This paper deals with insecticidal potentialities of Striga hermonthica (Del.) (Scrophulariaceae) in protection of cowpea Vigna unguculata (L.) Walp against Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae) during storage. Crude acetone extract at 0,5% w/w (100 mg of extract for 20 g of grain) exhibits 48% of ovicidal effect and then reduces by half emer...

  14. Improving Assessment of Work Related Mental Health Function Using the Work Disability Functional Assessment Battery (WD-FAB).

    Science.gov (United States)

    Marfeo, Elizabeth E; Ni, Pengsheng; McDonough, Christine; Peterik, Kara; Marino, Molly; Meterko, Mark; Rasch, Elizabeth K; Chan, Leighton; Brandt, Diane; Jette, Alan M

    2017-05-05

    Purpose To improve the mental health component of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Specifically our goal was to expand the WD-FAB scales of mood & emotions, resilience, social interactions, and behavioral control to improve the depth and breadth of the current scales and expand the content coverage to include aspects of cognition & communication function. Methods Data were collected from a random, stratified sample of 1695 claimants applying for the SSA work disability benefits, and a general population sample of 2025 working age adults. 169 new items were developed to replenish the WD-FAB scales and analyzed using factor analysis and item response theory (IRT) analysis to construct unidimensional scales. We conducted computer adaptive test (CAT) simulations to examine the psychometric properties of the WD-FAB. Results Analyses supported the inclusion of four mental health subdomains: Cognition & Communication (68 items), Self-Regulation (34 items), Resilience & Sociability (29 items) and Mood & Emotions (34 items). All scales yielded acceptable psychometric properties. Conclusions IRT methods were effective in expanding the WD-FAB to assess mental health function. The WD-FAB has the potential to enhance work disability assessment both within the context of the SSA disability programs as well as other clinical and vocational rehabilitation settings.

  15. A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

    Science.gov (United States)

    Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

    2014-03-01

    Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

  16. SWARM INTELLIGENCE BASED DYNAMIC REAL-TIME SCHEDULING APPROACH FOR SEMICONDUCTOR WAFER FAB

    Institute of Scientific and Technical Information of China (English)

    Li Li; Fei Qiao; Wu Qidi

    2005-01-01

    Based on the analysis of collective activities of ant colonies, the typical example of swarm intelligence, a new approach to construct swarm intelligence based multi-agent-system (SMAS) for dynamic real-time scheduling for semiconductor wafer fab is proposed. The relevant algorithm,pheromone-based dynamic real-time scheduling algorithm (PBDR), is given. MIMAC test bed data set mini-fab is used to compare PBDR with FIFO (first in first out), SRPT(shortest remaining processing time) and CR(critical ratio) under three different release rules, i.e. deterministic rule, Poisson rule and CONWIP (constant WIP). It is shown that PBDR is prior to FIFO, SRPT and CR with better performance of cycle time, throughput, and on-time delivery, especially for on-time delivery performance.

  17. The FermiFab Toolbox for Fermionic Many-Particle Quantum Systems

    CERN Document Server

    Mendl, Christian B

    2011-01-01

    This paper introduces the FermiFab toolbox for many-particle quantum systems. It is mainly concerned with the representation of (symbolic) fermionic wavefunctions and the calculation of corresponding reduced density matrices (RDMs). The toolbox transparently handles the inherent antisymmetrization of wavefunctions and incorporates the creation/annihilation formalism. Thus, it aims at providing a solid base for a broad audience to use fermionic wavefunctions with the same ease as matrices in Matlab, say. Leveraging symbolic computation, the toolbox can greatly simply tedious pen-and-paper calculations for concrete quantum mechanical systems, and serves as "sandbox" for theoretical hypothesis testing. FermiFab (including full source code) is freely available as a plugin for both Matlab and Mathematica.

  18. Development of tools to study personal weight control strategies: OxFAB taxonomy

    OpenAIRE

    Hartmann‐Boyce, Jamie; Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A

    2016-01-01

    Objective To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. Methods The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think‐aloud interviews were...

  19. Development of tools to study personal weight control strategies: OxFAB taxonomy.

    OpenAIRE

    Hartmann-Boyce, J; Aveyard, P; Koshiaris, C; Jebb, SA

    2016-01-01

    To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts.The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think-aloud interviews were conducted to test ...

  20. Fab the coming revolution on your desktop : from personal computers to personal fabrication

    CERN Document Server

    Gershenfeld, Neil

    2005-01-01

    What if you could someday put the manufacturing power of an automobile plant on your desktop? According to Neil Gershenfeld, the renowned MIT scientist and inventor, the next big thing is personal fabrication-the ability to design and produce your own products, in your own home, with a machine that combines consumer electronics and industrial tools. Personal fabricators are about to revolutionize the world just as personal computers did a generation ago, and Fab shows us how.

  1. A status analysis of current digital marketing: a case study of Kauneusstudio FAB

    OpenAIRE

    Tran, Trong

    2015-01-01

    The subject of this thesis is a small company's current digital marketing status. This study was con-ducted in order for the owners of the beauty and hair salon Kauneusstudio FAB to improve their understanding of their customers’ behavior online and the significance of each digital channel they are using in the present marketing strategy. The goal of this study is to provide information for the company to recognize the strengths and the development points of the current digital marketing stra...

  2. Aerial image measurement technique for automated reticle defect disposition (ARDD) in wafer fabs

    Science.gov (United States)

    Zibold, Axel M.; Schmid, Rainer M.; Stegemann, B.; Scheruebl, Thomas; Harnisch, Wolfgang; Kobiyama, Yuji

    2004-08-01

    The Aerial Image Measurement System (AIMS)* for 193 nm lithography emulation has been brought into operation successfully worldwide. A second generation system comprising 193 nm AIMS capability, mini-environment and SMIF, the AIMS fab 193 plus is currently introduced into the market. By adjustment of numerical aperture (NA), illumination type and partial illumination coherence to match the conditions in 193 nm steppers or scanners, it can emulate the exposure tool for any type of reticles like binary, OPC and PSM down to the 65 nm node. The system allows a rapid prediction of wafer printability of defects or defect repairs, and critical features, like dense patterns or contacts on the masks without the need to perform expensive image qualification consisting of test wafer exposures followed by SEM measurements. Therefore, AIMS is a mask quality verification standard for high-end photo masks and established in mask shops worldwide. The progress on the AIMS technology described in this paper will highlight that besides mask shops there will be a very beneficial use of the AIMS in the wafer fab and we propose an Automated Reticle Defect Disposition (ARDD) process. With smaller nodes, where design rules are 65 nm or less, it is expected that smaller defects on reticles will occur in increasing numbers in the wafer fab. These smaller mask defects will matter more and more and become a serious yield limiting factor. With increasing mask prices and increasing number of defects and severability on reticles it will become cost beneficial to perform defect disposition on the reticles in wafer production. Currently ongoing studies demonstrate AIMS benefits for wafer fab applications. An outlook will be given for extension of 193 nm aerial imaging down to the 45 nm node based on emulation of immersion scanners.

  3. Functional capacity of immunoglobulin G preparations and the F(ab')2 split product.

    OpenAIRE

    Steele, R W

    1989-01-01

    Five immunoglobulin G preparations, including one 5S F(ab')2 split product, were compared for activity against common bacterial, viral, and protozoan pathogens. Standard assays were used to quantitate antibodies to tetanus, diphtheria, cytomegalovirus, herpes simplex virus types 1 and 2, rubella virus, and Toxoplasma gondii. Opsonization and killing of bacteria were examined by chemiluminescence methods using Streptococcus pneumoniae types 5, 12F, and 14 and Staphylococcus aureus. Antibodies ...

  4. Family of Advanced Beyond Line-of-Sight Terminals (FAB-T)

    Science.gov (United States)

    2015-12-01

    Conferencing (PNVC) function; the Telemetry , Tracking & Control for the Milstar and AEHF constellations, for Nuclear Command, Control, & Communications...Coexistences: 4 ​Footnotes: 1/ For FAB-T, access to privileged Tracking Telemetry and Control (TT&C) capabilities and resource controller capabilities is...DIACAP - DoD Information Assurance Certification & Accreditation Process DIRECT - Defense IEMATS Replacement Command and Control Terminal DMU - Dual

  5. Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

    Directory of Open Access Journals (Sweden)

    Hiroshi Hanagata

    2014-05-01

    Full Text Available Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production.

  6. An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

    Science.gov (United States)

    Demissie, Robel D; Kabre, Pauline; Tuntland, Micheal L; Fung, Leslie W-M

    2016-04-01

    Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

  7. Productivity improvement through automated operation of reticle defect inspection tools in a wafer fab environment

    Science.gov (United States)

    Holfeld, Christian; Wagner, Heiko; Tchikoulaeva, Anna; Loebeth, Steffen; Melzig, Stephan; Zhang, Yulin; Tanabe, Shinichi; Katoh, Takenori; Moriizumi, Koichi

    2013-04-01

    Traditionally, product development for reticle defect inspection mostly addressed operational requirements of the mask shops with highly individualized manufacturing. As a result, limited automation capability was available as compared to the standards in wafer production. Wafer fabs are guided by completely different conditions. Thousands of active reticles exist in a single fab requiring frequent re-inspections without interruption of wafer exposures. This requires high throughput of inspection tools, smart management of tool fleet, sophisticated scheduling and in-time execution of reticle inspections linked to the wafer manufacturing. The paper reports about the successful implementation of fully automated reticle defect inspection in a high-volume advanced logic fab. Automation scenarios - created based on existing SEMI standards - included inspection scheduling, reticle transport and inspection tool operation. A considerable productivity gain for the operation of Lasertec MATRICS X700 series inspection tools was obtained. Based on the learning throughout implementation, the requirements to the automation capability and tool operation as well as adjustments to working procedures are discussed.

  8. Fabricating quench condensed lead thin film circuits using MEMS Fab on a Chip technology

    Science.gov (United States)

    Imboden, Matthias; Han, Han; Del Corro, Pablo; Pardo, Flavio; Bolle, Cristian; Bishop, David

    2015-03-01

    We have developed a MEMS Fab on a Chip consisting of micro-sources, mass sensors, heaters/thermometers, shutters and a dynamic stencil. The fab only occupies a volume of a few cubic millimeters and consumes milliwatts of power, and hence can be operated in a cryostat. Thin film patterns of arbitrary shapes using multiple materials can be manufactured, while strongly suppressing thermal annealing effects. We demonstrate deposition of quench condensed lead films with fractions of a monolayer thickness control. Furthermore, using low deposition rates it is estimated that the surface temperature of the target heats by only 1.7 K. We study the effects of growing quench condensed films with different evaporation rates to demonstrate thermal annealing effects which occur during deposition. We measure the minimum conduction thickness (insulator to metal transition) as well as the superconducting transition temperature as a function of film thickness in order to shed light on growth of amorphous films and the transition to nanocluster formations. The Fab on a Chip will allow us to build nanocircuits made of ultra-thin materials. Annealing and doping is controlled and measurements occur in situ, without exposing the fabricated circuits to thermal fluctuations or foreign contaminants. This enables new types of experiments based on quantum circuits which cannot be fabricated using standard lithography techniques.

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

    Science.gov (United States)

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

    2004-09-01

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

  10. String fragmentation; La fragmentation des cordes

    Energy Technology Data Exchange (ETDEWEB)

    Drescher, H.J.; Werner, K. [Laboratoire de Physique Subatomique et des Technologies Associees - SUBATECH, Centre National de la Recherche Scientifique, 44 - Nantes (France)

    1997-10-01

    The classical string model is used in VENUS as a fragmentation model. For the soft domain simple 2-parton strings were sufficient, whereas for higher energies up to LHC, the perturbative regime of the QCD gives additional soft gluons, which are mapped on the string as so called kinks, energy singularities between the leading partons. The kinky string model is chosen to handle fragmentation of these strings by application of the Lorentz invariant area law. The `kinky strings` model, corresponding to the perturbative gluons coming from pQCD, takes into consideration this effect by treating the partons and gluons on the same footing. The decay law is always the Artru-Menessier area law which is the most realistic since it is invariant to the Lorentz and gauge transformations. For low mass strings a manipulation of the rupture point is necessary if the string corresponds already to an elementary particle determined by the mass and the flavor content. By means of the fragmentation model it will be possible to simulate the data from future experiments at LHC and RHIC 3 refs.

