Cell surface expression of single chain antibodies with applications to imaging of gene expression in vivo

International Nuclear Information System (INIS)

Northrop, Jeffrey P.; Bednarski, Mark; Li, King C.; Barbieri, Susan O.; Lu, Amy T.; Nguyen, Dee; Varadarajan, John; Osen, Maureen; Star-Lack, Josh

2003-01-01

Imaging of gene expression in vivo has many potential uses for biomedical research and drug discovery, ranging from the study of gene regulation and cancer to the non-invasive assessment of gene therapies. To streamline the development of imaging marker gene technologies for nuclear medicine, we propose a new approach to the design of reporter/probe pairs wherein the reporter is a cell surface-expressed single chain antibody variable fragment that has been raised against a low molecular weight imaging probe with optimized pharmacokinetic properties. Proof of concept of the approach was achieved using a single chain antibody variable fragment that binds with high affinity to fluorescein and an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine penta-acetic acid labeled with the gamma-emitter 111 In. We demonstrate specific high-affinity binding of this probe to the cell surface-expressed reporter in vitro and assess the in vivo biodistribution of the probe both in wild-type mice and in mice harboring tumor xenografts expressing the reporter. Specific uptake of the probe by, and in vivo imaging of, tumors expressing the reporter are shown. Since ScFvs with high affinities can be raised to almost any protein or small molecule, the proposed methodology may offer a new flexibility in the design of imaging tracer/reporter pairs wherein both probe pharmacokinetics and binding affinities can be readily optimized. (orig.)

Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

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Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

2012-08-22

We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

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Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-04-28

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

RECOMBINATION OF ANTIBODY POLYPEPTIDE CHAINS IN THE PRESENCE OF ANTIGEN

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Metzger, Henry; Mannik, Mart

1964-01-01

Conditions were developed by which the separated H and L chains of gamma2 globulins recombined to form four-chained molecules in good yields. In the absence of antigen, anti-2,4-dinitrophenyl (anti-DNP) H chains randomly reassociated with a mixture of antibody and non-specific gamma2 globulin L chains. In the presence of a specific hapten, however, the antibody H chains preferentially interacted with the anti-DNP L chains. Antibody H chain-antibody L chain recombinants formed in the presence of hapten were more active than the corresponding recombinants formed in the absence of hapten. Speculations are made regarding the possible mechanisms and biological significance of these effects. PMID:14247718

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

African Journals Online (AJOL)

Abstract. Purpose: To develop specific single chain variable fragments (scFv) against ... libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was ..... Hermelink H, Koscielniak E. A human recombinant.

Arg9 facilitates the translocation and downstream signal inhibition of an anti-HER2 single chain antibody

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Hu Yi

2012-07-01

Full Text Available Abstract Background HER2 plays a critical role in the pathogenesis of many cancers and is linked to poor prognosis or cancer metastases. Monoclonal antibodies, such as Herceptin against HER2-overexpressing cancers, have showed satisfactory clinical therapeutic effect. However, they have difficulty to surmount obstacles to enter cells or blood–brain barrier. Results In this study, a cell-penetrating peptide Arg9 was linked to the C-terminus of anti-HER2 single chain antibody (MIL5scFv. Flow cytometry, confocal microscopy and electron microscopy analysis all revealed that Arg9 peptide facilitated the penetration of MIL5scFv into HER2-negative cell line NIH3T3 and orientate in mitochondria. More interestingly, Western blot assay showed the potential enhanced bioactivity of MIL5scFv-Arg9 in HER2+ cell line SKOV3, indicating that Arg9 could help large molecules (e.g. antibody to penetrate into cells and therefore enhance its anti-neoplastic function. Conclusions Our work represented an attractive by preliminary strategy to enhance the therapeutic effect of existing antibodies by entering cells easier, or more desirable, surmounting the physical barriers, especially in hard-to-reach cancers such as brain metastases cases.

Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

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Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

2002-01-01

AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

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Liu, Yang; Zhou, Huihao; Zhu, Juanjuan; Gao, Yongxiang; Niu, Liwen; Liu, Jing; Teng, Maikun

2009-01-01

An antibody–antigen complex consisting of a single-chain variable fragment of the potential therapeutic antibody chA21 and an N-terminal fragment (residues 1–192) of the human ErbB2 extracellular domain was expressed, purified and crystallized. X-ray diffraction data were collected to 2.45 Å resolution. ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody–antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1–192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 Å resolution from a single flash-cooled crystal; the crystal belonged to space group P2 1 2 1 2 1

Modelling antibody side chain conformations using heuristic database search.

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Ritchie, D W; Kemp, G J

1997-01-01

We have developed a knowledge-based system which models the side chain conformations of residues in the variable domains of antibody Fv fragments. The system is written in Prolog and uses an object-oriented database of aligned antibody structures in conjunction with a side chain rotamer library. The antibody database provides 3-dimensional clusters of side chain conformations which can be copied en masse into the model structure. The object-oriented database architecture facilitates a navigational style of database access, necessary to assemble side chains clusters. Around 60% of the model is built using side chain clusters and this eliminates much of the combinatorial complexity associated with many other side chain placement algorithms. Construction and placement of side chain clusters is guided by a heuristic cost function based on a simple model of side chain packing interactions. Even with a simple model, we find that a large proportion of side chain conformations are modelled accurately. We expect our approach could be used with other homologous protein families, in addition to antibodies, both to improve the quality of model structures and to give a "smart start" to the side chain placement problem.

Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

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Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

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Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

2018-01-01

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

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Kazuhisa Sugimura

2013-02-01

Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

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Cui, Xiping; Vasylieva, Natalia; Wu, Panpan; Barnych, Bogdan; Yang, Jun; Shen, Ding; He, Qiyi; Gee, Shirley J; Zhao, Suqing; Hammock, Bruce D

2017-10-17

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC 50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

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

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

2013-08-01

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

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

African Journals Online (AJOL)

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

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies

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Katherine E. Harris

2018-04-01

Full Text Available We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1. This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Expression, production and renaturation of a functional single-chain ...

African Journals Online (AJOL)

The single-chain variable antibody fragment (scFv) against human intercellular adhesion molecule-1 (ICAM-1) was expressed at a high level in Escherichia coli as inclusion bodies. We attempted to refold the scFv by ion-exchange chromatography (IEC), dialysis and dilution. The results show that the column ...

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

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Barth Stefan

2005-01-01

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

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

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

2014-01-01

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

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

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Scott A Walper

Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

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

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George P Anderson

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

Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

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Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

2011-08-05

Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

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

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

2012-01-01

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

SINGLE DOMAIN ANTIBODIES AND BIOENGINEERING DRUGS ON THEIR BASIS: NEW OPPORTUNITIES FOR DIAGNOSTICS AND THERAPY

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E. N. Gorshkova

2016-01-01

Full Text Available Almost 20 years ago, a unique class of antibodies devoid of L chains was discovered in Camelidae blood serum. Only one variable domain is responsible for antigen recognition in these unusual antibodies. A recombinant protein, which is analogue to such antigen-recognizing variable domain was called the single domain antibody (sdAb, “nanobody” or “nanoantibody”. The single-domain antibodies and their derivatives have been widely used in the field of biology, toxicology and medicine offering new opportunities for diagnosis and treatment of cancer, autoimmune diseases, infectious diseases, and for toxin neutralization. This review focuses on latest researches in the field and concerns some prospectives for creation of nanoantibody-based diagnostic and therapeutic drugs.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

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Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

2013-04-01

Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by

High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

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Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

2014-03-01

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

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

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Ali Riazi

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

Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin

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Goldman, Ellen R.; Anderson, George P.; Zabetakis, Dan; Walper, Scott; Liu, Jinny L.; Bernstein, Rachael; Calm, Alena; Carney, James P.; O’Brien, Thomas W.; Walker, Jennifer L.; Garber, Eric A. E.

2011-01-01

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. PMID:22174977

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

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

2015-10-01

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

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

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Dorota Smolarek

2012-06-01

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

Using llama derived single domain antibodies to target botulinum neurotoxins

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

2010-04-01

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

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

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Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

2015-01-01

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

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Timo Heidt

Full Text Available BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111Indium ((111In via bifunctional DTPA ( = (111In-LIBS/(111In-control. Autoradiography after incubation with (111In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2, 4010 ± 630 DLU/mm(2 and 4520 ± 293 DLU/mm(2 produced a significantly higher ligand uptake compared to (111In-control (2101 ± 76 DLU/mm(2, 1181 ± 96 DLU/mm(2 and 1866 ± 246 DLU/mm(2 indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2 vs. 17390 ± 7470 DLU/mm(2; P<0.05. These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01. CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of

Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

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Yang J

2014-05-01

Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

Advances in single chain technology.

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Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

2015-10-07

The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses.

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

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

2017-01-01

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

Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement

International Nuclear Information System (INIS)

Grennan, Kathleen; Strachan, Gillian; Porter, Andrew J.; Killard, Anthony J.; Smyth, Malcolm R.

2003-01-01

This work describes the development of an electrochemical immunosensor for the analysis of atrazine using recombinant single-chain antibody (scAb) fragments. The sensors are based on carbon paste screen-printed electrodes incorporating the conducting polymer polyaniline (PANI)/poly(vinylsulphonic acid) (PVSA), which enables direct mediatorless coupling to take place between the redox centres of antigen-labelled horseradish peroxidase (HRP) and the electrode surface. Competitive immunoassays can be performed in real-time using this separation-free system. Analytical measurements based on the pseudo-linear relationship between the slope of a real-time amperometric signal and the concentration of analyte, yield a novel immunosensor set-up capable of regenerationless amperometric analysis. Multiple, sequential measurements of standards and samples can be performed on a single scAb-modified surface in a matter of minutes. No separation of bound and unbound species was necessary prior to detection. The system is capable of measuring atrazine to a detection limit of 0.1 ppb (0.1 μg l -1 ). This system offers the potential for rapid, cost-effective immunosensing for the analysis of samples of environmental, medical and pharmaceutical significance

The use of a cocktail of single chain Fv antibody fragments to improve the in vitro and in vivo targeting of melanoma

International Nuclear Information System (INIS)

Pacifico, M.D.; Pearl, R.A.; Kupsch, J.M.

2006-01-01

Radio scintigraphy using single chain antibody fragments (scFvs) offers a potenti al means of early detection of melanoma metastases. However, previous studies have shown suboptimal levels of tumour localization and nonspecific background accumulation which may be due to antigen heterogeneity. We aimed to improve tumour localization by using a cocktail of different scFvs targeting different epitopes on melanoma cells. We have previously developed three scFvs against distinct and highly tumour-specific melanoma cell-surface antigens by chain shuffling and antibody phage selection on melanoma cells. Three scFvs, RAFT3, B3 and B4 were labeled with 1 25I odine and tested both individually and as a cocktail in a nude mouse xenograft model far human melanoma. Results demonstrated improved tumour localization in vivo when compared to the individual scFvs. Tumour uptake of the cocktail at l hour was 24.220% ID/g (injected dose/gram) compared with 2.854%, 2.263% and 1.355% far B4, RAFT3 and B3, respectively, when injected individually. In addition, the cocktail exhibited significantly superior tumour to normal tissue ratios far muscle and spleen (p<0.05). A combination or cocktail of scFv clones may have an advantage aver individual scFvs far melanoma targeting in patients because of heterogeneity in the expression of different epitopes of antigens on melanoma cells

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

International Nuclear Information System (INIS)

Hu, Zu-Quan; Li, He-Ping; Zhang, Jing-Bo; Huang, Tao; Liu, Jin-Long; Xue, Sheng; Wu, Ai-Bo; Liao, Yu-Cai

2013-01-01

Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

Energy Technology Data Exchange (ETDEWEB)

Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

2013-02-18

Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

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María Elena Iezzi

2018-02-01

Full Text Available Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs, in particular those engineered from the variable heavy-chain fragment (VHH gene found in Camelidae heavy-chain antibodies (or IgG2 and IgG3, are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.

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van der Steen, Sophieke C H A; van Tilborg, Angela A G; Vallen, Myrtille J E; Bulten, Johan; van Kuppevelt, Toin H; Massuger, Leon F A G

2016-03-01

The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology. The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status. The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status. Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

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Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

2017-09-02

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

2017-12-01

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

[Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

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Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

2009-06-01

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

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Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

Science.gov (United States)

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

Science.gov (United States)

Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

2014-10-01

Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond.