  11. Fragmentation trees reloaded.

    Science.gov (United States)

    Böcker, Sebastian; Dührkop, Kai

    2016-01-01

    Untargeted metabolomics commonly uses liquid chromatography mass spectrometry to measure abundances of metabolites; subsequent tandem mass spectrometry is used to derive information about individual compounds. One of the bottlenecks in this experimental setup is the interpretation of fragmentation spectra to accurately and efficiently identify compounds. Fragmentation trees have become a powerful tool for the interpretation of tandem mass spectrometry data of small molecules. These trees are determined from the data using combinatorial optimization, and aim at explaining the experimental data via fragmentation cascades. Fragmentation tree computation does not require spectral or structural databases. To obtain biochemically meaningful trees, one needs an elaborate optimization function (scoring). We present a new scoring for computing fragmentation trees, transforming the combinatorial optimization into a Maximum A Posteriori estimator. We demonstrate the superiority of the new scoring for two tasks: both for the de novo identification of molecular formulas of unknown compounds, and for searching a database for structurally similar compounds, our method SIRIUS 3, performs significantly better than the previous version of our method, as well as other methods for this task. SIRIUS 3 can be a part of an untargeted metabolomics workflow, allowing researchers to investigate unknowns using automated computational methods.Graphical abstractWe present a new scoring for computing fragmentation trees from tandem mass spectrometry data based on Bayesian statistics. The best scoring fragmentation tree most likely explains the molecular formula of the measured parent ion.

  12. Fragmentation of monoclonal antibodies

    Science.gov (United States)

    Vlasak, Josef

    2011-01-01

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

  13. Design of synthetic autonomous VH domain libraries and structural analysis of a VH domain bound to vascular endothelial growth factor.

    Science.gov (United States)

    Ma, Xiaolei; Barthelemy, Pierre A; Rouge, Lionel; Wiesmann, Christian; Sidhu, Sachdev S

    2013-06-26

    We compared the capacity of an autonomous heavy chain variable (VH) domain (VH-B1a) to support diversity within its antigen-binding site relative to the conventional antigen-binding fragment (Fab) from which it was derived. We find that VH-B1a can tolerate significant diversity within all three complementarity-determining regions (CDRs) and also within framework 3, and thus, VH-B1a and the Fab are similar in terms of the regions of the antigen-binding site that can tolerate diversity without compromising stability. We constructed libraries of synthetic VH domains and isolated binders with moderate affinity for vascular endothelial growth factor (VEGF) from a library in which only CDR3 was randomized. One binder was subjected to affinity maturation to derive an autonomous VH domain (VH-V1a) that recognized both human and mouse VEGF with high affinity (KD=16nM or 10nM, respectively). Structural analysis revealed that VH-V1a binds to an epitope that is distinct from the epitopes of a natural VEGF receptor and six different anti-VEGF Fabs. Moreover, VH-V1a recognizes VEGF by using an unusual paratope consisting predominantly of CDR3 but with significant contributions from framework residues within the former light chain interface. These results suggest that VH-B1a and other autonomous VH domains may be useful scaffolds to support both conventional libraries with antigen-binding sites built from the three CDR loops and, also, nonconventional libraries with antigen-binding sites built from CDR3 and the former light chain interface.

  14. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S. (GSK)

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  15. Purificação e caracterização do fragmento Fab anti-digoxina obtido pela técnica de phage display.

    OpenAIRE

    2016-01-01

    A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clo...

  16. Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans

    OpenAIRE

    Faustoferri, R.C.; Hubbard, C.J.; Santiago, B.; Buckley, A.A.; Seifert, T.B.; Quivey, R.G.

    2014-01-01

    SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTSm) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA seq...

  17. Embedded Fragments Registry (EFR)

    Data.gov (United States)

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

  18. Fragmentation in Biaxial Tension

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, G H; Archbold, G C; Hurricane, O A; Miller, P L

    2006-06-13

    We have carried out an experiment that places a ductile stainless steel in a state of biaxial tension at a high rate of strain. The loading of the ductile metal spherical cap is performed by the detonation of a high explosive layer with a conforming geometry to expand the metal radially outwards. Simulations of the loading and expansion of the metal predict strain rates that compare well with experimental observations. A high percentage of the HE loaded material was recovered through a soft capture process and characterization of the recovered fragments provided high quality data, including uniform strain prior to failure and fragment size. These data were used with a modified fragmentation model to determine a fragmentation energy.

  19. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  20. Fragmentation Main Model

    Data.gov (United States)

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

  1. Peptides from the variable region of specific antibodies are shared among lung cancer patients.

    Directory of Open Access Journals (Sweden)

    Dominique de Costa

    Full Text Available Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding-fragments (Fab of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass-spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients.

  2. Thermodynamics of fragment binding.

    Science.gov (United States)

    Ferenczy, György G; Keserű, György M

    2012-04-23

    The ligand binding pockets of proteins have preponderance of hydrophobic amino acids and are typically within the apolar interior of the protein; nevertheless, they are able to bind low complexity, polar, water-soluble fragments. In order to understand this phenomenon, we analyzed high resolution X-ray data of protein-ligand complexes from the Protein Data Bank and found that fragments bind to proteins with two near optimal geometry H-bonds on average. The linear extent of the fragment binding site was found not to be larger than 10 Å, and the H-bonding region was found to be restricted to about 5 Å on average. The number of conserved H-bonds in proteins cocrystallized with multiple different fragments is also near to 2. These fragment binding sites that are able to form limited number of strong H-bonds in a hydrophobic environment are identified as hot spots. An estimate of the free-energy gain of H-bond formation versus apolar desolvation supports that fragment sized compounds need H-bonds to achieve detectable binding. This suggests that fragment binding is mostly enthalpic that is in line with their observed binding thermodynamics documented in Isothermal Titration Calorimetry (ITC) data sets and gives a thermodynamic rationale for fragment based approaches. The binding of larger compounds tends to more rely on apolar desolvation with a corresponding increase of the entropy content of their binding free-energy. These findings explain the reported size-dependence of maximal available affinity and ligand efficiency both behaving differently in the small molecule region featured by strong H-bond formation and in the larger molecule region featured by apolar desolvation.

  3. PREPARATION AND IDENTIFICATION OF ANTI-PROGESTERONE Fab'-SBP%抗孕酮Fab'-大豆过氧化物酶交联物的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    龚振明; 李蓓; 陈鲁勇; 沈明泉

    2003-01-01

    用硫酸铵沉淀法对兔抗孕酮血清进行粗提后用DEAE Sepharose Fast Fl0W对IgG进行层析纯化,然后用胃蛋白酶切除Fc片段而制备F(ab')2,用β-巯基乙醇还原F(ab')2后得到Fab',再将Fab'与热稳定性较高的大豆过氧化物酶(SBP)偶联制得抗孕酮Fab'-SBP.将抗孕酮Fab'-SBP用于二级竞争酶联免疫法测定孕酮有以下优点:(1)减少因Fc片段与固相载体非特异性吸附而引起的高背景干扰;(2)由于Fab'和SBP在很温和的条件下偶联,可使偶联产物保持较高的免疫活性和酶活性.

  4. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  5. Deletion of fabN in Enterococcus faecalis results in unsaturated fatty acid auxotrophy and decreased release of inflammatory cytokines.

    Science.gov (United States)

    Diederich, Ann-Kristin; Duda, Katarzyna A; Romero-Saavedra, Felipe; Engel, Regina; Holst, Otto; Huebner, Johannes

    2016-05-01

    The Gram-positive bacterium Enterococcus faecalis can cause life-threatening infections and is resistant to several commonly used antibiotics. The type II fatty acid pathway in bacteria is discussed as a potential target for antimicrobial therapy. However, it was shown that inhibition or deletion of its enzymes can be rescued in Gram-positive bacteria by supplementation with fatty acids. Here we show that by deletion of the fabN gene, which is essential for unsaturated fatty acid (UFA) synthesis in E. faecalis, growth is impaired but can be rescued by supplementation with oleic acid or human serum. Nonetheless, we demonstrate alterations of the UFA profile after supplementation with oleic acid in the ΔfabN mutant using a specific glycolipid. In addition, we demonstrate that cytokine release in vitro is almost abolished after stimulation of mouse macrophages by the mutant in comparison to the wild type. The results indicate that fabN is not a suitable target for antimicrobials as UFA auxotrophy can be overcome. However, deletion of fabN resulted in a decreased inflammatory response indicating that fabN and resulting UFA synthesis are relevant for virulence.

  6. 抗HBs Fab-IFNα融合蛋白的制备与初步鉴定%Preparation and preliminary identification of anti HBs Fab-IFNa fusion protein

    Institute of Scientific and Technical Information of China (English)

    陆慧琦; 宋杰; 叶伟民; 韩焕兴

    2011-01-01

    目的 构建抗HBsAg的pComb Fab-IFNα载体,原核表达具双重生物活性的抗HBs Fab-IFNα融合蛋白.方法 以pBAD-Fab和pBAD IFNα作为模板,分别扩增Fd、λ和IENα基因,经相应的限制性内切酶酶切后分3次克隆人pComBHss质粒,转化XL-1 Blue大肠埃希菌.限制性酶切和测序鉴定重组质粒,免疫蛋向印迹(Western blotting)和斑点印迹(Dot blotting)鉴定融合蛋白的表达及抗原结合活性.结果 重组载体的酶切、电泳及测序表明抗HBs Fab-IFNα基因克隆正确.表达产物经12%SDS-PAGE电泳、转印,Western blotting显示该融合蛋白分子量约为65kD,Dot blotting显示其与HBsAg具有结合能力.细胞病变抑制法测定IFNα生物学活性为7.8×104~5.1×105U/ml.结论 该原核系统成功表达了抗HBs Fab-IFNα融合蛋白,表明其既具有抗HBsAg结合能力,又具备IFNα的生物活性,为进一步的系统表达和应用研究提供了条件.%Objective To construct the anti HBsAg pComb Fab-IFNe vector, and to express the fusion protein consisting of IFN and anti-HBs Fab in prokaryon. Methods Using pBAD-IFN plasmid and pBAD-Fab plasmid as template, the anti-HBs Fd, and IFN were amplified separately with corresponding endonuclease sites by polymerase chain reaction (PCR). Each PCR product was digested with specific endonuclease and inserted into pComBHss vector, and then transformed into XL-1 Blue. The recombinant plasmid was isolated by miniprep for restriction analysis and sequencing. Fusion protein was identified by Western blotting and Dot blotting. Results The recombinant plasmid was confirmed by restriction electrophoresis and sequencing. Aliquots of human anti-HBs Fab-IFNα were concentrated and size fractionated by 12% SDS-PAGE and stained with Coomassie. The proteins were transferred to nitrocellulose, incubated with HRP- conjugated goat anti-human IgG Fab and Rah anti-human IFN. The molecular weight of the fusion protein was about 65kD. Anti HBs Fab-IFNa fusion

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

    Science.gov (United States)

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

    2014-05-21

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

  8. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  9. The FermiFab toolbox for fermionic many-particle quantum systems

    Science.gov (United States)

    Mendl, Christian B.

    2011-06-01

    This paper introduces the FermiFab toolbox for many-particle quantum systems. It is mainly concerned with the representation of (symbolic) fermionic wavefunctions and the calculation of corresponding reduced density matrices (RDMs). The toolbox transparently handles the inherent antisymmetrization of wavefunctions and incorporates the creation/annihilation formalism. Thus, it aims at providing a solid base for a broad audience to use fermionic wavefunctions with the same ease as matrices in Matlab, say. Leveraging symbolic computation, the toolbox can greatly simply tedious pen-and-paper calculations for concrete quantum mechanical systems, and serves as "sandbox" for theoretical hypothesis testing. FermiFab (including full source code) is freely available as a plugin for both Matlab and Mathematica. Program summaryProgram title:FermiFab Catalogue identifier: AEIN_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEIN_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Special license provided by the author No. of lines in distributed program, including test data, etc.: 1 165 461 No. of bytes in distributed program, including test data, etc.: 15 557 308 Distribution format: tar.gz Programming language: MATLAB 7.9, Mathematica 7.0, C Computer: PCs, Sun Solaris workstation Operating system: Any platform supporting MATLAB or Mathematica; tested with Windows (32 and 64 bit) and Sun Solaris. RAM: Case dependent Classification: 4.15 Nature of problem: Representation of fermionic wavefunctions, computation of RDMs (reduced density matrices) and handing of the creation/annihilation operator formalism. Solution method: Mapping of Slater determinants to bitfields, implementation of the creation/annihilation and RDM formalism by bit operations. Running time: Depends on the problem size; several seconds for the provided demonstration files.

  10. Fluctuations of fragment observables

    CERN Document Server

    Gulminelli, F

    2006-01-01

    This contribution presents a review of our present theoretical as well as experimental knowledge of different fluctuation observables relevant to nuclear multifragmentation. The possible connection between the presence of a fluctuation peak and the occurrence of a phase transition or a critical phenomenon is critically analyzed. Many different phenomena can lead both to the creation and to the suppression of a fluctuation peak. In particular, the role of constraints due to conservation laws and to data sorting is shown to be essential. From the experimental point of view, a comparison of the available fragmentation data reveals that there is a good agreement between different data sets of basic fluctuation observables, if the fragmenting source is of comparable size. This compatibility suggests that the fragmentation process is largely independent of the reaction mechanism (central versus peripheral collisions, symmetric versus asymmetric systems, light ions versus heavy ion induced reactions). Configurationa...

  11. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  12. Critical Stage Rule-Based Real Time Dispatch(RTD)System in Highly-Mixed-Products (HMP) FAB

    Institute of Scientific and Technical Information of China (English)

    YUXiao-hua; XIANGYu-qun

    2005-01-01

    An improving utilization and efficiency of critical equipments in semiconductor wafer fabrication facilities are concerned. Semiconductor manufacturing FAB is one of the most complicated and cost sensitive environments. A good dispatching tool will make big difference in equipment utilization and FAB output as a whole. The equipment in this paper is In-Line DUV Scanner.There are many factors impacting utilization and output on this equipment group. In HMP environment one of the issues is changing of reticule in this area and idle counts due to load unbalance between equipments. Here we'll introduce a rule-based RTD system which aiming at decreasing the number of recipe change and idle counts among a group of scanner equipment in a high-mixedproducts FAB.

  13. Critical Stage Rule-Based Real Time Dispatch(RTD) System in Highly-Mixed-Products (HMP) FAB

    Institute of Scientific and Technical Information of China (English)

    YU Xiao-hua; XIANG Yu-qun

    2005-01-01

    An improving utilization and efficiency of critical equipments in semiconductor wafer fabrication facilities are concerned. Semiconductor manufacturing FAB is one of the most omplicated and cost sensitive environments. A good dispatching tool will make big difference in equipment utilization and FAB output as a whole. The equipment in this paper is In-Line DUV Scanner.There are many factors impacting utilization and output on this equipment group. In HMP environment one of the issues is changing of reticule in this area and idle counts due to load unbalance between equipments. Here we'll introduce a rule-based RTD system which aiming at decreasing the number of recipe change and idle counts among a group of scanner equipment in a high-mixedproducts FAB.

  14. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Bernhard [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany); Lecker, Laura S. M.; Zoltner, Martin [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Jaenicke, Elmar [Johannes Gutenberg-Universität, Jakob Welder Weg 26, 55128 Mainz (Germany); Schnell, Robert [Karolinska Institutet, 17 177 Stockholm (Sweden); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Brenk, Ruth, E-mail: w.n.hunter@dundee.ac.uk [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany)

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  15. 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...... made almost completely of still images, recounting the end of the world. These stills can be viewed as fragments that have survived the end of the world and now provide the only access to the events that occured. Shane Carruth's Primer has a different approach to time travel, the narrative diegesis...

  16. 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...... 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...

  17. IMPACT fragmentation model developments

    Science.gov (United States)

    Sorge, Marlon E.; Mains, Deanna L.

    2016-09-01

    The IMPACT fragmentation model has been used by The Aerospace Corporation for more than 25 years to analyze orbital altitude explosions and hypervelocity collisions. The model is semi-empirical, combining mass, energy and momentum conservation laws with empirically derived relationships for fragment characteristics such as number, mass, area-to-mass ratio, and spreading velocity as well as event energy distribution. Model results are used for several types of analysis including assessment of short-term risks to satellites from orbital altitude fragmentations, prediction of the long-term evolution of the orbital debris environment and forensic assessments of breakup events. A new version of IMPACT, version 6, has been completed and incorporates a number of advancements enabled by a multi-year long effort to characterize more than 11,000 debris fragments from more than three dozen historical on-orbit breakup events. These events involved a wide range of causes, energies, and fragmenting objects. Special focus was placed on the explosion model, as the majority of events examined were explosions. Revisions were made to the mass distribution used for explosion events, increasing the number of smaller fragments generated. The algorithm for modeling upper stage large fragment generation was updated. A momentum conserving asymmetric spreading velocity distribution algorithm was implemented to better represent sub-catastrophic events. An approach was developed for modeling sub-catastrophic explosions, those where the majority of the parent object remains intact, based on estimated event energy. Finally, significant modifications were made to the area-to-mass ratio distribution to incorporate the tendencies of different materials to fragment into different shapes. This ability enabled better matches between the observed area-to-mass ratios and those generated by the model. It also opened up additional possibilities for post-event analysis of breakups. The paper will discuss

  18. A study on EUV reticle surface molecular contamination under different storage conditions in a HVM foundry fab

    Science.gov (United States)

    Singh, SherJang; Yatzor, Brett; Taylor, Ron; Wood, Obert; Mangat, Pawitter

    2017-03-01

    The prospect of EUVL (Extreme Ultraviolet Lithography) insertion into HVM (High Volume Manufacturing) has never been this promising. As technology is prepared for "lab to fab" transition, it becomes important to comprehend challenges associated with integrating EUVL infrastructure within existing high volume chip fabrication processes in a foundry fab. The existing 193nm optical lithography process flow for reticle handling and storage in a fab atmosphere is well established and in-fab reticle contamination concerns are mitigated with the reticle pellicle. However EUVL reticle pellicle is still under development and if available, may only provide protection against particles but not molecular contamination. HVM fab atmosphere is known to be contaminated with trace amounts of AMC's (Atmospheric Molecular Contamination). If such contaminants are organic in nature and get absorbed on the reticle surface, EUV photon cause photo-dissociation resulting into carbon generation which is known to reduce multilayer reflectivity and also degrades exposure uniformity. Chemical diffusion and aggregation of other ions is also reported under the e-beam exposure of a EUV reticle which is known to cause haze issues in optical lithography. Therefore it becomes paramount to mitigate absorbed molecular contaminant concerns on EUVL reticle surface. In this paper, we have studied types of molecular contaminants that are absorbed on an EUVL reticle surface under HVM fab storage and handling conditions. Effect of storage conditions (gas purged vs atmospheric) in different storage pods (Dual pods, Reticle Clamshells) is evaluated. Absorption analysis is done both on ruthenium capping layer as well as TaBN absorber. Ru surface chemistry change as a result of storage is also studied. The efficacy of different reticle cleaning processes to remove absorbed contaminant is evaluated as well.

  19. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  20. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...... surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...

  1. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    Energy Technology Data Exchange (ETDEWEB)

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.

  2. Fragmented Work Stories

    DEFF Research Database (Denmark)

    Humle, Didde Maria; Reff Pedersen, Anne

    2015-01-01

    by exploring how different types of fragmentation create meanings. This is done by studying the work stories of job and personnel consultants and by drawing on the results of a narrative, ethnographic study of a consultancy. The analysis demonstrates how work stories are social practices negotiated, retold...

  3. Picking Up (On) Fragments

    NARCIS (Netherlands)

    Ellis, Phil

    2015-01-01

    abstractThis article discusses the implications for archival and media archaeological research and reenactment artwork relating to a recent arts practice project: reenacttv: 30 lines / 60 seconds. It proposes that archival material is unstable but has traces and fragments that are full of creative p

  4. Fragments of the Past

    Directory of Open Access Journals (Sweden)

    Peter Szende

    2016-10-01

    Full Text Available With travel being made more accessible throughout the decades, the hospitality industry constantly evolved their practices as society and technology progressed. Hotels looked for news ways up service their customers, which led to the invention of the Servidor in 1918. Once revolutionary innovations have gone extinct, merely becoming fragments of the past.

  5. Cryobiology of coral fragments.

    Science.gov (United States)

    Hagedorn, Mary; Farrell, Ann; Carter, Virginia L

    2013-02-01

    Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1M dimethyl sulfoxide at 1.0, 1.5 and 2.0h exposures, and 1.5M dimethyl sulfoxide at 1.0 and 1.5h exposures, P>0.05, ANOVA), whereas 2M concentrations did not (Pcoral tissue, but not in the zooxanthellae. During the winter when the fragments were chilled, the coral tissue remained relatively intact (∼25% loss) post-treatment, but the zooxanthellae numbers in the tissue declined after 5min of chilling (Pcoral tissue (∼75% loss) and zooxanthellae numbers declined in response to chilling alone (Pcoral against tissue loss after 45min of cryoprotectant exposure (P>0.05, ANOVA), but it did not protect against the loss of zooxanthellae (Pcoral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Picking Up (On) Fragments

    NARCIS (Netherlands)

    Ellis, Phil

    2015-01-01

    abstractThis article discusses the implications for archival and media archaeological research and reenactment artwork relating to a recent arts practice project: reenacttv: 30 lines / 60 seconds. It proposes that archival material is unstable but has traces and fragments that are full of creative p

  7. Wildlife habitat fragmentation.

    Science.gov (United States)

    John. Lehmkuhl

    2005-01-01

    A primary issue in forest wildlife management is habitat fragmentation and its effects on viability, which is the "bottom line" for plant and animal species of conservation concern. Population viability is the likelihood that a population will be able to maintain itself (remain viable) over a long period of time-usually 100 years or more. Though it is true...

  8. Nurturing Creativity and Innovation Through FabKids: A Case Study

    Science.gov (United States)

    Beyers, Ronald Noel

    2010-10-01

    This paper will report on a case study that was conducted involving Grade 10 learners who were exposed to a high-tech rapid-prototyping environment of a Fabrication Laboratory as part of a FabKids experience. This project must be viewed in the context of a global shortage of key skills placing a higher priority on the initiation and development of a pipeline to attract youth into science, engineering and technology careers. Creativity and innovation feature high on the skills agenda but more importantly preliminary results indicate that learners from a broad range of schools were able to operate effectively in this post constructivist environment. Participants had to apply their knowledge, skill, attitudes and values in order to produce a solution to the challenges provided. The fundamentals of the design process of investigate, design, make, evaluate and communicate were emphasized where the FabKids had to draw on their own collective knowledge using a range of technologies available to them.