Science.gov (United States)

Liu, Jinny L; Goldman, Ellen R; Zabetakis, Dan; Walper, Scott A; Turner, Kendrick B; Shriver-Lake, Lisa C; Anderson, George P

2015-10-09

Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity. Introduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production. The ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced

Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

Science.gov (United States)

Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

2012-10-01

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

Science.gov (United States)

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

NARCIS (Netherlands)

Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

2003-01-01

In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

OpenAIRE

Matsukizono, Miho; Kamegawa, Mariko; Tanaka, Koichi; Kohra, Shinya; Arizono, Koji; Hamazoe, Yuta; Sugimura, Kazuhisa

2013-01-01

An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competi...

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

Science.gov (United States)

Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

2015-01-01

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

Science.gov (United States)

Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

2016-11-01

Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

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

Science.gov (United States)

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

2017-01-01

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

Affinity of antibody secreted by a single cell

International Nuclear Information System (INIS)

Doran, D.M.

1978-01-01

It was the intention of this research to measure the affinity of antibody secreted by a single cell, and to describe the spectrum of affinities displayed in response to antigenic stimulation. The single cell secreting specific antibody was isolated by means of the hemolytic plaque assay. The amount of antibody secreted by the cell was to be measured through the use of a solid phase radioimmunoassay. The affinity of the antibody would be estimated by comparing the diameter of the plaque, and the amount of antibody secreted, with a mathematical theory of the formation of a plaque in agar. As a test system, a solid phase radioimmunoassay was developed for human serum albumin using antibody coupled to Sephadex. A sensitivity of 1 nanogram was attained with this assay. A solid phase radioimmunoassay for mouse immunoglobulin M was developed, using antibody coupled to Sepharose. The sensitivity attained with this assay was only on the order of 10 micrograms. The mouse immunoglobulin M radioimmunoassay was not sensitive enough to measure the amount of antibody secreted by a single cell. From a theoretical equation, the relationship between antibody affinity, plaque diameter and antibody secretion rate was calculated for the experimental conditions used in this research. By assuming a constant antibody secretion rate, an effective binding constant for the antibody was estimated from the average plaque diameters. This effective binding constant was observed to increase during the immune response

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

Science.gov (United States)

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

2015-12-01

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

Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook

Directory of Open Access Journals (Sweden)

Mehdi Arbabi-Ghahroudi

2017-11-01

Full Text Available Tremendous effort has been expended over the past two and a half decades to understand many aspects of camelid heavy chain antibodies, from their biology, evolution, and immunogenetics to their potential applications in various fields of research and medicine. In this article, I present a historical perspective on the development of camelid single-domain antibodies (sdAbs or VHHs, also widely known as nanobodies since their discovery and discuss the advantages and disadvantages of these unique molecules in various areas of research, industry, and medicine. Commercialization of camelid sdAbs exploded in 2001 with a flurry of patents issued to the Vrije Universiteit Brussel (VUB and later taken on by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB and, after 2002, the VIB-founded spin-off company, Ablynx. While entrepreneurial spirit has certainly catalyzed the exploration of nanobodies as marketable products, IP restrictions may be partially responsible for the relatively long time span between the discovery of these biomolecules and their entry into the pharmaceutical market. It is now anticipated that the first VHH-based antibody drug, Caplacizumab, a bivalent anti-vWF antibody for treating rare blood clotting disorders, may be approved and commercialized in 2018 or shortly thereafter. This elusive first approval, along with the expiry of key patents, may substantially alter the scientific and biomedical landscape surrounding camelid sdAbs and pave the way for their emergence as mainstream biotherapeutics.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

Science.gov (United States)

De Nardis, Camilla; Hendriks, Linda J A; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B H; de Kruif, John

2017-09-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

Science.gov (United States)

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

Science.gov (United States)

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Baculovirus display of single chain antibody (scFv using a novel signal peptide

Directory of Open Access Journals (Sweden)

Gonzalez Gaëlle

2010-11-01

Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

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

DEFF Research Database (Denmark)

Duan, Zhi; Siegumfeldt, Henrik

2010-01-01

We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

Structure of single-chain single crystals of isotactic polystyrene and their radiation resistance

International Nuclear Information System (INIS)

Bu Haishan; Cao Jie; Xu Shengyong; Zhang Ze

1997-01-01

The structure of the single-chain single crystals of isotactic polystyrene (i-PS) was investigated by electron diffraction (ED) and high resolution electron microscopy (HREM). The nano-scale single-chain single crystals were found to be very stable to electron irradiation. According to the unit cell of i-PS crystals, the reflection rings in ED pattern and the lattice fringes in HREM images could be indexed, but the lower-index diffractions were not found. It is proposed that the single-chain single crystals are very small, thus secondary electrons may be allowed to escape and radiation damage is highly reduced, and that there are less lower-index lattice planes in the single-chain single crystals to provide sufficient diffraction intensity for recording. HREM images can be achieved at room temperature in the case of single-chain single crystals because of its stability to electron irradiation, therefore, this might be a novel experimental approach to the study of crystal structure of macromolecules

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

Science.gov (United States)

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Antibody Heavy Chain Variable Domains of Different Germline Gene Origins Diversify through Different Paths

Directory of Open Access Journals (Sweden)

Ufuk Kirik

2017-11-01

Full Text Available B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.

Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

1983-02-25

A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

Nanobodies - the new concept in antibody engineering

African Journals Online (AJOL)

STORAGESEVER

2009-06-17

Jun 17, 2009 ... These heavy-chain antibodies contain a single variable domain (VHH) and two ... clonal antibody products were on the market and more than 100 in ..... genous showing no sign of spontaneous dimerisation in contrast to scFv ...

Improved affinity of a chicken single-chain antibody to avian ...

African Journals Online (AJOL)

linyuan

2011-12-12

Dec 12, 2011 ... Jianhua Wang. 1. , Ying Zhang ... et al., 1991; Dougan et al., 1998; Chang et al., 2009). The site-directed ... Analysis of the three-dimensional structure of the antigen-antibody ..... Altering the fine specificity of an anti-Legionella ...

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

NARCIS (Netherlands)

Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

Science.gov (United States)

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Directory of Open Access Journals (Sweden)

Andrea Pecora

2015-03-01

Full Text Available Bovine viral diarrhea virus (BVDV is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2 was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

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.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

Science.gov (United States)

Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

2014-01-01

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

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

Directory of Open Access Journals (Sweden)

Els Beirnaert

2017-07-01

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

Effects of genetic engineering on the pharmacokinetics of antibodies

International Nuclear Information System (INIS)

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

1999-01-01

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

Anti-DNA antibodies: Sequencing, cloning, and expression

Energy Technology Data Exchange (ETDEWEB)

Barry, M.M.

1992-01-01

To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

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.

Modeling single cell antibody excretion on a biosensor

NARCIS (Netherlands)

Stojanovic, Ivan; Baumgartner, W.; van der Velden, T.J.G.; Terstappen, Leonardus Wendelinus Mathias Marie; Schasfoort, Richardus B.M.

2016-01-01

We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed

The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

KAUST Repository

Chailyan, Anna

2011-06-28

The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

International Nuclear Information System (INIS)

Sikora, K.; Alderson, T.St.J.; Ellis, J.

1983-01-01

A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants. (Auth.)

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

Science.gov (United States)

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Science.gov (United States)

Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

2015-01-01

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Single-Chain Folding of Synthetic Polymers: A Critical Update.

Science.gov (United States)

Altintas, Ozcan; Barner-Kowollik, Christopher

2015-11-23

The current contribution serves as a critical update to a previous feature article from us (Macromol. Rapid Commun. 2012, 33, 958-971), and highlights the latest advances in the preparation of single chain polymeric nanoparticles and initial-yet promising-attempts towards mimicking the structure of natural biomacromolecules via single-chain folding of well-defined linear polymers via so-called single chain selective point folding and repeat unit folding. The contribution covers selected examples from the literature published up to ca. September 2015. Our aim is not to provide an exhaustive review but rather highlight a selection of new and exciting examples for single-chain folding based on advanced macromolecular precision chemistry. Initially, the discussion focuses on the synthesis and characterization of single-chain folded structures via selective point folding. The second part of the feature article addresses the folding of well-defined single-chain polymers by means of repeat unit folding. The current state of the art in the field of single-chain folding indicates that repeat unit folding-driven nanoparticle preparation is well-advanced, while initial encouraging steps towards building selective point folding systems have been taken. In addition, a summary of the-in our view-open key questions is provided that may guide future biomimetic design efforts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

Science.gov (United States)

Pakdeepak, Kanet; Pata, Supansa; Chiampanichayakul, Sawitree; Kasinrerk, Watchara; Tatu, Thanusak

2016-01-01

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

Immunoprophylaxis in fish by injection of mouse antibody genes

DEFF Research Database (Denmark)

Lorenzen, Niels; Cupit, P.M.; Einer-Jensen, Katja

2000-01-01

Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals, The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of inf...

Stable single helical C- and I-chains inside single-walled carbon nanotubes

International Nuclear Information System (INIS)

Yao Z; Li Y; Jing X D; Meng F S; Zhao X; Li J H; Qiu Z Y; Yuan Q; Wang W X; Bi L; Liu H; Zhang Y P; Liu C J; Zheng S P; Liu B B

2016-01-01

The helicity of stable single helical carbon chains and iodine chains inside single-walled carbon nanotubes (SWCNTs) is studied by calculating the systematic van der Waals interaction energy. The results show that the optimal helical radius increases linearly with increasing tube radius, which produces a constant separation between the chain structure and the tube wall. The helical angle exhibits a ladder-like decrease with increasing tube radius, indicating that a large tube can produce a small helicity in the helical structures. (paper)

Single Chain Fv Constructs of Anti-Ganglioside GD2 Antibodies for Radioimaging and Radioimmunotherapy

International Nuclear Information System (INIS)

Cheung, Nai-Kong

2003-01-01

because of its broad and usually homogeneous distribution in human solid tumors, and most importantly, their absence on cell membranes of normal human tissues. In separate experiments, we have shown that T-cells transduced with the herpes simplex viral thymidine kinase (HSV-tk) gene can be radiolabeled with 131 I-FIAU to a safe nuclear radiation dose. Using a dicistronic construct we are inserting chimeric immune receptor plus HSV-tk into T-cells to allow such their trafficking to be radioactively monitored. We plan to study the role of cytokines, chemoreceptors and CD4 helper T-cells in recruiting CD8+ transduced T-cells to the tumor site. These studies should provide us with an adoptive cell therapy approach to target cytotoxicity to human tumors, and a lymphocyte tracking tool to study delivery to the tumor sites, to determine if they proliferate locally and/or recirculate. Such pharmacologic information is crucial for optimizing gene-modified T-cells in future clinical trials. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

Directory of Open Access Journals (Sweden)

Merima Bublin

Full Text Available Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1, carp (Cyp c 1 and rainbow trout (Onc m 1 parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

Science.gov (United States)

Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

2015-01-01

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

Science.gov (United States)

Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

2015-12-01

Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

Science.gov (United States)

Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

2003-05-01

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

Supramolecular Nanoparticles via Single-Chain Folding Driven by Ferrous Ions.

Science.gov (United States)

Wang, Fei; Pu, Hongting; Jin, Ming; Wan, Decheng

2016-02-01

Single-chain nanoparticles can be obtained via single-chain folding assisted by intramolecular crosslinking reversibly or irreversibly. Single-chain folding is also an efficient route to simulate biomacromolecules. In present study, poly(N-hydroxyethylacrylamide-co-4'-(propoxy urethane ethyl acrylate)-2,2':6',2''-terpyridine) (P(HEAm-co-EMA-Tpy)) is synthesized via reversible addition fragmentation chain transfer polymerization. Single-chain folding and intramolecular crosslinking of P(HEAm-co-EMA-Tpy) are achieved via metal coordination chemistry. The intramolecular interaction is characterized on ultraviolet/visible spectrophotometer (UV-vis spectroscopy), proton nuclear magnetic resonance ((1)H NMR), and differential scanning calorimetry (DSC). The supramolecular crosslinking mediated by Fe(2+) plays an important role in the intramolecular collapsing of the single-chain and the formation of the nanoparticles. The size and morphology of the nanoparticles can be controlled reversibly via metal coordination chemistry, which can be characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic force microscope (AFM). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel scenario of the folding transition of a single chain

International Nuclear Information System (INIS)

Yoshikawa, Kenichi; Yoshinaga, Natsuhiko

2005-01-01

Unique characteristics of a single polymer chain with the effects of stiffness and charge are discussed. It has been well established that a flexible polymer chain undergoes a continuous transition from an elongated coil to a compact globule, corresponding to the transition between disordered gas-like and disordered liquid-like states. Here, we will show that a semiflexible chain exhibits a discrete transition from coil to compact states, corresponding to a disorder-order transition to an ordered crystalline state. We will propose a novel strategy to obtain various kinds of nano-ordered structures from single chains connecting a pair of chains of different stiffness. We will also discuss the effect of charge, putting emphasis on intramolecular segregation in a single polyelectrolyte chain

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

Science.gov (United States)

Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

1994-12-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions

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Jinny L. Liu

2016-06-01

Full Text Available Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.