  9. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  10. Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

    Science.gov (United States)

    Zhang, Huimin; Zheng, Beiwen; Gao, Rongsui; Feng, Youjun

    2015-09-01

    The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid

  11. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...

  12. Electroeluting DNA fragments.

    Science.gov (United States)

    Zarzosa-Alvarez, Ana L; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L; Bermudez-Cruz, Rosa M

    2010-09-05

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

  13. Structural models of antibody variable fragments: A method for investigating binding mechanisms

    Science.gov (United States)

    Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

    1998-03-01

    The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

  14. FabSim: Facilitating computational research through automation on large-scale and distributed e-infrastructures

    Science.gov (United States)

    Groen, Derek; Bhati, Agastya P.; Suter, James; Hetherington, James; Zasada, Stefan J.; Coveney, Peter V.

    2016-10-01

    We present FabSim, a toolkit developed to simplify a range of computational tasks for researchers in diverse disciplines. FabSim is flexible, adaptable, and allows users to perform a wide range of tasks with ease. It also provides a systematic way to automate the use of resources, including HPC and distributed machines, and to make tasks easier to repeat by recording contextual information. To demonstrate this, we present three use cases where FabSim has enhanced our research productivity. These include simulating cerebrovascular bloodflow, modelling clay-polymer nanocomposites across multiple scales, and calculating ligand-protein binding affinities. Catalogue identifier: AFAO_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFAO_v1_0.html Program obtainable from: CPC Programme Library, Queen's University, Belfast, N. Ireland Licensing provisions: BSD 3-Clause No. of lines in distributed program, including test data, etc.: 268282 No. of bytes in distributed program, including test data, etc.: 2791792 Distribution format: tar.gz Programming language: Python. Computer: PC or Mac. Operating system: Unix, OSX. RAM: 1 Gbytes Classification: 3, 4, 6.5. External routines: NumPy, SciPy, Fabric (1.5 or newer), PyYaml Nature of problem: Running advanced computations using remote resources is an activity that requires considerable time and human attention. These activities, such as organizing data, configuring software and setting up individual runs often vary slightly each time they are performed. To lighten this burden, we required an approach that introduced little burden of its own to set up and adapt, beyond which very substantial productivity ensues. Solution method: We present a toolkit which helps to simplify and automate the activities which surround computational science research. FabSim is aimed squarely at the experienced computational scientist, who can use the command line interface and a system of modifiable content to quickly automate sets of

  15. Fab'-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.

    Science.gov (United States)

    Laroui, Hamed; Viennois, Emilie; Xiao, Bo; Canup, Brandon S B; Geem, Duke; Denning, Timothy L; Merlin, Didier

    2014-07-28

    Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.

  16. Bispecific antibody fragments with CD20 X CD28 specificity allow effective autologous and allogeneic T-cell activation against malignant cells in peripheral blood and bone marrow cultures from patients with B-cell lineage leukemia and lymphoma.

    Science.gov (United States)

    Brandl, M; Grosse-Hovest, L; Holler, E; Kolb, H J; Jung, G

    1999-08-01

    Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation, such as the TCR/CD3 complex and the costimulatory receptor CD28, are capable of mediating T-cell activation resulting in tumor cell killing. In this study, we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2, when used alone rather than in combination, markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore, these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and, in particular, of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients.

  17. Heavy meson fragmentation at LHC

    Directory of Open Access Journals (Sweden)

    M. A. Gomshi Nobary

    2003-06-01

    Full Text Available   Large Hadron Collider (LHC at CERN will provide excellent opportunity to study the production and decay of heavy mesons and baryons with high statistics. We aim at the heavy mesons in this work and calculate their fragmentation functions consistent with this machine and present their total fragmentation probabilities and average fragmentation parameters.

  18. SCALING AND 4-QUARK FRAGMENTATION

    NARCIS (Netherlands)

    SCHOLTEN, O; BOSVELD, GD

    1991-01-01

    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

  19. Building blocks X-FAB SOI 0.18 μm

    Science.gov (United States)

    Cizel, J.-B.; Ahmad, S.; Callier, S.; Cornat, R.; Dulucq, F.; Fleury, J.; Martin-Chassard, G.; Raux, L.; de La Taille, C.; Thienpont, D.

    2015-02-01

    This work has been done in order to study a new technology provided by X-FAB named xt018. It is an SOI (Silicon On Insulator) technology with a minimal gate length of 180 nm. Building blocks have been done to test the advantages and drawbacks of this technology compared to the one currently used (AMS SiGe 0.35 μm). These building blocks have been designed to fit in an existing experience housed by the CALICE collaboration: the read-out chip for the Electromagnetic CALorimeter (ECAL) of the foreseen International Linear Collider (ILC). Performances will be compared to those of the SKIROC2 chip designed by the OMEGA laboratory, trying to fit the same requirements. The chip is being manufactured and will be back for measurements in December, the displayed results are only simulation results and thus the conclusions concerning the performances of these building blocks are subject to change.

  20. Photomask film degradation effects in the wafer fab: how to detect and monitor over time

    Science.gov (United States)

    Whittey, John; Hess, Carl; Garcia, Edgardo; Wagner, Mark; Duffy, Brian

    2012-11-01

    As a result of repeated cleanings and exposure effects such as chrome migration or MoSi oxidation some photomasks in the semiconductor fabs exhibit changes in critical dimension uniformity (CDU) over time. Detecting these effects in a timely manner allows for better risk management and process control in manufacturing. By monitoring changes in film reflectance intensity due to the various degradation mechanisms it is possible to predict when they may begin to influence across chip line width variations (ACLV). By accurately predicting the magnitude of these changes it is possible for semiconductor manufacturers to replace the photomasks before they have an impact on yields. This paper looks at possible causes of CDU variations on reticles during use and how this information might be used to improve or monitor reticle CDU changes over time.

  1. Efecto de la temperatura, humedad y dieta sobre el desarrollo de Gnathocerus cornutus Fab. (Coleopera: Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Núñez Fernando

    1989-06-01

    Full Text Available The influence of temperature, relative humídíty and diet composítíon in the duration and velocity of the development cicle of Gnathoccnis comutus Fab., was studied. Each of two diets (Aand Bl was tried in presence of 5 different combinations of temperature and humídíty. Optimal conditions fOI development were found to be: 28°C and 85-90% R.H.. in which the complete cycle lasts 54 days. the larval stage is the critical one in the development in which a htgh variation among individuals in both, velocity and span of development, are notorious. The cycle duration is virtually the same for both diets, but in the diet A. a better adaptability, expressed as a proportion of descendents, was observed. Life tables and surviving curves were made.
    Se estudió la influencia de la temperatura. la humedad relativa y la composición de la dieta sobre la duración del ciclo de desarrollo y la velocidad del mismo en Gnathocerus comutus Fab, Para el efecto. cada una de las dos dietas (A y B] se ensayó en presencia de 5 combinaciones distintas de temperatura y humedad. Se encontró que las condiciones óptimas para el desarrollo. son: 28°C y 85-90% H.R.. en las cuales. el ciclo completo se desenvuelve en 54 días. El estado crítico en cuanto al desarrollo. es el larval en el cual es notoria una alta variación entre los individuos en velocidad y tiempo de desarrollo. La duración del ciclo es virtualmente la misma en ambas dietas. pero en la A se observa una mejor adaptabilidad expresada en proporctón de descendientes. Se construyeron tablas de vida y curvas de supervivencia.

  2. Picking Up (On Fragments

    Directory of Open Access Journals (Sweden)

    Phil Ellis

    2015-09-01

    Full Text Available This 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 potential to re-think and re-examine past media historical events through a media archaeological approach to reenactment. The article contains images and links to videos from the final reenactment artworks as well as from rehearsals in Vienna and Bradford.

  3. An Archeology of Fragments

    Directory of Open Access Journals (Sweden)

    Gerald L. Bruns

    2014-10-01

    Full Text Available This is a short (fragmentary history of fragmentary writing from the German Romantics (F. W. Schlegel, Friedrich Hölderlin to modern and contemporary concrete or visual poetry. Such writing is (often deliberately a critique of the logic of subsumption that tries to assimilate whatever is singular and irreducible into totalities of various categorical or systematic sorts. Arguably, the fragment (parataxis is the distinctive feature of literary Modernism, which is a rejection, not of what precedes it, but of what Max Weber called “the rationalization of the world” (or Modernity whose aim is to keep everything, including all that is written, under surveillance and control.

  4. 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)

    2008-02-15

    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)

  5. Stone fragmentation by ultrasound

    Indian Academy of Sciences (India)

    S K Shrivastava; Kailash

    2004-08-01

    The presence of kidney stone in the kidney causes discomfort to patients. Hence, removal of such stones is important which is commonly done these days, non-destructively, with lithotripters without surgery. Commercially, lithotripters like extra-corporeal shock wave lithotripters (ESWL) made by Siemens etc are in routine use. These methods are very cumbersome and expensive. Treatment of the patients also takes comparatively more time because of more number of sittings. 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 disintegration efficiency of the lithotripters, which give better treatment to the patients.

  6. Structure-based design,synthesis of novel inhibitors of Mycobacterium tuberculosis FabH as potential anti-tuberculosis agents

    Institute of Scientific and Technical Information of China (English)

    Xue Hui Zhang; Hong Yu; Wu Zhong; Li Li Wang; Song Li

    2009-01-01

    Mycobacterium tuberculosis FabH,an essential enzyme in mycolic acids biosynthetic pathway,is an attractive target for novel anti-tuberculosis agents.Structure-based design,synthesis of novel inhibitors of mrFabH was reported in this paper.A novel scaffold structure was designed,and 12 candidate compounds that displayed favorable binding with the active site were identified and synthesized.

  7. Active site modification of the β-ketoacyl-ACP synthase FabF3 of Streptomyces coelicolor affects the fatty acid chain length of the CDA lipopeptides.

    Science.gov (United States)

    Lewis, Richard A; Nunns, Laura; Thirlway, Jenny; Carroll, Kathleen; Smith, Colin P; Micklefield, Jason

    2011-02-14

    Using site directed mutagenesis we altered an active site residue (Phe107) of the enzyme encoded by fabF3 (SCO3248) in the Streptomyces coelicolor gene cluster required for biosynthesis of the calcium dependent antibiotics (CDAs), successfully generating two novel CDA derivatives comprising truncated (C4) lipid side chains and confirming that fabF3 encodes a KAS-II homologue that is involved in determining CDA fatty acid chain length.

  8. CONTROL OF FRAGMENTATION BY BLASTING

    Directory of Open Access Journals (Sweden)

    Branko Božić

    1998-12-01

    Full Text Available The degree of fragmentation influences the economy of the excavation operations. Characteristics of blasted rock such as fragment size, volume and mass are fundamental variables effecting the economics of a mining operation and are in effect the basis for evaluating the quality of a blast. The properties of fragmentation, such as size and shape, are very important information for the optimization of production. Three factors control the fragment size distribution: the rock structure, the quantity of explosive and its distribution within the rock mass. Over the last decade there have been considerable advances in our ability to measure and analyze blasting performance. These can now be combined with the continuing growth in computing power to develop a more effective description of rock fragmentation for use by future blasting practitioners. The paper describes a view of the fragmentation problem by blasting and the need for a new generation of engineering tools to guide the design and implementation of blasting operations.

  9. Transcription of the Escherichia coli fatty acid synthesis operon fabHDG is directly activated by FadR and inhibited by ppGpp.

    Science.gov (United States)

    My, Laetitia; Rekoske, Brian; Lemke, Justin J; Viala, Julie P; Gourse, Richard L; Bouveret, Emmanuelle

    2013-08-01

    In Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.

  10. Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics.

    Science.gov (United States)

    Yao, Jiangwei; Rock, Charles O

    2016-03-01

    Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single-base-pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed pathogen-specific antibiotics have the potential to overcome this liability.

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

    Institute of Scientific and Technical Information of China (English)

    Zuanning Yuan; Minge Du; Yiwen Chen; Fei Dou

    2013-01-01

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

  12. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

    Science.gov (United States)

    Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

    2007-10-01

    Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

  13. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    Science.gov (United States)

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  14. Crystallization and preliminary X-ray analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Jun, E-mail: jun-saito@meiji.co.jp; Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo [Pharmaceutical Research Center, Meiji Seika Kaisha Ltd, 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567 (Japan)

    2006-06-01

    Enoyl-acyl carrier protein (ACP) reductases are responsible for bacterial type II fatty-acid biosynthesis and are attractive targets for developing novel antibiotics. The S. pneumoniae enoyl-ACP reductase (FabK) was crystallized and selenomethionine MAD data were collected to 2 Å resolution. The enoyl-acyl carrier protein (ACP) reductase from Streptococcus pneumoniae (FabK; EC 1.3.1.9) is responsible for catalyzing the final step in each elongation cycle of fatty-acid biosynthesis. Selenomethionine-substituted FabK was purified and crystallized by the hanging-drop vapour-diffusion method at 277 K. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.26, b = 126.70, c = 53.63 Å, β = 112.46°. Diffraction data were collected to 2.00 Å resolution using synchrotron beamline BL32B2 at SPring-8. Two molecules were estimated to be present in the asymmetric unit, with a solvent content of 45.1%.

  15. Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

    Science.gov (United States)

    Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

    2013-09-01

    Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods.

  16. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    Science.gov (United States)

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  17. Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

    Science.gov (United States)

    Schlievert, Patrick M.; Chuang-Smith, Olivia N.; Peterson, Marnie L.; Cook, Laura C. C.; Dunny, Gary M.

    2010-01-01

    Background Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS+ E. faecalis endocarditis. Methodology/Principal Findings When challenged with AS+ E. faecalis, 10 rabbits actively immunized against AS+ E. faecalis developed more significant vegetations than 9 animals immunized against AS− E. faecalis, and 9/10 succumbed compared to 2/9 (pendocarditis lesion) microbial counts (7.9×106 versus 2.0×105, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS+ E. faecalis illness. PMID:20957231

  18. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  19. 抗轮状病毒IgY和Fab的研制%Preparation of IgY and Fab against HRV

    Institute of Scientific and Technical Information of China (English)

    孙淑清; 孟岩; 段春燕; 胡彦涛

    2005-01-01

    目的对抗轮状病毒(RV)IgY和胃蛋白酶水解片断Fab进行分离与纯化.方法免疫鸡得到抗HRVIgY,用两步盐析结合凝胶过滤将其从蛋黄中分离出来,纯的IgY经胃蛋白酶水解得抗体片断Fab.结果抗HRVIgY用SDS-PAGE检测纯度可达到95%以上.抗轮状病毒(RV)IgY和抗体片断Fab经SDS-PAGE和MALDIMS法测定,其纯度达到99%以上,经ELISA法检测,Fab'的活性保持在IgY原始活性的70%以上.结论我们所设计的分离和纯化抗HRVIgY和Fab'的方法简单、有效.

  20. Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen.

    Science.gov (United States)

    Wang, Naidong; Yuan, Anwen; Ma, Jun; Deng, Zhibang; Xue, Liqun

    2015-06-01

    The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos.

  1. The response regulator YycF inhibits expression of the fatty acid biosynthesis repressor FabT in Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Maria Luz Mohedano

    2016-08-01

    Full Text Available The YycFG (also known as WalRK, VicRK, MicAB or TCS02 two-component system (TCS is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  2. Generation and characterization of a human neutralizing bivalent antibody Fab094-DDD against rabies virus%人源特异性抗狂犬病毒二价Fab094-DDD抗体的制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    目的:利用蛋白激酶A(protein kinase A,PKA)调节亚基(R亚基)的二聚体化功能结构域(DDD)的二聚化功能,制备具有中和活性的人源特异性抗狂犬病毒二价抗体.方法:优化合成linker-C-DDD序列,设计引物扩增抗狂犬病毒抗体Fab094的Fd段和轻链可变区序列(Vκ),通过overlap PCR将Fab094的Fd段和linker-C-DDD基因重组为Fd-DDD,将其和Vκ分别克隆到真核表达载体中,共转染293Free style细胞,表达纯化该抗体.应用SDS-PAGE 、Western blot、ELISA、免疫共沉淀、亲和力测定及免疫荧光试验检测该抗体的免疫学特性,用荧光抗体病毒中和试验检测其中和活性.结果:成功构建了全人源抗狂犬病毒二价抗体Fab094-DDD的真核表达载体,获得的二价Fab094-DDD抗体与抗原保持着较好的亲和力,能特异性结合狂犬病毒,中和效价为213.2 U/mg.结论:成功制备了抗狂犬病毒二价Fab094-DDD抗体,具有较高的中和活性,为进一步研发狂犬病治疗性抗体药物奠定了基础,对于其他疾病双表位人源特异性抗体的制备也具有借鉴作用.

  3. Thermodynamical string fragmentation

    Science.gov (United States)

    Fischer, Nadine; Sjöstrand, Torbjörn

    2017-01-01

    The observation of heavy-ion-like behaviour in pp collisions at the LHC suggests that more physics mechanisms are at play than traditionally assumed. The introduction e.g. of quark-gluon plasma or colour rope formation can describe several of the observations, but as of yet there is no established paradigm. In this article we study a few possible modifications to the Pythia event generator, which describes a wealth of data but fails for a number of recent observations. Firstly, we present a new model for generating the transverse momentum of hadrons during the string fragmentation process, inspired by thermodynamics, where heavier hadrons naturally are suppressed in rate but obtain a higher average transverse momentum. Secondly, close-packing of strings is taken into account by making the temperature or string tension environment-dependent. Thirdly, a simple model for hadron rescattering is added. The effect of these modifications is studied, individually and taken together, and compared with data mainly from the LHC. While some improvements can be noted, it turns out to be nontrivial to obtain effects as big as required, and further work is called for.

  4. Thermodynamical String Fragmentation

    CERN Document Server

    Fischer, Nadine

    2016-01-01

    The observation of heavy-ion-like behaviour in pp collisions at the LHC suggests that more physics mechanisms are at play than traditionally assumed. The introduction e.g. of quark-gluon plasma or colour rope formation can describe several of the observations, but as of yet there is no established paradigm. In this article we study a few possible modifications to the Pythia event generator, which describes a wealth of data but fails for a number of recent observations. Firstly, we present a new model for generating the transverse momentum of hadrons during the string fragmentation process, inspired by thermodynamics, where heavier hadrons naturally are suppressed in rate but obtain a higher average transverse momentum. Secondly, close-packing of strings is taken into account by making the temperature or string tension environment-dependent. Thirdly, a simple model for hadron rescattering is added. The effect of these modifications is studied, individually and taken together, and compared with data mainly from...

  5. Fragmentation Considered Poisonous

    CERN Document Server

    Herzberg, Amir

    2012-01-01

    We present practical poisoning and name-server block- ing attacks on standard DNS resolvers, by off-path, spoofing adversaries. Our attacks exploit large DNS responses that cause IP fragmentation; such long re- sponses are increasingly common, mainly due to the use of DNSSEC. In common scenarios, where DNSSEC is partially or incorrectly deployed, our poisoning attacks allow 'com- plete' domain hijacking. When DNSSEC is fully de- ployed, attacker can force use of fake name server; we show exploits of this allowing off-path traffic analy- sis and covert channel. When using NSEC3 opt-out, attacker can also create fake subdomains, circumvent- ing same origin restrictions. Our attacks circumvent resolver-side defenses, e.g., port randomisation, IP ran- domisation and query randomisation. The (new) name server (NS) blocking attacks force re- solver to use specific name server. This attack allows Degradation of Service, traffic-analysis and covert chan- nel, and also facilitates DNS poisoning. We validated the attac...

  6. Unique features of monoclonal IgG2b in the cleavage reaction with pepsin.

    Directory of Open Access Journals (Sweden)

    Sumii,Hiroshi

    1989-06-01

    Full Text Available Preparations of IgG2b purified from several mouse hybridoma clones were highly susceptible, compared to other subclasses, to peptic digestion under conditions usually used to prepare F (ab'2 fragments. Analyses of the digestion products revealed that no F (ab'2 was produced and that the main product was a Fab-like fragment. Demonstration of the hinge disulfides in the Fc portion clearly indicated that in IgG2b the primary peptic cleavage occurs on the NH2-terminal side of the inter-heavy chain disulfide bridge. The resulting Fab failed to bind with antigen, suggesting the importance of the CH1-hinge region in maintaining the native conformation of the antigen-binding site.

  7. Fast atom bombardment (FAB) mass spectrometry: a mass spectral investigation of some of the insulins.

    Science.gov (United States)

    Barber, M; Bordoli, R S; Elliott, G J; Tyler, A N; Bill, J C; Green, B N

    1984-04-01

    Mass measurements of the protonated molecules [M + H]+ of four insulins are presented. In addition, structurally significant fragment ions are observed in the mass spectrum and metastable scanning has been used to link these ions to the protonated molecule.

  8. Route of infection and hematological effect of Metarhizium anisopliae (Metsch.) Sorokin on Dysdercus cingulatus (Fab.) adult.

    Science.gov (United States)

    Sahayaraj, Kitherian; Borgio, Jesu Francis; Lucini, Luigi

    2014-01-01

    The primary objective of this work was to identify, under laboratory conditions, the route of infection and hemogram of Dysdercus cingulatus (Fab.) adults by Metarhizium anisopliae. The infection process in D. cingulatus by M. anisopliae involved the conidia adherence to the host cuticle and germination after 24 h post-infection, accompanied by falling of bristles. The subsequent step, within 24-48 h post-infection, comprised penetration of fungus through spiracles, root of bristles, hemolymph, and the three dorsal sacs. Subsequently, within 72-96 h post-infection, the fungus penetrated into trachea and sacs, then emerged on cuticular surface and was found to be maximum in hemolymph. A great decrease in hemocytes count was observed within 96 h from infection. The hemosomic index (HSI) decreased gradually as the incubation period increased. As far as we know, this is the first study to know the mechanism of action of M. anisopliae to D. cingulatus.

  9. Characterization of silicon photomultipliers at National Nano-Fab Center for PET-MR.

    Science.gov (United States)

    Kim, Hyoungtaek; Sul, Woo Suk; Cho, Gyuseong

    2014-10-01

    The silicon photomultipliers (SiPMs) were fabricated for magnetic resonance compatible positron emission tomography (PET) applications using customized CMOS processes at National NanoFab Center. Each micro-cell consists of a shallow n+/p well junction on a p-type epitaxial wafer and passive quenching circuit was applied. The size of the SiPM is 3 × 3 mm(2) and the pitch of each micro-cell is 65 μm. In this work, several thousands of SiPMs were packaged and tested to build a PET ring detector which has a 60 mm axial and 390 mm radial field of view. I-V characteristics of the SiPMs are shown good uniformity and breakdown voltage is around 20 V. The photon detection efficiency was measured via photon counting method and the maximum value was recorded as 16% at 470 nm. The gamma ray spectrum of a Ge-68 isotope showed nearly 10% energy resolution at 511 keV with a 3 × 3 × 20 mm(3) LYSO crystal.