The research progress and medicine application of the ScFv antibody

International Nuclear Information System (INIS)

Qin Lili; Zhang Chunming

2005-01-01

Since the scholar of England and Japan had found the diphtheria antitoxin, the research of the antibody has experienced three phases: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. In recent years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-lives in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte. (authors)

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

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Goldman Ellen R

2007-11-01

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

Effect of Chain Conformation on the Single-Molecule Melting Force in Polymer Single Crystals: Steered Molecular Dynamics Simulations Study.

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Feng, Wei; Wang, Zhigang; Zhang, Wenke

2017-02-28

Understanding the relationship between polymer chain conformation as well as the chain composition within the single crystal and the mechanical properties of the corresponding single polymer chain will facilitate the rational design of high performance polymer materials. Here three model systems of polymer single crystals, namely poly(ethylene oxide) (PEO), polyethylene (PE), and nylon-66 (PA66) have been chosen to study the effects of chain conformation, helical (PEO) versus planar zigzag conformation (PE, PA66), and chain composition (PE versus PA66) on the mechanical properties of a single polymer chain. To do that, steered molecular dynamics simulations were performed on those polymer single crystals by pulling individual polymer chains out of the crystals. Our results show that the patterns of force-extension curve as well as the chain moving mode are closely related to the conformation of the polymer chain in the single crystal. In addition, hydrogen bonds can enhance greatly the force required to stretch the polymer chain out of the single crystal. The dynamic breaking and reformation of multivalent hydrogen bonds have been observed for the first time in PA66 at the single molecule level.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

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Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

Improved production and function of llama heavy chain antibody fragments by molecular evolution

NARCIS (Netherlands)

Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.

2000-01-01

The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

The biodistribution and pretargeting radioimmunoimaging of the fusion protein of anti-CEA single-chain antibody and core-streptavidin in human rectocolonic tumor bearing nude mice

International Nuclear Information System (INIS)

Yang Weidong; Li Biao; Zhu Chengmo; Jiang Xufeng; Feng Guowei; Wu Xiangpu

2002-01-01

Objective: To investigate the biodistribution and two-step pretargeting radioimmunoimaging of the fusion protein of anti-carcinoembryonic antigen (CEA) single-chain antibody (ScFv) and core-streptavidin in human rectocolonic tumor bearing nude mice. Methods: Before the injection of 153 Sm-biotin, the fusion protein of ScFv-core-streptavidin was pretargeted for 24 h (200 μg every nude mouse), 24 h later 153 Sm-biotin was injected. The uptake of radioactivity in tumor and normal tissues in 20 nude mice was measured at 1, 4, 8 and 24 h and the other 3 nude mice was scanned at 8 and 24 h after peritoneal injection of 153 Sm-biotin. Results: The tumor to blood ratio (tumor/blood) reached 0.49 , 1.21, 1.56 and 3.09 at 1, 4, 8 and 24 h respectively. Radioactivity concentration peaked at 8 h in tumor site and demonstrated a 'hot' area, with significant decreasing its background at 24 h. Conclusion: The fusion protein can elevate the tumor/blood ratio, shorten pretargeting and imaging process and also improve image quality

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

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McElhiney, Jacqui; Drever, Mathew; Lawton, Linda A.; Porter, Andy J.

2002-01-01

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples. PMID:12406716

Unexpectedly normal phase behavior of single homopolymer chains

International Nuclear Information System (INIS)

Paul, W.; Strauch, T.; Rampf, F.; Binder, K.

2007-01-01

Employing Monte Carlo simulations, we show that the topology of the phase diagram of a single flexible homopolymer chain changes in dependence on the range of an attractive square well interaction between the monomers. For a range of attraction larger than a critical value, the equilibrium phase diagram of the single polymer chain and the corresponding polymer solution phase diagram exhibit vapor (swollen coil, dilute solution), liquid (collapsed globule, dense solution), and solid phases. Otherwise, the liquid-vapor transition vanishes from the equilibrium phase diagram for both the single chain and the polymer solution. This change in topology of the phase diagram resembles the behavior known for colloidal dispersions. The interplay of enthalpy and conformational entropy in the polymer case thus can lead to the same topology of phase diagrams as the interplay of enthalpy and translational entropy in simple liquids

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

NARCIS (Netherlands)

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

2005-01-01

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

Domain walls in single-chain magnets

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Pianet, Vivien; Urdampilleta, Matias; Colin, Thierry; Clérac, Rodolphe; Coulon, Claude

2017-12-01

The topology and creation energy of domain walls in different magnetic chains (called Single-Chain Magnets or SCMs) are discussed. As these domain walls, that can be seen as "defects", are known to control both static and dynamic properties of these one-dimensional systems, their study and understanding are necessary first steps before a deeper discussion of the SCM properties at finite temperature. The starting point of the paper is the simple regular ferromagnetic chain for which the characteristics of the domain walls are well known. Then two cases will be discussed (i) the "mixed chains" in which isotropic and anisotropic classical spins alternate, and (ii) the so-called "canted chains" where two different easy axis directions are present. In particular, we show that "strictly narrow" domain walls no longer exist in these more complex cases, while a cascade of phase transitions is found for canted chains as the canting angle approaches 45∘. The consequence for thermodynamic properties is briefly discussed in the last part of the paper.

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

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Sharad P Adekar

2008-08-01

Full Text Available Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

DEFF Research Database (Denmark)

Deufel, T; Grove, A; Kofod, Hans

1985-01-01

Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

Single-ion and single-chain magnetism in triangular spin-chain oxides

Science.gov (United States)

Seikh, Md. Motin; Caignaert, Vincent; Perez, Olivier; Raveau, Bernard; Hardy, Vincent

2017-05-01

S r4 -xC axM n2Co O9 oxides (x =0 and x =2 ) are found to exhibit magnetic responses typical of single-chain magnets (SCMs) and single-ion magnets (SIMs), two features generally investigated in coordination polymers or complexes. The compound x =0 appears to be a genuine SCM, in that blocking effects associated with slow spin dynamics yield remanence and coercivity in the absence of long-range ordering (LRO). In addition, SIM signatures of nearly identical nature are detected in both compounds, coexisting with SCM in x =0 and with LRO in x =2 . It is also observed that a SCM response can be recovered in x =2 after application of magnetic field. These results suggest that purely inorganic systems could play a valuable role in the topical issue of the interplay among SIM, SCM, and LRO phenomena in low-dimensional magnetism.

Fab is the most efficient format to express functional antibodies by yeast surface display.

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Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

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Markiv Anatoliy

2011-11-01

Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

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

Science.gov (United States)

Bauer, Georg; Motz, Manfred

2016-11-01

Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

Science.gov (United States)

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Elementary excitations in single-chain magnets

Science.gov (United States)

Lutz, Philipp; Aguilà, David; Mondal, Abhishake; Pinkowicz, Dawid; Marx, Raphael; Neugebauer, Petr; Fâk, Björn; Ollivier, Jacques; Clérac, Rodolphe; van Slageren, Joris

2017-09-01

Single-chain magnets (SCMs) are one-dimensional coordination polymers or spin chains that display slow relaxation of the magnetization. Typically their static magnetic properties are described by the Heisenberg model, while the description of their dynamic magnetic properties is based on an Ising-like model. The types of excitations predicted by these models (collective vs localized) are quite different. Therefore we probed the nature of the elementary excitations for two SCMs abbreviated Mn2Ni and Mn2Fe , as well as a mononuclear derivative of the Mn2Fe chain, by means of high-frequency electron paramagnetic resonance spectroscopy (HFEPR) and inelastic neutron scattering (INS). We find that the HFEPR spectra of the chains are clearly distinct from those of the monomer. The momentum transfer dependence of the INS intensity did not reveal significant dispersion, indicating an essentially localized nature of the excitations. At the lowest temperatures these are modified by the occurrence of short-range correlations.

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

Science.gov (United States)

2012-08-01

H.J. Properties , production, and applications of camelid single domain antibody fragments. Appl. Microbiol. Biot. 2007, 77, 13‒22. 2. Goldman...Conway, J.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of Botulinum A...antiparallel coiled-coil inserted. J. Mol. Bio. 2001, 306, 25‒35. 9. Liu, J.L.; Anderson, G.P.; Goldman, E.R. Isolation of anti- toxin single domain

Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

Science.gov (United States)

Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

2013-04-08

Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high

Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

Science.gov (United States)

Daley, L P; Gagliardo, L F; Duffy, M S; Smith, M C; Appleton, J A

2005-03-01

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

Development of two dimensional electrophoresis method using single chain DNA

International Nuclear Information System (INIS)

Ikeda, Junichi; Hidaka, So

1998-01-01

By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

Science.gov (United States)

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

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

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Shibaguchi H

2017-08-01

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

Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

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Masato Kiyoshi

Full Text Available The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1. We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101 is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody. Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu. The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

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Gualberto Gonzalez-Sapienza

2017-08-01

Full Text Available With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

Polyclonal and monoclonal antibodies in clinic.

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Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

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

IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

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Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

2017-06-26

IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

Generation and Characterization of Polyclonal Antibody Against Part of Immunoglobulin Constant Heavy υ Chain of Goose

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Zhao, Panpan; Guo, Yongli; Ma, Bo; Wang, Junwei

2014-01-01

Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin that is the major antibody in bird, reptile, and lungfish blood. IgY consists of two light (λ) and two heavy (υ) chains. In the present study, polyclonal antibody against IgYFc was generated and evaluated. rIgYCυ3/Cυ4 was expressed in Escherichia coli, purified and utilized to raise polyclonal antibody in rabbit. High affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:204,800 for ELISA assay. The antibody can specifically recognize both rIgYCυ3/Cυ4 and native IgY by Western bolt analysis. Furthermore, the serum of Grus japonensis or immunoglobulin of chicken, duck, turkey, and silkie samples and dynamic changes of serum GoIgY after immunogenicity with GPV-VP3-virus-like particles (GPV-VP3-VLPs) can be detected with the anti-GoIgYFc polyclonal antibody. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of GoIgY. PMID:25171010

Single-copy entanglement in critical quantum spin chains

International Nuclear Information System (INIS)

Eisert, J.; Cramer, M.

2005-01-01

We consider the single-copy entanglement as a quantity to assess quantum correlations in the ground state in quantum many-body systems. We show for a large class of models that already on the level of single specimens of spin chains, criticality is accompanied with the possibility of distilling a maximally entangled state of arbitrary dimension from a sufficiently large block deterministically, with local operations and classical communication. These analytical results--which refine previous results on the divergence of block entropy as the rate at which maximally entangled pairs can be distilled from many identically prepared chains--are made quantitative for general isotropic translationally invariant spin chains that can be mapped onto a quasifree fermionic system, and for the anisotropic XY model. For the XX model, we provide the asymptotic scaling of ∼(1/6)log 2 (L), and contrast it with the block entropy

Unconventional phase transitions in a constrained single polymer chain

International Nuclear Information System (INIS)

Klushin, L I; Skvortsov, A M

2011-01-01

Phase transitions were recognized among the most fascinating phenomena in physics. Exactly solved models are especially important in the theory of phase transitions. A number of exactly solved models of phase transitions in a single polymer chain are discussed in this review. These are three models demonstrating the second order phase transitions with some unusual features: two-dimensional model of β-structure formation, the model of coil–globule transition and adsorption of a polymer chain grafted on the solid surface. We also discuss models with first order phase transitions in a single macromolecule which admit not only exact analytical solutions for the partition function with explicit finite-size effects but also the non-equilibrium free energy as a function of the order parameter (Landau function) in closed analytical form. One of them is a model of mechanical desorption of a macromolecule, which demonstrates an unusual first order phase transition with phase coexistence within a single chain. Features of first and second order transitions become mixed here due to phase coexistence which is not accompanied by additional interfacial free energy. Apart from that, there exist several single-chain models belonging to the same class (adsorption of a polymer chain tethered near the solid surface or liquid–liquid interface, and escape transition upon compressing a polymer between small pistons) that represent examples of a highly unconventional first order phase transition with several inter-related unusual features: no simultaneous phase coexistence, and hence no phase boundary, non-concave thermodynamic potential and non-equivalence of conjugate ensembles. An analysis of complex zeros of partition functions upon approaching the thermodynamic limit is presented for models with and without phase coexistence. (topical review)

Screened ion-ion interaction in mercury-chain compounds: Single chain

International Nuclear Information System (INIS)

Mohan, M.M.; Griffin, A.