  10. Characterization of silicon photomultipliers at National Nano-Fab Center for PET-MR

    Science.gov (United States)

    Kim, Hyoungtaek; Sul, Woo Suk; Cho, Gyuseong

    2014-10-01

    The silicon photomultipliers (SiPMs) were fabricated for magnetic resonance compatible positron emission tomography (PET) applications using customized CMOS processes at National NanoFab Center. Each micro-cell consists of a shallow n+/p well junction on a p-type epitaxial wafer and passive quenching circuit was applied. The size of the SiPM is 3 × 3 mm2 and the pitch of each micro-cell is 65 μm. In this work, several thousands of SiPMs were packaged and tested to build a PET ring detector which has a 60 mm axial and 390 mm radial field of view. I-V characteristics of the SiPMs are shown good uniformity and breakdown voltage is around 20 V. The photon detection efficiency was measured via photon counting method and the maximum value was recorded as 16% at 470 nm. The gamma ray spectrum of a Ge-68 isotope showed nearly 10% energy resolution at 511 keV with a 3 × 3 × 20 mm3 LYSO crystal.

  11. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

    Science.gov (United States)

    Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L; Jiang, Haiyan

    2016-05-01

    Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

  12. Integration of the APC framework with AMD's Fab25 factory system

    Science.gov (United States)

    Bushman, Scott; Campbell, William J.; Miller, Michael L.

    1999-09-01

    This paper discusses the integration and development of advanced process control technologies with AMD's Fab25 factory systems using the Advance Process Control Framework. The Framework is an open software architecture that allows the integration of existing factory systems, such as the manufacturing execution systems, configurable equipment interfaces, recipe management systems, metrology tools, process tools, and add-on sensors, into a system which provides advanced process control specific functionality. The Advanced Process Control Framework project was formulated to enable effective integration of Advanced Process Control applications into a semiconductor facility to improve manufacturing productivity and product yields. The main communication link between the factory system and the Framework is the Configurable Equipment Interface. It interfaces through a specialized component in the framework, the Machine Interface, which converts the factory system communication protocol, ISIS, to the Framework protocol, CORBA. The Framework is a distributed architecture that uses CORBA as a communication protocol between specialized components. A generalized example of how the Framework is integrated into the semiconductor facility is provided, as well as a description of the overall architecture used for process control strategy development. The main development language, Tcl/Tk, provides for increased development and deployment over traditional coding methods.

  13. Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

    OpenAIRE

    1994-01-01

    The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with io...

  14. An Algebra for Program Fragments

    DEFF Research Database (Denmark)

    Kristensen, Bent Bruun; Madsen, Ole Lehrmann; Møller-Pedersen, Birger

    1985-01-01

    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...

  15. Complete axiomatizations for XPath fragments

    NARCIS (Netherlands)

    ten Cate, B.; Litak, T.; Marx, M.

    2010-01-01

    We provide complete axiomatizations for several fragments of Core XPath, the navigational core of XPath 1.0 introduced by Gottlob, Koch and Pichler. A complete axiomatization for a given fragment is a set of equivalences from which every other valid equivalence is derivable; equivalences can be thou

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

    Science.gov (United States)

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

    2003-09-01

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

  17. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    Science.gov (United States)

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Driven fragmentation of granular gases.

    Science.gov (United States)

    Cruz Hidalgo, Raúl; Pagonabarraga, Ignacio

    2008-06-01

    The dynamics of homogeneously heated granular gases which fragment due to particle collisions is analyzed. We introduce a kinetic model which accounts for correlations induced at the grain collisions and analyze both the kinetics and relevant distribution functions these systems develop. The work combines analytical and numerical studies based on direct simulation Monte Carlo calculations. A broad family of fragmentation probabilities is considered, and its implications for the system kinetics are discussed. We show that generically these driven materials evolve asymptotically into a dynamical scaling regime. If the fragmentation probability tends to a constant, the grain number diverges at a finite time, leading to a shattering singularity. If the fragmentation probability vanishes, then the number of grains grows monotonously as a power law. We consider different homogeneous thermostats and show that the kinetics of these systems depends weakly on both the grain inelasticity and driving. We observe that fragmentation plays a relevant role in the shape of the velocity distribution of the particles. When the fragmentation is driven by local stochastic events, the long velocity tail is essentially exponential independently of the heating frequency and the breaking rule. However, for a Lowe-Andersen thermostat, numerical evidence strongly supports the conjecture that the scaled velocity distribution follows a generalized exponential behavior f(c) approximately exp(-cn) , with n approximately 1.2 , regarding less the fragmentation mechanisms.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  20. 抗胃癌鼠单抗Fab段在大肠杆菌中的表达%EXPRESSION OF MONOCLONAL MOUSE ANTI-Fab FRAGMENT IN E. COLI

    Institute of Scientific and Technical Information of China (English)

    李时荣; 王琰; 孙勤暖; 翁立新

    2006-01-01

    目的:探索强启动子T7在可溶性抗体Fab表达中的作用.方法:利用DNA重组的方法将Fd段及K链基因转移到质粒pT7中,置于T7噬菌体启动子的下游,通过蛋白质印迹法证明pT7- 3G9是否有可溶性Fab的表达.结果:pT7- 3G9有可溶性Fab的表达;pT7- 3G9表达的可溶性Fab比p3G9-L高.结论:强启动子系统可作为可溶性抗体Fab的表达载体,T7启动子在可溶性抗体Fab表达中作用比Lac启动子强.

  1. Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

    Science.gov (United States)

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dhagat, Urmi; Owczarek, Catherine M; Hardy, Matthew P; Fabri, Louis J; Scotney, Pierre D; Nash, Andrew D; Wilson, Nicholas J; Lopez, Angel F; Parker, Michael W

    2014-03-01

    Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.

  2. 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

    1999-01-01

    The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of th......, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 A, alpha = 105.7, beta = 98.32, gamma = 97.62 degrees, and diffract to 2.9 A resolution when analyzed at 100 K using synchrotron-generated X--rays....

  3. Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

    Science.gov (United States)

    Hahn, M; Winkler, D; Welfle, K; Misselwitz, R; Welfle, H; Wessner, H; Zahn, G; Scholz, C; Seifert, M; Harkins, R; Schneider-Mergener, J; Höhne, W

    2001-11-23

    The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.

  4. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-12-01

    Full Text Available Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY, the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the sc

  5. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin.

    Science.gov (United States)

    Pleckaityte, Milda; Zvirbliene, Aurelija; Sezaite, Indre; Gedvilaite, Alma

    2011-12-15

    Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and

  6. 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)

    1998-12-31

    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.

  7. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    Energy Technology Data Exchange (ETDEWEB)

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  8. Deletion of the β-acetoacetyl synthase FabY in Pseudomonas aeruginosa induces hypoacylation of lipopolysaccharide and increases antimicrobial susceptibility.

    Science.gov (United States)

    Six, David A; Yuan, Yanqiu; Leeds, Jennifer A; Meredith, Timothy C

    2014-01-01

    The β-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.

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

    Science.gov (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  10. Best-practice evaluation-methods for wafer-fab photomask-requalification inspection tools

    Science.gov (United States)

    Cho, Chan Seob; Mungmode, Ashish; Taylor, Ron; Cho, David; Koh, Hui Peng

    2014-10-01

    Requalifying semiconductor photomasks remains critically important and is increasingly challenging for 20nm and 14nm node logic reticles. Patterns are becoming more complex on the photomask, and defect sensitivity requirements are more stringent than ever before. Reticle inspection tools are equally important for effective process development and the successful ramp and sustained yield for high volume manufacturing. The inspection stages considered were: incoming inspection to match with Mask Shop Outgoing result and to detect defects generated during transport; requalification by routine cycle inspection to detect Haze and any other defects; and inspection by in-house or Mask shop at the post cleaning. There are many critical capability and capacity factors for the decision for best inspection tool and strategy for high volume manufacturing, especially objective Lens NA, wavelength, power, pixel size, throughput, full-automation inspection linked with Overhead Transport, algorithm application, engineering application function, and inspection of PSM and OMOG . These tools are expensive but deliver differentiated value in terms of performance and throughput as well as extendibility. Performing a thorough evaluation and making a technically sound choice which explores these many factors is critical for success of a fab. This paper examines the methodology for evaluating two different photomask inspection tools. The focus is on ensuring production worthiness on real and advanced product photomasks requiring accurate evaluation of sensitivity, throughput, data analysis function and engineering work function on those product photomasks. Photomasks used for data collection are production reticles, PDM(Program defect Mask), SiN spray defect Reticle which is described that evaluates how the tools would perform on a contaminated plate.

  11. 抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选%Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    汪治华; 张国成; 李安茂; 周南; 陈一; 李小青; 苏字飞; 邓阳彬; 王治静

    2008-01-01

    Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children

  12. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.;

    2002-01-01

    The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  13. Crystallization and X-ray diffraction analysis of the β-ketoacyl-acyl carrier protein reductase FabG from Aquifex aeolicus VF5

    Energy Technology Data Exchange (ETDEWEB)

    Mao, Qilong [Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203 (United States); Duax, William L.; Umland, Timothy C., E-mail: umland@hwi.buffalo.edu [Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203 (United States); Department of Structural Biology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY (United States)

    2007-02-01

    FabG from A. aeolicus, a putative component of fatty-acid synthase II, has been overexpressed, purified and crystallized. Diffraction data have been collected to 1.8 Å resolution. The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 Å and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative β-ketoacyl-acyl carrier protein reductase. Structure–function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.

  14. Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients

    DEFF Research Database (Denmark)

    Cederkrantz, Elin; Andersson, Håkan; Bernhardt, Peter

    2015-01-01

    100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective......, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective...... dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation...

  15. Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of β-oxidation cycle.

    Science.gov (United States)

    Vick, Jacob E; Clomburg, James M; Blankschien, Matthew D; Chou, Alexander; Kim, Seohyoung; Gonzalez, Ramon

    2015-02-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli.

  16. Initial volume of a drug before it reaches the volume of distribution: pharmacokinetics of F(ab')2 antivenoms and other drugs.

    Science.gov (United States)

    Sevcik, Carlos; Salazar, Victor; Díaz, Patricia; D'Suze, Gina

    2007-10-01

    Fast disappearance of F(ab')2 antivenoms from the plasma compartment [Sevcik et al., 2004. Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues. Toxicon 44, 731-734; Vazquez et al., 2005. Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon 46, 797-805] suggests a quick time course to reach its final distribution volume. An equation was developed to describe how the volume occupied by a drug in the body grows with time. As discussed in the paper this equation is free of some shortcomings of an equation developed for the same purpose by Niazi [1976. Volume of distribution as a function of time. J. Pharm. Sci. 65, 452-454]. Fluorescence microscopy showed that the rapid initial decay in plasmatic F(ab')2 concentration may be related to uptake of F(ab')2 by vascular endothelium which, in combination with accumulation in the vascular wall connective tissue, may produce an intermediate plateau in F(ab')2 V(sl)(t), which reached its final value after 10 h. The V(sl)(t) equation predicts that the plasma concentration half-time of decay has little use to estimate how a drug distributes through the body to exert its action, and predicts that, in some instances, intermediate plateaus in the time course of V(sl)(t) exist. Data from the literature showed that the kinetic considerations for V(sl)(t) also apply to clevidipine, digoxin, digitoxin, lidocaine and thiopentone.

  17. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    Science.gov (United States)

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.

  18. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    Science.gov (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  19. Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

    Science.gov (United States)

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-11-19

    The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

  20. Crystallization and X-ray diffraction analysis of the beta-ketoacyl-acyl carrier protein reductase FabG from Aquifex aeolicus VF5.

    Science.gov (United States)

    Mao, Qilong; Duax, William L; Umland, Timothy C

    2007-02-01

    The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 A and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative beta-ketoacyl-acyl carrier protein reductase. Structure-function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.

  1. Activités insecticides de Striga hermonthica (Del. Benth (Scrophulariaceae sur Callosobrichus maculatus (Fab. (Coleoptera : Bruchidae

    Directory of Open Access Journals (Sweden)

    Nacoulma OG.

    2006-01-01

    Full Text Available Insecticidal activities of Striga hermonthica (Del. Benth (Scrophulariacecae on Callobruchus maculatus (Fab. (Coleptera Bruchidae. This paper deals with insecticidal potentialities of Striga hermonthica (Del. (Scrophulariaceae in protection of cowpea Vigna unguculata (L. Walp against Callosobruchus maculatus (Fab. (Coleoptera: Bruchidae during storage. Crude acetone extract at 0,5% w/w (100 mg of extract for 20 g of grain exhibits 48% of ovicidal effect and then reduces by half emergence rate of adult beetles at the first generation. This extract shows a weak insecticide activity against adults of C. maculatus. Petroleum ether fraction (0,4% w/w of the crude extract reveals ovicidal (51% and larvicidal (72% effects which reduce the emergence rate of adults to only 9%. LD50 and LD90 are monitored during crude extract fractionation to follow ovicidal and larvicidal compounds and to evaluate their efficacy during the isolation procedure. One fraction, mainly composed of two triterpenoid compounds has been identified as responsible of the ovicidal activity of S. hermonthica while the origin of the larvicidal activity hasn’t been identified.

  2. Nuclear energy release from fragmentation

    CERN Document Server

    Li, Cheng; Tsang, M B; Zhang, Feng-Shou

    2015-01-01

    Nuclear energy released by splitting Uranium and Thorium isotopes into two, three, four, up to eight fragments with nearly equal size are studied. We found that the energy released come from equally splitting the $^{235,238}$U and $^{230,232}$Th nuclei into to three fragments is largest. The statistical multifragmentation model is employed to calculate the probability of different breakup channels for the excited nuclei. Weighing the the probability distributions of fragments multiplicity at different excitation energies for the $^{238}$U nucleus, we found that an excitation energy between 1.2 and 2 MeV/u is optimal for the $^{235}$U, $^{238}$U, $^{230}$Th and $^{232}$Th nuclei to release nuclear energy of about 0.7-0.75 MeV/u.

  3. Hands as markers of fragmentation

    Directory of Open Access Journals (Sweden)

    A. Barnard

    2005-07-01

    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.

  4. Molecular characterization of monoclonal antibodies that inhibit acetylcholinesterase by targeting the peripheral site and backdoor region.

    Directory of Open Access Journals (Sweden)

    Yves Bourne

    Full Text Available The inhibition properties and target sites of monoclonal antibodies (mAbs Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE, have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.

  5. Phenomenology of Dihadron Fragmentation Function

    CERN Document Server

    Courtoy, A

    2016-01-01

    We report on the phenomenological results obtained through Dihadron Fragmentation Functions related processes. In 2015, an update on the fitting techniques for the Dihadron Fragmentation Functions has led to an improved extraction of the transversity PDF and, as a consequence, the nucleon tensor charge. We discuss the impact of the determination of the latter on search for physics Beyond the Standard Model, focusing on the error treatment. We also comment on the future of the extraction of the subleading-twist PDF $e(x)$ from JLab soon-to-be-released Beam Spin Asymmetry data.

  6. Transversity and dihadron fragmentation functions

    CERN Document Server

    Bacchetta, A; Bacchetta, Alessandro; Radici, Marco

    2005-01-01

    The observation of the quark transversity distribution requires another soft object sensitive to the quark's transverse spin. Dihadron fragmentation functions represent a convenient tool to analyze partonic spin, which can influence the angular distribution of the two hadrons. In particular, the so-called interference fragmentation functions can be used to probe transversity both in semi-inclusive deep inelastic scattering as well as proton-proton collisions. We discuss two single-spin asymmetries sensitive to transversity in the these two processes, at leading twist and leading order in alpha_S.

  7. RIA Fragmentation Line Beam Dumps

    Energy Technology Data Exchange (ETDEWEB)

    Stein, W

    2003-08-08

    The Rare Isotope Accelerator project involves generating heavy-element ion beams for use in a fragmentation target line to produce beams for physics research. The main beam, after passing through the fragmentation target, may be dumped into a beam dump located in the vacuum cavity of the first dipole magnet. For a dump beam power of 100 kW, cooling is required to avoid excessive high temperatures. The proposed dump design involves rotating cylinders to spread out the energy deposition and turbulent subcooled water flow through internal water cooling passages to obtain high, nonboiling, cooling rates.

  8. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  9. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G. (Purdue); (WU-MED); (CDC)

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  10. A thermodynamic theory of dynamic fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Yew, Ching H. [Texas Univ., Austin, TX (United States); Taylor, P.A. [Sandia National Labs., Albuquerque, NM (United States)

    1993-08-01

    We present a theory of dynamic fragmentation of brittle materials based on thermodynamic arguments. We recover the expressions for average fragment size and number as originally derived by Grady. We extend the previous work by obtaining descriptions of fragment size distribution and compressibility change due to the fragmentation process. The size distribution is assumed to be proportional to the spectral power of the strain history and a sample distribution is presented for a fragmentation process corresponding to a constant rate strain history. The description of compressibility change should be useful in computational studies of fragmentation. These results should provide insight into the process of fragmentation of brittle materials from hypervelocity impact.

  11. Population pressure and farm fragmentation:

    African Journals Online (AJOL)

    user

    small but farms are further fragmented into diminutive size fields due to ... terms of household characteristics; land use and performance indicators; technology adoption .... 'best' unit of measurement of farm size, and size of enterprises within farms will ..... less common, accounting for 18 percent (3 percent) and 10 percent (7.

  12. 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: fszhang@bnu.edu.cn [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)

    2016-08-15

    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.

  13. Nuclear energy release from fragmentation

    Science.gov (United States)

    Li, Cheng; Souza, S. R.; Tsang, M. B.; Zhang, Feng-Shou

    2016-08-01

    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 230,232Th and 235,238U 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 230,232Th and 235,238U 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.

  14. The Fragmentation of Literary Theory

    Science.gov (United States)

    Howard, Jennifer

    2005-01-01

    Syllabi from some 20 colleges and universities were reviewed with prominent English and literature departments and a discussion was held with a number of professors who teach literary theory. It is suggested that devolution and fragmentation of theory might be a survival strategy, an adaptation to the new realties of academic institutions.

  15. Fragmented nature : consequences for biodiversity

    NARCIS (Netherlands)

    Olff, Han; Ritchie, Mark E.

    2002-01-01

    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

  16. Fragmented nature: consequences for biodiversity

    NARCIS (Netherlands)

    Olff, H.; Ritchie, M.E.

    2002-01-01

    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

  17. Fragmented nature : consequences for biodiversity

    NARCIS (Netherlands)

    Olff, Han; Ritchie, Mark E.

    2002-01-01

    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

  18. Fragmented nature: consequences for biodiversity

    NARCIS (Netherlands)

    Olff, H.; Ritchie, M.E.

    2002-01-01

    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

  19. The VERDI fission fragment spectrometer

    Science.gov (United States)

    Frégeau, M. O.; Bryś, T.; Gamboni, Th.; Geerts, W.; Oberstedt, S.; Oberstedt, A.; Borcea, R.

    2013-12-01

    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.

  20. The VERDI fission fragment spectrometer

    Directory of Open Access Journals (Sweden)

    Frégeau M.O.

    2013-12-01

    Full Text Available 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.

  1. 苜蓿中华根瘤菌烯脂酰ACP还原酶基因fabI1功能研究%The enoyl-ACP reductase gene,fabI1, of Sinorhizobium meliloti is involved in salt tolerance, swarming mobility and nodulation efficiency

    Institute of Scientific and Technical Information of China (English)

    刘影; 朱家璧; 俞冠翘; 邹华松

    2009-01-01

    我们先前的工作表明,苜蓿中华根瘤菌的烯脂酰ACP还原酶基因fabI1在nifA突变根瘤中表达水平降低.本研究构建了fabI1的定点插入突变体.与野生型相比,突变菌株的生长速度变慢,在高浓度NaCl培养基上的生长能力降低.在半固体培养基上,该突变体的涌动能力完全丧失.在共生过程中,突变菌株在宿主植物上延迟结瘤,形成根瘤的能力下降.虽然苜蓿中华根瘤菌中的烯脂酰ACP还原酶基因fabI2的序列与fabI1有66%的一致性,但fabI2不能恢复fabI1突变体的表型,揭示了这两个基因在功能上的差异.

  2. Effect of degree of unsaturation of fatty acids on the activity of FabI (enoyl-acyl carrier protein reductase enzyme from Plasmodium falciparum: an enzoinformatics study

    Directory of Open Access Journals (Sweden)

    Sibhghatulla Shaikh

    2014-09-01

    Full Text Available Objective: To elucidate molecular interactions of enoyl-acyl carrier protein reductase (FabI with unsaturated fatty acids such as docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, octadecatrienoic acid, stearic acid and arachic acid to investigate the inhibitory activities of degree of unsaturation. Methods: Docking between these ligands and enzymes were performed using Autodock4.2. Results: Docosahexaenoic acid (a polyunsaturated fatty acid is more efficient inhibitor of enoylacyl carrier protein reductase (FabI compared to other unsaturated fatty acids with lesser double bonds and saturated fatty acid with reference to ∆G and Ki values. Hydrophobic interactions play an important role in the correct positioning of these fatty acids within the catalytic site of FabI enzyme to permit docking. Conclusions: It has been also observed that not only the degree of unsaturation affects the antiplasmodial activity, but the length of carbon chain also plays an important role in their inhibitory activity. Such information may aid in the design of versatile FabI-inhibitors.

  3. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

    NARCIS (Netherlands)

    A.F. Labrijn; A.O. Buijsse; E.T.J. van den Bremer; A.Y.W. Verwilligen; W.K. Bleeker; S.J. Thorpe; J. Killestein; C.H. Polman; R.C. Aalberse; J. Schuurman; J.G.J. van de Winkel; P.W.H.I. Parren

    2009-01-01

    Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules(1), Fab-arm exchange with therapeutic antibodies has not been demonstrat

  4. Structure-directed construction of a high-performance version of the enzyme FabG from the photosynthetic microorganism Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Liu, Yinghui; Feng, Yanbin; Cao, Xupeng; Li, Xia; Xue, Song

    2015-10-07

    PhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrier-protein reductase) catalyzes the β-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a high-performance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Sudden onset of EDTA-dependent pseudothrombocytopenia after therapy with the glycoprotein IIb/IIIa antagonist c7E3 Fab.

    Science.gov (United States)

    Stiegler, H; Fischer, Y; Steiner, S; Strauer, B E; Reinauer, H

    2000-03-01

    The development of thrombocytopenia following exposure to the platelet glycoprotein (GP) IIb/ IIIa receptor antagonist abciximab (c7E3 Fab, ReoPro) is associated with adverse clinical outcome and excessive bleeding. Pseudothrombocytopenia is an important differential diagnosis in sudden onset of thrombocytopenia in a patient treated with c7E3 Fab. We report on a case of documented sudden onset of EDTA-dependent pseudothrombocytopenia in a 63-year-old woman who was admitted for emergency coronary intervention. Four hours after bolus administration of c7E3 Fab, the platelet concentration in EDTA-anticoagulated blood decreased from 385 x 10(9)/l to 119 x 10(9)/l, and it showed a further decrease to 57 x 10(9)/l at the end of a 12-h infusion. Despite no warnings or abnormalities of the automated cell counter, platelet aggregates were observed by microscopic evaluation of the blood smear. Repeated platelet counts in citrate-anticoagulated samples revealed platelet concentrations within the reference range. EDTA-dependent pseudothrombocytopenia due to therapy with c7E3 Fab is an important differential diagnosis that needs to be excluded rapidly from other causes of thrombocytopenia to avoid unnecessary further examinations, discontinuation of therapy, or even initiation of inappropriate therapy.