1985-01-01

At room temperature, the mercury chains in Hg/sub 3-delta/AsF 6 exhibit phonons characteristic of a one-dimensional lattice. We calculate the screening of the Hg ion-ion interaction in a single chain by electrons moving in a cylindrical potential of finite radius, within the random-phase approximation. The resulting Bohm-Staver-type expression for the phonon velocity is (Z 2 mN/sub I//MN/sub e/)/sup 1/2/v/sub F/, where Z is the Hg ionic charge and N/sub I/ (N/sub e/) is the number of ions (electrons) per unit length. Use of the Tomonaga-Luttinger solution for the electronic response function (keeping only the small-momentum scattering processes) just renormalizes the Fermi velocity in this expression

Equivalence of chain conformations in the surface region of a polymer melt and a single Gaussian chain under critical conditions.

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Skvortsov, A M; Leermakers, F A M; Fleer, G J

2013-08-07

In the melt polymer conformations are nearly ideal according to Flory's ideality hypothesis. Silberberg generalized this statement for chains in the interfacial region. We check the Silberberg argument by analyzing the conformations of a probe chain end-grafted at a solid surface in a sea of floating free chains of concentration φ by the self-consistent field (SCF) method. Apart from the grafting, probe chain and floating chains are identical. Most of the results were obtained for a standard SCF model with freely jointed chains on a six-choice lattice, where immediate step reversals are allowed. A few data were generated for a five-choice lattice, where such step reversals are forbidden. These coarse-grained models describe the equilibrium properties of flexible atactic polymer chains at the scale of the segment length. The concentration was varied over the whole range from φ = 0 (single grafted chain) to φ = 1 (probe chain in the melt). The number of contacts with the surface, average height of the free end and its dispersion, average loop and train length, tail size distribution, end-point and overall segment distributions were calculated for a grafted probe chain as a function of φ, for several chain lengths and substrate∕polymer interactions, which were varied from strong repulsion to strong adsorption. The computations show that the conformations of the probe chain in the melt do not depend on substrate∕polymer interactions and are very similar to the conformations of a single end-grafted chain under critical conditions, and can thus be described analytically. When the substrate∕polymer interaction is fixed at the value corresponding to critical conditions, all equilibrium properties of a probe chain are independent of φ, over the whole range from a dilute solution to the melt. We believe that the conformations of all flexible chains in the surface region of the melt are close to those of an appropriate single chain in critical conditions, provided

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

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

2013-01-01

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley

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

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

2013-12-01

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

Neutralisation and binding of VHS virus by monovalent antibody fragments

DEFF Research Database (Denmark)

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

2001-01-01

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2...... 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...

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

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Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Uses of monoclonial antibody 8H9

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Cheung, Nai-Kong V.

2015-06-23

This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

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Zu-Quan Hu

2012-06-01

Full Text Available Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10⁻² μg/mL and contaminating mycelium at a quantity of 10⁻² mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

DEFF Research Database (Denmark)

Zhao, Heng; Stet, René J M; Skjødt, Karsten

2008-01-01

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

Effect of chain stiffness on the structure of single-chain polymer nanoparticles

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Moreno, Angel J.; Bacova, Petra; Lo Verso, Federica; Arbe, Arantxa; Colmenero, Juan; Pomposo, José A.

2018-01-01

Polymeric single-chain nanoparticles (SCNPs) are soft nano-objects synthesized by purely intramolecular cross-linking of single polymer chains. By means of computer simulations, we investigate the conformational properties of SCNPs as a function of the bending stiffness of their linear polymer precursors. We investigate a broad range of characteristic ratios from the fully flexible case to those typical of bulky synthetic polymers. Increasing stiffness hinders bonding of groups separated by short contour distances and increases looping over longer distances, leading to more compact nanoparticles with a structure of highly interconnected loops. This feature is reflected in a crossover in the scaling behaviour of several structural observables. The scaling exponents change from those characteristic for Gaussian chains or rings in θ-solvents in the fully flexible limit, to values resembling fractal or ‘crumpled’ globular behaviour for very stiff SCNPs. We characterize domains in the SCNPs. These are weakly deformable regions that can be seen as disordered analogues of domains in disordered proteins. Increasing stiffness leads to bigger and less deformable domains. Surprisingly, the scaling behaviour of the domains is in all cases similar to that of Gaussian chains or rings, irrespective of the stiffness and degree of cross-linking. It is the spatial arrangement of the domains which determines the global structure of the SCNP (sparse Gaussian-like object or crumpled globule). Since intramolecular stiffness can be varied through the specific chemistry of the precursor or by introducing bulky side groups in its backbone, our results propose a new strategy to tune the global structure of SCNPs.

Efficient Synthesis of Single-Chain Polymer Nanoparticles via Amide Formation

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Ana Sanchez-Sanchez

2015-01-01

Full Text Available Single-chain technology (SCT allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles. In this work we contribute to the enlargement of the SCT toolbox by demonstrating the efficient synthesis of single-chain polymer nanoparticles (SCNPs via intrachain amide formation. In particular, we exploit cross-linking between active methylene groups and isocyanate moieties as powerful “click” chemistry driving force for SCNP construction. By employing poly(methyl methacrylate- (PMMA- based copolymers bearing β-ketoester units distributed randomly along the copolymer chains and bifunctional isocyanate cross-linkers, SCNPs were successfully synthesized at r.t. under appropriate reaction conditions. Characterization of the resulting SCNPs was carried out by means of a combination of techniques including size exclusion chromatography (SEC, infrared (IR spectroscopy, proton nuclear magnetic resonance (1H NMR spectroscopy, dynamic light scattering (DLS, and elemental analysis (EA.

Porous silicon biosensor for detection of variable domain of heavy-chain of HCAb antibody

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Zhang, Hong-yan; Lü, Xiao-yi; Jia, Zhen-hong; Li, Jiang-wei; Zhang, Fu-chun

2012-03-01

In this paper, we produce porous silicon (PSi) by electrochemical etching, and it is the first time to evaluate the performance of label-free porous silicon biosensor for detection of variable domain of heavy chain of heavy-chain antibody (VHH). The binding of hen egg white lysozyme (HEWL) and VHH causes a red shift in the reflection spectrum of the biosensor. The red shift is proportional to the VHH concentration in the range from 14 g·ml-1 to 30 g·ml-1 with a detection limit of 0.648 ng·ml-1. The research is useful for the development of label-free biosensor applied in the rapid and sensitive determination of small molecules.

Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

DEFF Research Database (Denmark)

Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

2011-01-01

We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

Single-Domain Antibodies As Therapeutics against Human Viral Diseases

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Yanling Wu

2017-12-01

Full Text Available In full-size formats, monoclonal antibodies have been highly successful as therapeutics against cancer and immune diseases. However, their large size leads to inaccessibility of some epitopes and relatively high production costs. As an alternative, single-domain antibodies (sdAbs offer special advantages compared to full-size antibodies, including smaller size, larger number of accessible epitopes, relatively low production costs and improved robustness. Currently, sdAbs are being developed against a number of viruses, including human immunodeficiency virus-1 (HIV-1, influenza viruses, hepatitis C virus (HCV, respiratory syncytial virus (RSV, and enteric viruses. Although sdAbs are very potent inhibitors of viral infections, no sdAbs have been approved for clinical use against virial infection or any other diseases. In this review, we discuss the current state of research on sdAbs against viruses and their potential as therapeutics against human viral diseases.

Mechanics of biopolymer materials: Single chains to bulk properties

NARCIS (Netherlands)

Amuasi, H.E.; Storm, C.

2010-01-01

We outline the first stages in the multiscale modeling of biopolymer materials, starting with the statistical mechanics of single stiff chains. In the first coarse graining step, we demonstrate how to integrate out the single polymer degrees of freedom in supramolecular assemblies of such

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Science.gov (United States)

Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

2016-06-10

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

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D’Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; Erasmus, M. Frank; Hraber, Peter; Bradbury, Andrew R. M.

2018-01-01

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. PMID:29568296

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

Science.gov (United States)

Bergwerff, A A; Stroop, C J; Murray, B; Holtorf, A P; Pluschke, G; Van Oostrum, J; Kamerling, J P; Vliegenthart, J F

1995-06-01

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two

A Single Domain–Based Anti-Her2 Antibody Has Potent Antitumor Activities

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Xiaoqiong Wu

2018-04-01

Full Text Available Human epidermal growth factor receptor 2 (HER2 is overexpressed in approximately 20% to 30% of breast cancers and various other types of cancers, which plays a vital role in the cancer progression. Monoclonal antibodies targeting Her2 are now used in the clinic to treat Her2 overexpression cancer patients. However, relapse or resistance is frequent with the current therapies. To generate a new treatment avenue against Her2, we immunized and selected a specific anti-Her2 single domain antibody C3 for further studies. The C3-Fc antibody drove antibody-dependent cell-mediated cytotoxicity against Her2-positive tumor cells in vitro and resulted in potent antitumor growth in vivo. These data suggest that the C3-Fc antibody may provide an alternative avenue for Her2-positive cancer therapy.

Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

Directory of Open Access Journals (Sweden)

Wei-Gang Hu

Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

International Nuclear Information System (INIS)

Vindigni, Alessandro; Pini, Maria Gloria

2009-01-01

The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

Energy Technology Data Exchange (ETDEWEB)

Vindigni, Alessandro [Laboratorium fuer Festkoerperphysik, ETH Zuerich, CH-8093 Zuerich (Switzerland); Pini, Maria Gloria [Istituto dei Sistemi Complessi, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019 Sesto Fiorentino (Italy)], E-mail: vindigni@phys.ethz.ch

2009-06-10

The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

Science.gov (United States)

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

2014-01-01

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Covalent bond force profile and cleavage in a single polymer chain

Science.gov (United States)

Garnier, Lionel; Gauthier-Manuel, Bernard; van der Vegte, Eric W.; Snijders, Jaap; Hadziioannou, Georges

2000-08-01

We present here the measurement of the single-polymer entropic elasticity and the single covalent bond force profile, probed with two types of atomic force microscopes (AFM) on a synthetic polymer molecule: polymethacrylic acid in water. The conventional AFM allowed us to distinguish two types of interactions present in this system when doing force spectroscopic measurements: the first interaction is associated with adsorption sites of the polymer chains onto a bare gold surface, the second interaction is directly correlated to the rupture process of a single covalent bond. All these bridging interactions allowed us to stretch the single polymer chain and to determine the various factors playing a role in the elasticity of these molecules. To obtain a closer insight into the bond rupture process, we moved to a force sensor stable in position when measuring attractive forces. By optimizing the polymer length so as to fulfill the elastic stability conditions, we were able for the first time to map out the entire force profile associated with the cleavage of a single covalent bond. Experimental data coupled with molecular quantum mechanical calculations strongly suggest that the breaking bond is located at one end of the polymer chain.

Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

Directory of Open Access Journals (Sweden)

Begoña Barriuso

2016-06-01

Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

Exploring single chain amphiphile self-assembly and their possible roles in light transduction

DEFF Research Database (Denmark)

Monnard, Pierre-Alain

2011-01-01

Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain......: the medium composition in terms of ionic strengths and the medium physical parameters, such as temperature, significantly influence the formation of structures, as well as their subsequent stability. In addition, membranes composed of a single amphiphile type seem to be implausible as no potential amphiphile...... source studied to date can supply one single type of amphiphile at concentrations conducive to self-assembly. Mixtures of single-chain amphiphiles were therefore proposed to better model primitive membranes and potentially enhance their structural integrity1-3. Recently, we have established that complex...

Real-time observation of conformational switching in single conjugated polymer chains.

Science.gov (United States)

Tenopala-Carmona, Francisco; Fronk, Stephanie; Bazan, Guillermo C; Samuel, Ifor D W; Penedo, J Carlos

2018-02-01

Conjugated polymers (CPs) are an important class of organic semiconductors that combine novel optoelectronic properties with simple processing from organic solvents. It is important to study CP conformation in solution to understand the physics of these materials and because it affects the properties of solution-processed films. Single-molecule techniques are unique in their ability to extract information on a chain-to-chain basis; however, in the context of CPs, technical challenges have limited their general application to host matrices or semiliquid environments that constrain the conformational dynamics of the polymer. We introduce a conceptually different methodology that enables measurements in organic solvents using the single-end anchoring of polymer chains to avoid diffusion while preserving polymer flexibility. We explore the effect of organic solvents and show that, in addition to chain-to-chain conformational heterogeneity, collapsed and extended polymer segments can coexist within the same chain. The technique enables real-time solvent-exchange measurements, which show that anchored CP chains respond to sudden changes in solvent conditions on a subsecond time scale. Our results give an unprecedented glimpse into the mechanism of solvent-induced reorganization of CPs and can be expected to lead to a new range of techniques to investigate and conformationally manipulate CPs.

Quantum Heisenberg antiferromagnetic chains with exchange and single-ion anisotropies

International Nuclear Information System (INIS)

Peters, D; Selke, W; McCulloch, I P

2010-01-01

Using density matrix renormalization group calculations, ground state properties of the spin-1 Heisenberg chain with exchange and quadratic single-ion anisotropies in an external field are studied, for special choices of the two kinds of anisotropies. In particular, the phase diagram includes antiferromagnetic, spin-liquid (or spin-flop), IS2, and supersolid (or biconical) phases. Especially, new features of the spin-liquid and supersolid phases are discussed. Properties of the quantum chains are compared to those of corresponding classical spin chains.

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.

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

Science.gov (United States)

Rumfelt, L L; Avila, D; Diaz, M; Bartl, S; McKinney, E C; Flajnik, M F

2001-02-13

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

Science.gov (United States)

Alcorn, S.W.; Pascho, R.J.

2000-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

OpenAIRE

Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Bracci, Luisa; Lozzi, Luisa; Neri, Paolo; Adriani, Daniela; De Bernardis, Flavia; Cassone, Antonio

2003-01-01

Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exe...

Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

Science.gov (United States)

Ueberberg, Sandra; Meier, Juris J.; Waengler, Carmen; Schechinger, Wolfgang; Dietrich, Johannes W.; Tannapfel, Andrea; Schmitz, Inge; Schirrmacher, Ralf; Köller, Manfred; Klein, Harald H.; Schneider, Stephan

2009-01-01

OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS We report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS Our data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo. PMID:19592622

Force Spectroscopy of Hyaluronan by AFM; From H-bonded Networks Towards Single Chain Behavior

NARCIS (Netherlands)

Giannotti, M.I.; Rinaudo, Marguerite; Vancso, Gyula J.

2007-01-01

The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This comunication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures,

INCREASING OF THE EXPRESSION OF RECOMBINANT scFv-ANTIBODIES EFFICIENCY

Directory of Open Access Journals (Sweden)

O.V. Galkin

2017-10-01

Full Text Available Obtaining single-chain variable fragments (scFv of recombinant antibodies in E. coli cells is often associated with numerous problems causing low yields or inactive conformation of the product. The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins. Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

International Nuclear Information System (INIS)

Leger, J.; Chevalier, J.; Larue, C.; Gautier, P.; Planchenault, J.; Aumaitre, E.; Messner, P.; Puech, P.; Saccavini, J.C.; Pau, B.

1991-01-01

The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis

Pressure effects on single chain magnets

Energy Technology Data Exchange (ETDEWEB)

Mito, M. E-mail: mitoh@elcs.kyutech.ac.jp; Shindo, N.; Tajiri, T.; Deguchi, H.; Takagi, S.; Miyasaka, H.; Yamashita, M.; Clerac, R.; Coulon, C

2004-05-01

Pressure effects on a single chain magnet [Mn{sub 2}(saltmen){sub 2}Ni(pao){sub 2}(py){sub 2}](ClO{sub 4}){sub 2} (saltmen{sup 2-}=N,N'-(1,1,2,2-tetramethylethylene)bis(salicylideneiminate), and pao{sup -}=pyridine-2-aldoximate) have been investigated through AC magnetic measurements under pressure (P). The slow relaxation of the magnetization depends on pressure. Both the blocking temperature (T{sub B}) and energy barrier ({delta}) increase by pressurization, and those enhancements saturate at around P=7 kbar.

Single-Domain Antibodies as Tools to Perturb and Study RNA Viruses

NARCIS (Netherlands)

Hanke, Leo

2017-01-01

In this thesis, I describe the generation and characterization of alpaca-derived, antiviral, single-domain antibody fragments (VHHs). The antiviral targets of the described VHHs are the nuclear proteins of influenza A virus (IAV) and vesicular stomatitis virus (VSV). The described VHHs protect cells

Stereoelectronic Effect-Induced Conductance Switching in Aromatic Chain Single-Molecule Junctions.

Science.gov (United States)

Xin, Na; Wang, Jinying; Jia, Chuancheng; Liu, Zitong; Zhang, Xisha; Yu, Chenmin; Li, Mingliang; Wang, Shuopei; Gong, Yao; Sun, Hantao; Zhang, Guanxin; Liu, Zhirong; Zhang, Guangyu; Liao, Jianhui; Zhang, Deqing; Guo, Xuefeng

2017-02-08

Biphenyl, as the elementary unit of organic functional materials, has been widely used in electronic and optoelectronic devices. However, over decades little has been fundamentally understood regarding how the intramolecular conformation of biphenyl dynamically affects its transport properties at the single-molecule level. Here, we establish the stereoelectronic effect of biphenyl on its electrical conductance based on the platform of graphene-molecule single-molecule junctions, where a specifically designed hexaphenyl aromatic chain molecule is covalently sandwiched between nanogapped graphene point contacts to create stable single-molecule junctions. Both theoretical and temperature-dependent experimental results consistently demonstrate that phenyl twisting in the aromatic chain molecule produces different microstates with different degrees of conjugation, thus leading to stochastic switching between high- and low-conductance states. These investigations offer new molecular design insights into building functional single-molecule electrical devices.

Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG

International Nuclear Information System (INIS)

Hibma, M.; Griffin, J.F.T.

1992-01-01

Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species

Earlinet single calculus chain: new products overview

Science.gov (United States)

D'Amico, Giuseppe; Mattis, Ina; Binietoglou, Ioannis; Baars, Holger; Mona, Lucia; Amato, Francesco; Kokkalis, Panos; Rodríguez-Gómez, Alejandro; Soupiona, Ourania; Kalliopi-Artemis, Voudouri

2018-04-01

The Single Calculus Chain (SCC) is an automatic and flexible tool to analyze raw lidar data using EARLINET quality assured retrieval algorithms. It has been already demonstrated the SCC can retrieve reliable aerosol backscatter and extinction coefficient profiles for different lidar systems. In this paper we provide an overview of new SCC products like particle linear depolarization ratio, cloud masking, aerosol layering allowing relevant improvements in the atmospheric aerosol characterization.

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

Science.gov (United States)

Maggi, Maristella; Scotti, Claudia

2017-08-01

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

Population pharmacokinetics of recombinant coagulation factor VIII-SingleChain in patients with severe hemophilia A.

Science.gov (United States)

Zhang, Y; Roberts, J; Tortorici, M; Veldman, A; St Ledger, K; Feussner, A; Sidhu, J

2017-06-01

Essentials rVIII-SingleChain is a unique recombinant factor VIII (FVIII) molecule. A population pharmacokinetic model was based on FVIII activity of severe hemophilia A patients. The model was used to simulate factor VIII activity-time profiles for various dosing scenarios. The model supports prolonged dosing of rVIII-SingleChain with intervals of up to twice per week. Background Single-chain recombinant coagulation factor VIII (rVIII-SingleChain) is a unique recombinant coagulation factor VIII molecule. Objectives To: (i) characterize the population pharmacokinetics (PK) of rVIII-SingleChain in patients with severe hemophilia A; (ii) identify correlates of variability in rVIII-SingleChain PK; and (iii) simulate various dosing scenarios of rVIII-SingleChain. Patients/Methods A population PK model was developed, based on FVIII activity levels of 130 patients with severe hemophilia A (n = 91 for ≥ 12-65 years; n = 39 for  85% and > 93% of patients were predicted to maintain FVIII activity level above 1 IU dL -1 , at all times with three-times-weekly dosing (given on days 0, 2, and 4.5) at the lowest (20 IU kg -1 ) and highest (50 IU kg -1 ) doses, respectively. For twice weekly dosing (days 0 and 3.5) of 50 IU kg -1 rVIII-SingleChain, 62-80% of patients across all ages were predicted to maintain a FVIII activity level above 1 IU dL -1 at day 7. Conclusions The population PK model adequately characterized rVIII-SingleChain PK, and the model can be utilized to simulate FVIII activity-time profiles for various dosing scenarios. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Recombinant fragment of an antibody tailored for direct radioiodination

Czech Academy of Sciences Publication Activity Database

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

2012-01-01

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

Modification of Antibody Function by Mutagenesis.

Science.gov (United States)

Dasch, James R; Dasch, Amy L

2017-09-01

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

Molecular dynamics simulation of AFM studies of a single polymer chain

International Nuclear Information System (INIS)

Wang Wenhai; Kistler, Kurt A.; Sadeghipour, Keya; Baran, George

2008-01-01

Single polymer chain force-extension behavior measured by Atomic Force Microscopy (AFM) was interpreted by molecular dynamics (MD) simulation performed by applying a bead-spring (coarse-graining) model in which the bond potential function between adjacent beads is described by a worm-like chain (WLC) model. Simulation results indicate that caution should be applied when interpreting experimental AFM data, because the data vary depending on the point of AFM tip-polymer chain attachment. This approach offers an effective way for eventual analysis of the mechanical behavior of complex polymer networks

Molecular dynamics simulation of AFM studies of a single polymer chain

Energy Technology Data Exchange (ETDEWEB)

Wang Wenhai [Center for Bioengineering and Biomaterials, College of Engineering, Temple University, 1947 N. 12th Street, Philadelphia, PA 19122 (United States); Kistler, Kurt A. [Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, PA 19122 (United States); Sadeghipour, Keya [Center for Bioengineering and Biomaterials, College of Engineering, Temple University, 1947 N. 12th Street, Philadelphia, PA 19122 (United States); Baran, George [Center for Bioengineering and Biomaterials, College of Engineering, Temple University, 1947 N. 12th Street, Philadelphia, PA 19122 (United States)], E-mail: grbaran@temple.edu

2008-11-24

Single polymer chain force-extension behavior measured by Atomic Force Microscopy (AFM) was interpreted by molecular dynamics (MD) simulation performed by applying a bead-spring (coarse-graining) model in which the bond potential function between adjacent beads is described by a worm-like chain (WLC) model. Simulation results indicate that caution should be applied when interpreting experimental AFM data, because the data vary depending on the point of AFM tip-polymer chain attachment. This approach offers an effective way for eventual analysis of the mechanical behavior of complex polymer networks.

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

Science.gov (United States)

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

2015-05-01

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

Engineered antibodies for monitoring of polynuclear aromatic hydrocarbons

International Nuclear Information System (INIS)

Karu, A.E.; Li, Q.X.; Roberts, V.A.

1998-01-01

'The long-term goal of this project is to develop antibodies and antibody-based methods for detection and recovery of polynuclear aromatic hydrocarbons (PAHs) and PAH adducts that are potential biomarkers in environmental and biological samples. The inherent cross-reactivity will be exploited by pattern recognition methods. Dr. Karu''s laboratory uses new haptens representing key PAHs to derive recombinant Fab (rFab) and single-chain Fv (scFv) antibodies from hybridoma lines and combinatorial phage display libraries. Computational models of the haptens and combining sites made by Dr. Roberts''s group are used to guide antibody engineering by mutagenesis. Dr. Li''s laboratory develops enzyme immunoassays (EIAs), sensors, and immunoaffinity methods that make use of the novel haptens and antibodies for practical analytical applications in support of DOE''s mission. This report summarizes work completed in one and one-half years of a 3-year project, with close collaboration between the three research groups. Dr. Alexander Karu''s laboratory: the authors proceeded with the two strategies described in the original proposal. Site-directed mutagenesis was used to correct differences in the rFab N-terminal amino acids that were introduced by the degenerate PCR primers used for gene amplification. The binding constants of the rFabs with the corrected sequences will be compared with those of the parent MAbs, and should be very similar. The 4D5 and 10C10 heavy and light chain sequences are being moved to the pCOMB3H phagemid vector to facilitate selection of new engineered mutants.'

Single-Molecule Imaging Reveals Topology Dependent Mutual Relaxation of Polymer Chains

KAUST Repository

Abadi, Maram; Serag, Maged F.; Habuchi, Satoshi

2015-01-01

The motion and relaxation of linear and cyclic polymers under entangled conditions are investigated by means of a newly developed single-molecule tracking technique, cumulative-area (CA) tracking. CA tracking enables simultaneous quantitative characterization of the diffusion mode, diffusion rate, and relaxation time that have been impossible with a widely used conventional single-molecule localization and tracking method, by analyzing cumulative areas occupied by the moving molecule. Using the novel approach, we investigate the motion and relaxation of entangled cyclic polymers, which have been an important but poorly understood question. Fluorescently labeled 42 kbp linear or cyclic tracer dsDNAs in concentrated solutions of unlabeled linear or cyclic DNAs are used as model systems. We show that CA tracking can explicitly distinguish topology-dependent diffusion mode, rate, and relaxation time, demonstrating that the method provides an invaluable tool for characterizing topological interaction between the entangled chains. We further demonstrate that the current models proposed for the entanglement between cyclic polymers which are based on cyclic chains moving through an array of fixed obstacles cannot correctly describe the motion of the cyclic chain under the entangled conditions. Our results rather suggest the mutual relaxation of the cyclic chains, which underscore the necessity of developing a new model to describe the motion of cyclic polymer under the entangled conditions based on the mutual interaction of the chains.