  6. Preparation of single chain variable fragment of MG7 mAb by phage display technology

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Yong-Zhan Nie; Dai-Ming Fan; Xue-Yong Zhang

    2001-01-01

    AIM To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS mRNA was isolated from MG7-producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOⅢ of highly expressing MG7binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigenbinding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE. RESULTS The VH, VL and ScFv DNAs were about 340bp,320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATO Ⅲ cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATO Ⅲ cells.SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32. CONCLUSION The MG7 ScFv was successfully produced by phage antibody technology, which may

  7. Changes in membrane fatty acid composition through proton-induced fabF mutation enhancing 1-butanol tolerance in E. coli

    Science.gov (United States)

    Jeong, Haeyoung; Kim, Sun Hong; Han, Sang Soo; Kim, Myung Hee; Lee, Keun Chul

    2012-07-01

    While a rational approach based on genomic data has become the preferred method for microbial strain development, radiation-induced random mutagenesis is still a robust method for organisms such as plants whose genome or target gene information is unavailable. We previously reported on a combined approach that consists of proton irradiation and a long-term experimental evolution to enhance 1-butanol tolerance of the E. coli C strain so that it can be used as a basal strain for the production of 1-butanol, a potential biofuel along with ethanol. Genome sequencing of one randomly chosen clone (PKH5000) from the endpoint population revealed eleven mutations occurring in the coding regions, and we found that a mutation (F74C) in fabF gene encoding β-ketoacyl-ACP synthases II is associated with a twofold increase in the major unsaturated fatty acid, cis-vaccenic acid. The increase of cis-vaccenic acid by wild-type FabF, which is more active at low temperatures or in the presence of organic compounds, is considered to be a protective mechanism against cold stress. A structural analysis of the FabF protein suggests that the F74C mutation may affect the enzyme activity through a change in flexibility around the catalytic site. The expression of a plasmid that harbors mutant fabF gene in the fabF knockout strain enhanced growth in a medium containing butanol with a concomitant elevation of the cis-vaccenic acid level. Among the eight available Keio knockout strains for genes that have amino acid substitution in the PKH5000 strain, the fabF mutant showed the slowest growth in the presence of 0.7% butanol. We propose that fabF, as probably the gene most responsible for butanol tolerance in wild-type form, contributes further when converted into a F74C missense mutation, which is beneficial as it increases the level of cis-vaccenic acid.

  8. Fragmentation

    Science.gov (United States)

    K.H. Riitters

    2009-01-01

    Effective resource management takes into account the administrative and biophysical settings within which natural resources occur. A setting may be described in many ways; for example, by forest land ownership, by reserved and roadless designation, or by the distribution of human populations in relation to forest (chapter 3). The physical arrangement of forest in a...

  9. 抗内毒素Fab'对严重烧伤早期炎症细胞因子的影响%Effects of Fab' against LPS on inflanunatory cytokines during the early stage of severe burns in mice

    Institute of Scientific and Technical Information of China (English)

    庄颖; 张雅萍; 马思远

    2008-01-01

    目的 观察抗内毒素Fab'对严重烧伤早期肠源性内毒素血症小鼠肠道损伤的保护作用.方法 采用严重烧伤早期肠源性内毒素血症小鼠模型,分为烧伤组、治疗组及对照组,分别于6、12、24、48 h四个时相点测定血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-lβ、IL-10的浓度.结果 与正常对照组比较,烧伤后血清TNF-α、IL-1β、IL-10水平增高,差异有统计学意义(P<0.01);治疗组血清TNF-α、IL-1β、IL-10水平较烧伤组显著降低(P<0.01).病理检查结果提示治疗组较烧伤组肠黏膜损伤明显减轻.结论 抗内毒素Fab'能抑制内毒素所诱导的TNF-α、IL-1β产生,同时调节血清中的IL-10水平,减轻内毒素对机体的损害,从而起到对严重烧伤后肠源性脓毒症的防治作用.%Objective To investigate the effect of the Fab' against LPS on intestinal mucosa during the early stage of severe bums in mice. Methods The gut-derived endotoximia mice model in the early stage of severe burn was created. The mice were randomly divided into three groups: burn group, control group and treatment group. At 6,12,24 and 48 h after burns ,levels of serum TNF-a,IL-1β and IL-10 were detenninated by enzyme linked immunosorbant assay. Results After burns,levels of serum TNF-α,IL-1 β and IL-10 in burn group were significantly higher than those in the control group (P<0.01). The levels of serum TNF-α,IL-β and IL-10 in treatment group were significantly lowere than in the burn group (P < 0.01 ). Pathological examinations revealed that the damage to the intestinal mucosa in treatment group was obviously alleviated as compared with bum group. Conclusion The Fab' against LPS can significantly decrease the levels of serum TNF-α,IL-1β and IL-10 during the early stage in severely burned gut-derived endotoximia mice, and inhibit injury by LPS,then prevent and cure burn sepsis.

  10. FAB-MS quantitatively analyze ligustrazine with the non-linear curve fitting of total ion current%FAB-MS总离子流非线性拟合法分析川芎嗪

    Institute of Scientific and Technical Information of China (English)

    郭志峰; 宋彩瑜; 郭婷婷; 何乾妹

    2009-01-01

    目的:用快原子轰击质谱(FAB-MS)建立一种川芎嗪定量分析的新方法.方法:以氘代甲醇为氘代试剂,激光照射0.5h得到的氘代川芎嗪为内标物;内标物与被测样品分子离子峰总离子流作非线性拟合,其初始峰强的比值与样品浓度呈线性,藉此对样品定量分析.质谱条件:快原子轰击源(FAB),仪器分辨率1000,枪压8kV,原子枪气Ar,底物为甘油.结果:氘代试剂用氘代甲醇,激光照射0.5 h下,氘代率为2.43%;以其作内标物,得到川芎嗪定最分析的工作曲线,线性方程为Y=6.75+2.24 X(r=0.9908),精密度RSD=2.9%.用此方法对磷酸川芎嗪片剂进行分析,给出了2种不同前处理方法的分析结果43.0 mg·片-1和45.0 mg·片-1.结论:快原子轰击质谱的非线性拟合法,是一种川芎嗪定量分析的新方法,该方法只需微克级样品,且干扰少,精度高.

  11. Equine Immunoglobulin and Equine Neutralizing F(ab?)2 Protect Mice from West Nile Virus Infection

    OpenAIRE

    Jiannan Cui; Yongkun Zhao; Hualei Wang; Boning Qiu; Zengguo Cao; Qian Li; Yanbo Zhang; Feihu Yan; Hongli Jin; Tiecheng Wang; Weiyang Sun; Na Feng; Yuwei Gao; Jing Sun; Yanqun Wang

    2016-01-01

    West Nile virus (WNV) is prevalent in Africa, Europe, the Middle East, West Asia, and North America, and causes epidemic encephalitis. To date, no effective therapy for WNV infection has been developed; therefore, there is urgent need to find an efficient method to prevent WNV disease. In this study, we prepared and evaluated the protective efficacy of immune serum IgG and pepsin-digested F(ab′)2 fragments from horses immunized with the WNV virus-like particles (VLP) expressing the WNV M and ...

  12. Efficient and accurate fragmentation methods.

    Science.gov (United States)

    Pruitt, Spencer R; Bertoni, Colleen; Brorsen, Kurt R; Gordon, Mark S

    2014-09-16

    Conspectus Three novel fragmentation methods that are available in the electronic structure program GAMESS (general atomic and molecular electronic structure system) are discussed in this Account. The fragment molecular orbital (FMO) method can be combined with any electronic structure method to perform accurate calculations on large molecular species with no reliance on capping atoms or empirical parameters. The FMO method is highly scalable and can take advantage of massively parallel computer systems. For example, the method has been shown to scale nearly linearly on up to 131 000 processor cores for calculations on large water clusters. There have been many applications of the FMO method to large molecular clusters, to biomolecules (e.g., proteins), and to materials that are used as heterogeneous catalysts. The effective fragment potential (EFP) method is a model potential approach that is fully derived from first principles and has no empirically fitted parameters. Consequently, an EFP can be generated for any molecule by a simple preparatory GAMESS calculation. The EFP method provides accurate descriptions of all types of intermolecular interactions, including Coulombic interactions, polarization/induction, exchange repulsion, dispersion, and charge transfer. The EFP method has been applied successfully to the study of liquid water, π-stacking in substituted benzenes and in DNA base pairs, solvent effects on positive and negative ions, electronic spectra and dynamics, non-adiabatic phenomena in electronic excited states, and nonlinear excited state properties. The effective fragment molecular orbital (EFMO) method is a merger of the FMO and EFP methods, in which interfragment interactions are described by the EFP potential, rather than the less accurate electrostatic potential. The use of EFP in this manner facilitates the use of a smaller value for the distance cut-off (Rcut). Rcut determines the distance at which EFP interactions replace fully quantum

  13. Rational optimization of drug-target residence time: Insights from inhibitor binding to the S. aureus FabI enzyme-product complex

    Science.gov (United States)

    Chang, Andrew; Schiebel, Johannes; Yu, Weixuan; Bommineni, Gopal R.; Pan, Pan; Baxter, Michael V.; Khanna, Avinash; Sotriffer, Christoph A.; Kisker, Caroline; Tonge, Peter J.

    2013-01-01

    Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme. PMID:23697754

  14. Fragmentation in the biopharmaceutical industry.

    Science.gov (United States)

    Goldsmith, Andrew D; Varela, Francisco E

    2017-02-01

    The large number of biopharmaceutical mergers and acquisitions (M&A) that occurred over the past decade has generated questions about whether the industry is consolidating around too-few players, negatively impacting both the number of medicines developed and overall innovation. However, closer examination of the level of biopharmaceutical consolidation by prescription sales shows that the industry was more fragmented in 2015 than in 2003. The trend towards increasing fragmentation is also observed across noncommercial and independent metrics over the same time period. The number and size of M&A deals has masked an active and competitive marketplace in which market growth and the number of companies entering the market exceeded the apparent reduction in the number of players caused by acquisitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Fragment separator momentum compression schemes

    Energy Technology Data Exchange (ETDEWEB)

    Bandura, Laura, E-mail: bandura@anl.gov [Facility for Rare Isotope Beams (FRIB), 1 Cyclotron, East Lansing, MI 48824-1321 (United States); National Superconducting Cyclotron Lab, Michigan State University, 1 Cyclotron, East Lansing, MI 48824-1321 (United States); Erdelyi, Bela [Argonne National Laboratory, Argonne, IL 60439 (United States); Northern Illinois University, DeKalb, IL 60115 (United States); Hausmann, Marc [Facility for Rare Isotope Beams (FRIB), 1 Cyclotron, East Lansing, MI 48824-1321 (United States); Kubo, Toshiyuki [RIKEN Nishina Center, RIKEN, Wako (Japan); Nolen, Jerry [Argonne National Laboratory, Argonne, IL 60439 (United States); Portillo, Mauricio [Facility for Rare Isotope Beams (FRIB), 1 Cyclotron, East Lansing, MI 48824-1321 (United States); Sherrill, Bradley M. [National Superconducting Cyclotron Lab, Michigan State University, 1 Cyclotron, East Lansing, MI 48824-1321 (United States)

    2011-07-21

    We present a scheme to use a fragment separator and profiled energy degraders to transfer longitudinal phase space into transverse phase space while maintaining achromatic beam transport. The first order beam optics theory of the method is presented and the consequent enlargement of the transverse phase space is discussed. An interesting consequence of the technique is that the first order mass resolving power of the system is determined by the first dispersive section up to the energy degrader, independent of whether or not momentum compression is used. The fragment separator at the Facility for Rare Isotope Beams is a specific application of this technique and is described along with simulations by the code COSY INFINITY.

  16. Fragment separator momentum compression schemes.

    Energy Technology Data Exchange (ETDEWEB)

    Bandura, L.; Erdelyi,