Single-Molecule Imaging Reveals Topology Dependent Mutual Relaxation of Polymer Chains

KAUST Repository

Abadi, Maram

2015-08-24

The motion and relaxation of linear and cyclic polymers under entangled conditions are investigated by means of a newly developed single-molecule tracking technique, cumulative-area (CA) tracking. CA tracking enables simultaneous quantitative characterization of the diffusion mode, diffusion rate, and relaxation time that have been impossible with a widely used conventional single-molecule localization and tracking method, by analyzing cumulative areas occupied by the moving molecule. Using the novel approach, we investigate the motion and relaxation of entangled cyclic polymers, which have been an important but poorly understood question. Fluorescently labeled 42 kbp linear or cyclic tracer dsDNAs in concentrated solutions of unlabeled linear or cyclic DNAs are used as model systems. We show that CA tracking can explicitly distinguish topology-dependent diffusion mode, rate, and relaxation time, demonstrating that the method provides an invaluable tool for characterizing topological interaction between the entangled chains. We further demonstrate that the current models proposed for the entanglement between cyclic polymers which are based on cyclic chains moving through an array of fixed obstacles cannot correctly describe the motion of the cyclic chain under the entangled conditions. Our results rather suggest the mutual relaxation of the cyclic chains, which underscore the necessity of developing a new model to describe the motion of cyclic polymer under the entangled conditions based on the mutual interaction of the chains.

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer

Science.gov (United States)

Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang

2014-05-01

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

A binary plasmid system for shuffling combinatorial antibody libraries.

OpenAIRE

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-01-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind a...

A binary plasmid system for shuffling combinatorial antibody libraries.

Science.gov (United States)

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-11-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

Science.gov (United States)

Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

2014-01-01

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

Single-Chain Conformation for Interacting Poly(N-isopropylacrylamide in Aqueous Solution

Directory of Open Access Journals (Sweden)

Boualem Hammouda

2015-04-01

Full Text Available The demixing phase behavior of Poly(N-isopropylacrylamide (PNIPAM aqueous solution is investigated using small-angle neutron scattering. This polymer phase separates upon heating and demixes around 32 °C. The pre-transition temperature range is characterized by two scattering modes; a low-Q (large-scale signal and a high-Q dissolved chains signal. In order to get insight into this pre-transition region, especially the origin of the low-Q (large-scale structure, the zero average contrast method is used in order to isolate single-chain conformations even in the demixing polymers transition region. This method consists of measuring deuterated and non-deuterated polymers dissolved in mixtures of deuterated and non-deuterated water for which the polymer scattering length density matches the solvent scattering length density. A fixed 4% polymer mass fraction is used in a contrast variation series where the d-water/h-water fraction is varied in order to determine the match point. The zero average contrast (match point sample displays pure single-chain scattering with no interchain contributions. Our measurements prove that the large scale structure in this polymer solution is due to a transient polymer network formed through hydrophobic segment-segment interactions. Scattering intensity increases when the temperature gets close to the phase boundary. While the apparent radius of gyration increases substantially at the Lower Critical Solution Temperature (LCST transition due to strong interchain correlation, the single-chain true radius of gyration has been found to decrease slightly with temperature when approaching the transition.

Single-molecule study on polymer diffusion in a melt state: Effect of chain topology

KAUST Repository

Habuchi, Satoshi

2013-08-06

We report a new methodology for studying diffusion of individual polymer chains in a melt state, with special emphasis on the effect of chain topology. A perylene diimide fluorophore was incorporated into the linear and cyclic poly(THF)s, and real-time diffusion behavior of individual chains in a melt of linear poly(THF) was measured by means of a single-molecule fluorescence imaging technique. The combination of mean squared displacement (MSD) and cumulative distribution function (CDF) analysis demonstrated the broad distribution of diffusion coefficient of both the linear and cyclic polymer chains in the melt state. This indicates the presence of spatiotemporal heterogeneity of the polymer diffusion which occurs at much larger time and length scales than those expected from the current polymer physics theory. We further demonstrated that the cyclic chains showed marginally slower diffusion in comparison with the linear counterparts, to suggest the effective suppression of the translocation through the threading-entanglement with the linear matrix chains. This coincides with the higher activation energy for the diffusion of the cyclic chains than of the linear chains. These results suggest that the single-molecule imaging technique provides a powerful tool to analyze complicated polymer dynamics and contributes to the molecular level understanding of the chain interaction. © 2013 American Chemical Society.

Single-molecule study on polymer diffusion in a melt state: Effect of chain topology

KAUST Repository

Habuchi, Satoshi; Fujiwara, Susumu; Yamamoto, Takuya; Vá cha, Martin; Tezuka, Yasuyuki

2013-01-01

We report a new methodology for studying diffusion of individual polymer chains in a melt state, with special emphasis on the effect of chain topology. A perylene diimide fluorophore was incorporated into the linear and cyclic poly(THF)s, and real-time diffusion behavior of individual chains in a melt of linear poly(THF) was measured by means of a single-molecule fluorescence imaging technique. The combination of mean squared displacement (MSD) and cumulative distribution function (CDF) analysis demonstrated the broad distribution of diffusion coefficient of both the linear and cyclic polymer chains in the melt state. This indicates the presence of spatiotemporal heterogeneity of the polymer diffusion which occurs at much larger time and length scales than those expected from the current polymer physics theory. We further demonstrated that the cyclic chains showed marginally slower diffusion in comparison with the linear counterparts, to suggest the effective suppression of the translocation through the threading-entanglement with the linear matrix chains. This coincides with the higher activation energy for the diffusion of the cyclic chains than of the linear chains. These results suggest that the single-molecule imaging technique provides a powerful tool to analyze complicated polymer dynamics and contributes to the molecular level understanding of the chain interaction. © 2013 American Chemical Society.

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.

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

Science.gov (United States)

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

2011-08-01

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

Markov chain analysis of single spin flip Ising simulations

International Nuclear Information System (INIS)

Hennecke, M.

1997-01-01

The Markov processes defined by random and loop-based schemes for single spin flip attempts in Monte Carlo simulations of the 2D Ising model are investigated, by explicitly constructing their transition matrices. Their analysis reveals that loops over all lattice sites using a Metropolis-type single spin flip probability often do not define ergodic Markov chains, and have distorted dynamical properties even if they are ergodic. The transition matrices also enable a comparison of the dynamics of random versus loop spin selection and Glauber versus Metropolis probabilities

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display

Directory of Open Access Journals (Sweden)

Greg Hussack

2017-10-01

Full Text Available ABTAG is a camelid single-domain antibody (sdAb that binds to bovine serum albumin (BSA with low picomolar affinity. In surface plasmon resonance (SPR analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

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

Directory of Open Access Journals (Sweden)

Edyta Petters

2015-08-01

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

Structural and electronic properties of a single C chain doped zigzag BN nanoribbons

International Nuclear Information System (INIS)

Wu, Ping; Wang, Qianwen; Cao, Gengyu; Tang, Fuling; Huang, Min

2014-01-01

The effects of single C-chain on the stability, structural and electronic properties of zigzag BN nanoribbons (ZBNNRs) were investigated by first-principles calculations. C-chain was expected to dope at B-edge for all the ribbon widths N z considered. The band gaps of C-chain doped N z -ZBNNR are narrower than that of perfect ZBNNR due to new localized states induced by C-chain. The band gaps of N z -ZBNNR-C(n) are direct except for the case of C-chain position n=2. Band gaps of BN nanoribbons are tunable by C-chain and its position n, which may endow the potential applications of BNNR in electronics.

Well-defined single-chain polymer nanoparticles via thiol-Michael addition

NARCIS (Netherlands)

Kröger, A. Pia P.; Boonen, Roy J.E.A.; Paulusse, Jos M.J.

2017-01-01

A synthetic strategy has been developed giving facile access to well-defined single-chain polymer nanoparticles (SCNPs) from styrene-, acrylate- and methacrylate-based polymers. Random copolymers (polydispersity indices 1.10–1.15) of methyl (meth)acrylate, benzyl methacrylate or styrene containing

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

Science.gov (United States)

Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

2016-11-25

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

International Nuclear Information System (INIS)

Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

2016-01-01

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

Single-chain statistics and the upper wave-vector cutoff in polymer blends

International Nuclear Information System (INIS)

Holyst, R.; Vilgis, T.A.

1994-01-01

We derive the equation for the single-chain correlation function in polymer blends. The chains in the incompressible blend have a radius of gyration smaller than the radius of gyration for ideal chains. The chains shrink progressively as we approach the critical temperature T c . The correction responsible for shrinking is proportional to 1/ √N , where N is the polymerization index. At T=T c and for N=1000, the size of the chain has been estimated to be 10% smaller than the size of the ideal coil. The estimate relies on the appropriate cutoff. In the limit of N→∞ the chains approach the random walk limit. Additionally, we propose in this paper a self-consistent determination of the radius of gyration and the upper wave-vector cutoff. Our model is free from any divergences such as were encountered in the previous mean-field studies; we make an estimate of the chain size at the true critical temperature and not the mean-field one

Hepatitis C Virus Antibodies in Dialysis Patients in Tunisia: A Single Center Study

Directory of Open Access Journals (Sweden)

Sassi F

2000-01-01

Full Text Available Fifty-eight patients on maintenance hemodialysis in a dialysis unit at Tunis, Tunisia were tested for anti-hepatitis C virus (anti-HCV antibodies by second generation ELISA test, and for HCV-RNA by nested reverse transcriptase polymerase chain reaction (RT-PCR of 5′ non-coding region. Specificity of the antibodies was confirmed by immunoblot test. HCV genotype was defined using INNO-LIPA test. Twenty-seven out of 58 patients (46.5% were reactive by ELISA. Transaminase levels were assessed over a six-month period and showed normal average values. Fourteen of the 27 anti-HCV positive patients (51% were positive by RT-PCR. Type 1b HCV genotype was the most prevalent, seen in all the dialysis patients and one patient in addition, was co-infected with genotype 4. There was a significant correlation between the duration on dialysis (over five years and the prevalence of anti-HCV-positive patients (P< 0.005 while no correlation existed between the number of blood transfusions and the presence of anti-HCV antibodies. The present study illustrates the high prevalence of HCV infection among Tunisian dialysis patients (51% and indicates that the spread may be nosocomial rather than transfusion-related.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Science.gov (United States)

Goicochea, Nancy L; Garnovskaya, Maria; Blanton, Mary G; Chan, Grace; Weisbart, Richard; Lilly, Michael B

2017-12-01

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

CHAINS-PC, Decay Chain Atomic Densities

International Nuclear Information System (INIS)

1994-01-01

1 - Description of program or function: CHAINS computes the atom density of members of a single radioactive decay chain. The linearity of the Bateman equations allows tracing of interconnecting chains by manually accumulating results from separate calculations of single chains. Re-entrant loops can be treated as extensions of a single chain. Losses from the chain are also tallied. 2 - Method of solution: The Bateman equations are solved analytically using double-precision arithmetic. Poles are avoided by small alterations of the loss terms. Multigroup fluxes, cross sections, and self-shielding factors entered as input are used to compute the effective specific reaction rates. The atom densities are computed at any specified times. 3 - Restrictions on the complexity of the problem: Maxima of 100 energy groups, 100 time values, 50 members in a chain

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-08-13

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

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

Science.gov (United States)

Schmidt, Michael M.; Thurber, Greg M.

2010-01-01

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. PMID:18408925

Immunoglobulin G4: an odd antibody

NARCIS (Netherlands)

Aalberse, R. C.; Stapel, S. O.; Schuurman, J.; Rispens, T.

2009-01-01

Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects

NARCIS (Netherlands)

Braat, E. A.; Levi, M. [=Marcel M.; Bos, R.; Haverkate, F.; Lassen, M. R.; de Maat, M. P.; Rijken, D. C.

1999-01-01

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence

Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

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Xiaocong Yu

2016-01-01

Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

Primitive-path statistics of entangled polymers: mapping multi-chain simulations onto single-chain mean-field models

International Nuclear Information System (INIS)

Steenbakkers, Rudi J A; Schieber, Jay D; Tzoumanekas, Christos; Li, Ying; Liu, Wing Kam; Kröger, Martin

2014-01-01

We present a method to map the full equilibrium distribution of the primitive-path (PP) length, obtained from multi-chain simulations of polymer melts, onto a single-chain mean-field ‘target’ model. Most previous works used the Doi–Edwards tube model as a target. However, the average number of monomers per PP segment, obtained from multi-chain PP networks, has consistently shown a discrepancy of a factor of two with respect to tube-model estimates. Part of the problem is that the tube model neglects fluctuations in the lengths of PP segments, the number of entanglements per chain and the distribution of monomers among PP segments, while all these fluctuations are observed in multi-chain simulations. Here we use a recently proposed slip-link model, which includes fluctuations in all these variables as well as in the spatial positions of the entanglements. This turns out to be essential to obtain qualitative and quantitative agreement with the equilibrium PP-length distribution obtained from multi-chain simulations. By fitting this distribution, we are able to determine two of the three parameters of the model, which govern its equilibrium properties. This mapping is executed for four different linear polymers and for different molecular weights. The two parameters are found to depend on chemistry, but not on molecular weight. The model predicts a constant plateau modulus minus a correction inversely proportional to molecular weight. The value for well-entangled chains, with the parameters determined ab initio, lies in the range of experimental data for the materials investigated. (paper)

Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus)

DEFF Research Database (Denmark)

Kim, Young Kyu; Lee, Jung Seok; Jung, Jae Wook

2017-01-01

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used...... successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11......]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more...

Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

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Sung Sun Yim

Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

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

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Stewart Nuttall

2013-01-01

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

Synthesis and functionalization of dextran-based single-chain nanoparticles in aqueous media

OpenAIRE

Gracia R.; Marradi M.; Cossío U.; Benito A.; Pérez-San Vicente A.; Gómez-Vallejo V.; Grande H.-J.; Llop J.; and Loinaz I.

2017-01-01

Water-dispersible dextran-based single-chain polymer nanoparticles (SCPNs) were prepared in aqueous media and under mild conditions. Radiolabeling of the resulting biocompatible materials allowed the study of lung deposition of aqueous aerosols after intratracheal nebulization by means of single-photon emission computed tomography (SPECT), demonstrating their potential use as imaging contrast agents.

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

Science.gov (United States)

Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

2002-06-28

MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors.

Science.gov (United States)

Annenkov, Alexander; Rigby, Anne; Amor, Sandra; Zhou, Dun; Yousaf, Nasim; Hemmer, Bernhard; Chernajovsky, Yuti

2011-08-01

In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional. 2011 Elsevier B.V. All rights reserved.

Heavy-chain isotype patterns of human antibody-secreting cells induced by Haemophilus influenzae type b conjugate vaccines in relation to age and preimmunity

DEFF Research Database (Denmark)

Barington, T; Juul, Lars; Gyhrs, A

1994-01-01

The influence of preexisting immunity on the heavy-chain isotypes of circulating antibody-secreting cells (AbSC) induced by vaccination with Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP) coupled to tetanus toxoid (TT) or diphtheria toxoid (DT) and by vaccination with TT or D...... of natural HibCP antibodies (r = 0.59; P = 0.00002). A possible role of natural exposure for Hib or cross-reactive bacteria on the mucosal surfaces in the shaping of the isotype response to HibCP conjugate vaccines is discussed....

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

DEFF Research Database (Denmark)

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

2009-01-01

three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol......A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward...

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

Science.gov (United States)

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Choice between Single and Multiple Reinforcers in Concurrent-Chains Schedules

Science.gov (United States)

Mazur, James E.

2006-01-01

Pigeons responded on concurrent-chains schedules with equal variable-interval schedules as initial links. One terminal link delivered a single reinforcer after a fixed delay, and the other terminal link delivered either three or five reinforcers, each preceded by a fixed delay. Some conditions included a postreinforcer delay after the single…

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

Science.gov (United States)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies

International Nuclear Information System (INIS)

Leahy, D.J.; Rule, G.S.; Whittaker, M.M.; McConnell, H.M.

1988-01-01

Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produces for use in NMR studies. They have been named AN01 and ANO3-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, ANO2, have been isolated. The nucleic acid sequence of the 5' end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of ANO1 and ANO3 are derived from the same variable-region gene families as those of the ANO2 antibody. ANO7 has a heavy chain that is related to that of ANO2, and ANO9 has a related light chain. ANO5 and ANO6 are unrelated to ANO2 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness

Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

Science.gov (United States)

Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

2015-12-01

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

Microbial platform technology for recombinant antibody fragment production: A review.

Science.gov (United States)

Gupta, Sanjeev Kumar; Shukla, Pratyoosh

2017-02-01

Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

Epitope analysis of anti-myeloperoxidase antibodies in patients with ANCA-associated vasculitis.

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Shen-Ju Gou

Full Text Available OBJECTIVE: Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA directing myeloperoxidase (MPO in ANCA-associated vasculitis (AAV. The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV. METHODS: Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands. RESULTS: Sera from 45 of the 77 (58.4% patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset. CONCLUSION: The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis.

Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay.

Science.gov (United States)

Sorsa-Leslie, Tarja; Mason, Helen D; Harris, William J; Fowler, Paul A

2005-09-26

We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the

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

NARCIS (Netherlands)

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

2005-01-01

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

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

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Scott A Walper

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

Evolution of Therapeutic Antibodies, Influenza Virus Biology, Influenza, and Influenza Immunotherapy

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Urai Chaisri

2018-01-01

Full Text Available This narrative review article summarizes past and current technologies for generating antibodies for passive immunization/immunotherapy. Contemporary DNA and protein technologies have facilitated the development of engineered therapeutic monoclonal antibodies in a variety of formats according to the required effector functions. Chimeric, humanized, and human monoclonal antibodies to antigenic/epitopic myriads with less immunogenicity than animal-derived antibodies in human recipients can be produced in vitro. Immunotherapy with ready-to-use antibodies has gained wide acceptance as a powerful treatment against both infectious and noninfectious diseases. Influenza, a highly contagious disease, precipitates annual epidemics and occasional pandemics, resulting in high health and economic burden worldwide. Currently available drugs are becoming less and less effective against this rapidly mutating virus. Alternative treatment strategies are needed, particularly for individuals at high risk for severe morbidity. In a setting where vaccines are not yet protective or available, human antibodies that are broadly effective against various influenza subtypes could be highly efficacious in lowering morbidity and mortality and controlling unprecedented epidemic/pandemic. Prototypes of human single-chain antibodies to several conserved proteins of influenza virus with no Fc portion (hence, no ADE effect in recipients are available. These antibodies have high potential as a novel, safe, and effective anti-influenza agent.

Strong-coupling behaviour of two t - J chains with interchain single-electron hopping

International Nuclear Information System (INIS)

Zhang Guangming; Feng Shiping; Yu Lu.

1994-01-01

Using the fermion-spin transformation to implement spin-charge separation of constrained electrons, a model of two t - J chains with interchain single-electron hopping is studied by abelian bosonization. After spin-charge decoupling the charge dynamics can be trivially solved, while the spin dynamics is determined by a strong-coupling fixed point where the correlation functions can be calculated explicitly. This is a generalization of the Luther-Emery line for two-coupled t - J chains. The interchain single-electron hopping changes the asymptotic behaviour of the interchain spin-spin correlation functions and the electron Green function, but their exponents are independent of the coupling strength. (author). 25 refs

Dynamical behavior of a single polymer chain under nanometric confinement

Science.gov (United States)

Lagrené, K.; Zanotti, J.-M.; Daoud, M.; Farago, B.; Judeinstein, P.

2010-10-01

We address the dynamical behavior of a single polymer chain under nanometric confinement. We consider a polymer melt made of a mixture of hydrogenated and deuterated high molecular mass Poly(Ethylene Oxide) (PEO). The confining material is a membrane of Anodic Aluminum Oxide (AAO), a macroscopically highly ordered confining system made of parallel cylindrical channels. We use Neutron Spin-Echo (NSE) under the Zero Average Contrast (ZAC) condition to, all at once, i) match the intense porous AAO detrimental elastic SANS (Small Angle Neutron Scattering) contribution to the total intermediate scattering function I(Q,t) and ii) measure the Q dependence of the dynamical modes of a single chain under confinement. The polymer dynamics is probed on an extremely broad spacial ([2.2 10-2 Å-1, 0.2 Å-1]) and temporal ([0.1 ns, 600 ns]) ranges. We do not detect any influence of confinement on the polymer dynamics. This result is discussed in the framework of the debate on the existence of a "corset effect" recently suggested by NMR relaxometry data.

Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies

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Fa Yang

2016-12-01

Full Text Available With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

A single administration of LFA-1 antibody confers prolonged allograft survival.

Science.gov (United States)

Talento, A; Nguyen, M; Blake, T; Sirotina, A; Fioravanti, C; Burkholder, D; Gibson, R; Sigal, N H; Springer, M S; Koo, G C

1993-02-01

C57BL/6 (B6) thyroid gland transplanted to the left kidney capsule of an allogeneic (BALB/c) host was typically rejected in 14 days. A single administration of 500 micrograms of an antibody to the adhesion molecule, leucocyte function-associated antigen (LFA-1, CD11a), prevented all thyroid allograft rejection for at least 70 days. Fifty percent of the treated recipients retained intact allografts for 470 days. However, the same treatment with anti-CD11a could not protect a sensitized BALB/c mouse from rejecting a second B6 thyroid allograft. Production of donor-specific alloantibodies elicited by allograft rejection was also inhibited in this system. In this transplant model, the Ab therapy is more efficacious than that of FK506, administered daily for 14 days at 15 mg/kg. These results demonstrate the remarkable effect of an anti-LFA-1 antibody in promotion of allograft survival.

Interfacial free energy governs single polystyrene chain collapse in water and aqueous solutions.

Science.gov (United States)

Li, Isaac T S; Walker, Gilbert C

2010-05-12

The hydrophobic interaction is significantly responsible for driving protein folding and self-assembly. To understand it, the thermodynamics, the role of water structure, the dewetting process surrounding hydrophobes, and related aspects have undergone extensive investigations. Here, we examine the hypothesis that polymer-solvent interfacial free energy is adequate to describe the energetics of the collapse of a hydrophobic homopolymer chain at fixed temperature, which serves as a much simplified model for studying the hydrophobic collapse of a protein. This implies that changes in polymer-solvent interfacial free energy should be directly proportional to the force to extend a collapsed polymer into a bad solvent. To test this hypothesis, we undertook single-molecule force spectroscopy on a collapsed, single, polystyrene chain in water-ethanol and water-salt mixtures where we measured the monomer solvation free energy from an ensemble average conformations. Different proportions within the binary mixture were used to create solvents with different interfacial free energies with polystyrene. In these mixed solvents, we observed a linear correlation between the interfacial free energy and the force required to extend the chain into solution, which is a direct measure of the solvation free energy per monomer on a single chain at room temperature. A simple analytical model compares favorably with the experimental results. This knowledge supports a common assumption that explicit water solvent may not be necessary for cases whose primary concerns are hydrophobic interactions and hydrophobic hydration.

Designer genes. Recombinant antibody fragments for biological imaging

Energy Technology Data Exchange (ETDEWEB)

Wu, A.M.; Yazaki, P.J. [Beckman Research Institute of the City of Hope, Duarte, CA (United States). Dept. of Molecular Biology

2000-09-01

Monoclonal antibodies (MAbs), with high specificity and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C{sub H}3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering

Designer genes. Recombinant antibody fragments for biological imaging

International Nuclear Information System (INIS)

Wu, A.M.; Yazaki, P.J.

2000-01-01

Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-C H 3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and

Targeting Malignant Brain Tumors with Antibodies

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Rok Razpotnik

2017-09-01

Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

Escherichia coli F4 fimbriae specific lama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhea

NARCIS (Netherlands)

Harmsen, M.M.; Solt, van C.B.; Hoogendoorn, A.; Zijderveld, van F.G.; Niewold, T.A.; Meulen, van der J.

2005-01-01

Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs)

Pricing and ordering policies for price-dependent demand in a supply chain of a single retailer and a single manufacturer

Science.gov (United States)

Kim, Jungkyu; Hong, Yushin; Kim, Taebok

2011-01-01

This article discusses joint pricing and ordering policies for price-dependent demand in a supply chain consisting of a single retailer and a single manufacturer. The retailer places orders for products according to an EOQ policy and the manufacturer produces them on a lot-for-lot basis. Four mechanisms with differing levels of coordination are presented. Mathematical models are formulated and solution procedures are developed to determine the optimal retail prices and order quantities. Through extensive numerical experiments, we analyse and compare the behaviours and characteristics of the proposed mechanisms, and find that enhancing the level of coordination has important benefits for the supply chain.

A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

Science.gov (United States)

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

2015-03-20

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Varicella zoster virus myelitis in two elderly patients: diagnostic value of nested polymerase chain reaction assay and antibody index for cerebrospinal fluid specimens.

Science.gov (United States)

Takahashi, Teruyuki; Tamura, Masato; Miki, Kenji; Yamaguchi, Mai; Kanno, Akira; Nunomura, Satoshi; Ra, Chisei; Tamiya, Takashi; Kamei, Satoshi; Takasu, Toshiaki

2013-01-01

Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV) infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th)-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR) assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF) specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

Science.gov (United States)

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Enantiopure heterobimetallic single-chain magnets from the chiral Ru(III) building block.

Science.gov (United States)

Ru, Jing; Gao, Feng; Wu, Tao; Yao, Min-Xia; Li, Yi-Zhi; Zuo, Jing-Lin

2014-01-21

A pair of one-dimensional enantiomers based on the versatile chiral dicyanoruthenate(III) building block have been synthesized and they are chiral single-chain magnets with the effective spin-reversal barrier of 28.2 K.

Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

NARCIS (Netherlands)

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

2005-01-01

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

Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

Directory of Open Access Journals (Sweden)

Haddad Muhammad

2016-01-01

Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

Conformation of Single Pentablock Ionomer Chains in Dilute Solutions

Energy Technology Data Exchange (ETDEWEB)

Aryal, Dipak [Clemson Univ., SC (United States); Perahia, Dvora [Clemson Univ., SC (United States); Grest, Gary S. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

2015-04-01

The conformation of single chain pentablock ionomers (A-B-C-B-A) containing randomly sulfonated polystyrene in the center block, tethered to poly-ethylene-r-propylene end-capped by poly-t-butyl styrene is studied in dilute solutions by molecular dynamics simulations. Multi-block copolymers offer a means to tailor several properties into one molecule, taking advantage of their rich phase diagram together with unique properties of specific blocks. For this pentablock the ionic block facilitates transport while the A and B components are incorporated for mechanical stability. The present study investigates the confirmation of a single chain of pentablock ionomer of molecular weight M_w ~ 50,000 g/mol and sulfonated polystyrene of the same molecular weight as that of the center block for six sulfonation fractions f from f=0.0-0.55. For the sulfonated systems Na⁺ counterions are included. Results for the equilibrium conformation of the chains and the three blocks in water and 1:1 mixture of cyclohexane and n-heptane are compared to simulations in implicit poor solvents with dielectric constants ε =1.0 and 77.73. In water, the pentablock is collapsed with sulfonated groups on the outer surface. As the sulfonation fraction f increases, the ionic, center block is increasingly segregated from the hydrophobic regions. In the 1:1 mixture of cyclohexane and heptane both the flexible and end blocks are swollen while the center ionic block is collasped for f>0, while for f=0 all blocks are swollen. In both implicit poor solvents the pentablock is collapsed into a nearly spherical shape for all f. The sodium counterions are dispersed widely throughout the simulation cell for both water and ε =77.73 whereas for ε =1.0 the counterions are largely condensed on the collapsed pentablock.

AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.

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Jonathan S Wall

Full Text Available The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K(D of ∼10 nM. Binding was inhibited in the presence of the -Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas as evidenced by single photon emission (SPECT imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.

From single magnetic adatoms on superconductors to coupled spin chains

Science.gov (United States)

Franke, Katharina J.

Magnetic adsorbates on conventional s-wave superconductors lead to exchange interactions that induce Yu-Shiba-Rusinov (YSR) states inside the superconducting energy gap. Here, we employ tunneling spectroscopy at 1.1 K to investigate magnetic atoms and chains on superconducting Pb surfaces. We show that individual Manganese (Mn) atoms give rise to a distinct number of YSR-states. The single-atom junctions are stable over several orders of magnitude in conductance. We identify single-electron tunneling as well as Andreev processes. When the atoms are brought into sufficiently close distance, the Shiba states hybridize, thus giving rise to states with bonding and anti-bonding character. It has been shown that the Pb(110) surface supports the self-assembly of Fe chains, which exhibit fingerprints of Majorana bound states. Using superconducting tips, we resolve a rich subgap structure including peaks at zero energy and low-energy resonances, which overlap with the putative Majorana states. We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft through collaborative research Grant Sfb 658, and through Grant FR2726/4, as well by the European Research Council through Consolidator Grant NanoSpin.

Analysis of the paired TCR α- and β-chains of single human T cells.

Directory of Open Access Journals (Sweden)

Song-Min Kim

Full Text Available Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV and multiple sclerosis (MS. In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+ T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+ T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.

Optical Properties of a Single Carbon Chain-Doped Silicene Nanoribbon

Science.gov (United States)

Lu, Dao-Bang; Song, Yu-Ling; Huang, Xiao-yu; Wang, Chong

2018-05-01

Using first-principles spin polarization density function theory calculations, we have studied the electronic and optical properties of zigzag-edge silicene nanoribbons (ZSiNRs) doped with a single carbon chain. Because of the doped carbon chain, there are several defect states in the band structures of ZSiNRs across the Fermi level, and the ferromagnetic ground state is metallic. The dielectric functions in all three dimensions are completely different from each other, and thus the system exhibits strong optical anisotropism. The carbon chain influenced the dielectric functions most at low energy. The first peak in the E//x direction of the dielectric spectrum exhibits a significant blueshift, and its value has changed as well. The main absorption wavelength depends on the polarization direction of the incident light, but occurs within the UV region for all polarization directions. The peaks of the energy loss spectra correspond to the trailing edges in the reflectivity spectrum, and the highest peak corresponds to a plasmon frequency. Our results could be useful for investigating nanodevices based on silicene nanoribbons.

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

NARCIS (Netherlands)

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

2012-01-01

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

Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

Science.gov (United States)

Kumada, Yoichi; Ishikawa, Yasuyuki; Fujiwara, Yusuke; Takeda, Rui; Miyamoto, Ryosuke; Niwa, Daisuke; Momose, Shun; Kang, Bongmun; Kishimoto, Michimasa

2014-09-01

In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Science.gov (United States)

van Lith, Sanne A M; van Duijnhoven, Sander M J; Navis, Anna C; Leenders, William P J; Dolk, Edward; Wennink, Jos W H; van Nostrum, Cornelus F; van Hest, Jan C M

2017-02-15

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu + -independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.

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

Directory of Open Access Journals (Sweden)

J. Mala

2017-06-01

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

Tumour targeting with monovalent fragments of anti-neuroblastoma antibody chCE7

International Nuclear Information System (INIS)

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

1997-01-01

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

Spin canting in a Dy-based single-chain magnet with dominant next-nearest-neighbor antiferromagnetic interactions

Science.gov (United States)

Bernot, K.; Luzon, J.; Caneschi, A.; Gatteschi, D.; Sessoli, R.; Bogani, L.; Vindigni, A.; Rettori, A.; Pini, M. G.

2009-04-01

We investigate theoretically and experimentally the static magnetic properties of single crystals of the molecular-based single-chain magnet of formula [Dy(hfac)3NIT(C6H4OPh)]∞ comprising alternating Dy3+ and organic radicals. The magnetic molar susceptibility χM displays a strong angular variation for sample rotations around two directions perpendicular to the chain axis. A peculiar inversion between maxima and minima in the angular dependence of χM occurs on increasing temperature. Using information regarding the monomeric building block as well as an ab initio estimation of the magnetic anisotropy of the Dy3+ ion, this “anisotropy-inversion” phenomenon can be assigned to weak one-dimensional ferromagnetism along the chain axis. This indicates that antiferromagnetic next-nearest-neighbor interactions between Dy3+ ions dominate, despite the large Dy-Dy separation, over the nearest-neighbor interactions between the radicals and the Dy3+ ions. Measurements of the field dependence of the magnetization, both along and perpendicularly to the chain, and of the angular dependence of χM in a strong magnetic field confirm such an interpretation. Transfer-matrix simulations of the experimental measurements are performed using a classical one-dimensional spin model with antiferromagnetic Heisenberg exchange interaction and noncollinear uniaxial single-ion anisotropies favoring a canted antiferromagnetic spin arrangement, with a net magnetic moment along the chain axis. The fine agreement obtained with experimental data provides estimates of the Hamiltonian parameters, essential for further study of the dynamics of rare-earth-based molecular chains.

Antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor.

Science.gov (United States)

Manjappa, Arehalli S; Chaudhari, Kiran R; Venkataraju, Makam P; Dantuluri, Prudhviraju; Nanda, Biswarup; Sidda, Chennakesavulu; Sawant, Krutika K; Murthy, Rayasa S Ramachandra

2011-02-28

A great deal of effort has been made over the years to develop liposomes that have targeting vectors (oligosaccharides, peptides, proteins and vitamins) attached to the bilayer surface. Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies are well established. Antibody conjugated liposomes have recently attracted a great deal of interest, principally because of their potential use as targeted drug delivery systems and in diagnostic applications. A number of methods have been reported for coupling antibodies to the surface of stealth liposomes. The objective of this review is to enumerate various strategies which are employed in the modification and conjugation of antibodies to the surface of stealth liposomes. This review also describes various derivatization techniques of lipids prior and after their use in the preparation of liposomes. The use of single chain variable fragments and affibodies as targeting ligands in the preparation of immunoliposomes is also discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

Antigen-targeting strategies using single-domain antibody fragments

NARCIS (Netherlands)

Duarte, Joao Nuno Silva

2017-01-01

Antibodies display high selectivity and affinity and have been the preferred platform for antigen targeting. Despite the development of antigen-delivery systems that enable T cell activation, targeting approaches that enhance antibody responses need improvement. This need specially applies to poorly

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

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Zoltán Balogh

2015-06-01

Full Text Available We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

Jet and ultrasonic nebulization of single chain urokinase plasminogen activator (scu-PA)

DEFF Research Database (Denmark)

Münster, Anna-Marie; Bendstrup, E; Jensen, J.I.

2000-01-01

locally by nebulization in a recombinant zymogen form as single chain urokinase plasminogen activator (scu-PA). We aimed to characterize the particle size distribution, drug output, and enzymatic activity of scu-PA after nebulization with a Ventstream jet nebulizer (Medic-Aid, Bognor Regis, UK) and a Syst...

Finite-size effects on the static properties of a single-chain magnet

Science.gov (United States)

Bogani, L.; Sessoli, R.; Pini, M. G.; Rettori, A.; Novak, M. A.; Rosa, P.; Massi, M.; Fedi, M. E.; Giuntini, L.; Caneschi, A.; Gatteschi, D.

2005-08-01

We study the role of defects in the “single-chain magnet” CoPhOMe by inserting a controlled number of diamagnetic impurities. The samples are analyzed with unprecedented accuracy with the particle induced x-ray emission technique, and with ac and dc magnetic measurements. In an external applied field the system shows an unexpected behavior, giving rise to a double peak in the susceptibility. The static thermodynamic properties of the randomly diluted Ising chain with alternating g values are then exactly obtained via a transfer matrix approach. These results are compared to the experimental behavior of CoPhOMe, showing qualitative agreement.

From single Debye-Hückel chains to polyelectrolyte solutions: Simulation results

Science.gov (United States)

Kremer, Kurt

1996-03-01

This lecture will present results from simulations of single weakly charged flexible chains, where the electrostatic part of the interaction is modeled by a Debye-Hückel potential,( with U. Micka, IFF, Forschungszentrum Jülich, 52425 Jülich, Germany) as well as simulations of polyelectrolyte solutions, where the counterions are explicitly taken into account( with M. J. Stevens, Sandia Nat. Lab., Albuquerque, NM 87185-1111) ( M. J. Stevens, K. Kremer, JCP 103), 1669 (1995). The first set of the simulations is meant to clear a recent contoversy on the dependency of the persistence length LP on the screening length Γ. While the analytic theories give Lp ~ Γ^x with either x=1 or x=2, the simulations find for all experimentally accessible chain lengths a varying exponent, which is significantly smaller than 1. This causes serious doubts on the applicability of this model for weakly charged polyelectrolytes in general. The second part deals with strongly charged flexible polyelectrolytes in salt free solution. These simulations are performed for multichain systems. The full Coulomb interactions of the monomers and counterions are treated explicitly. Experimental measurements of the osmotic pressure and the structure factor are reproduced and extended. The simulations reveal a new picture of the chain structure based on calculations of the structure factor, persistence length, end-to-end distance, etc. Even at very low density, the chains show significant bending. Furthermore, the chains contract significantly before they start to overlap. We also show that counterion condensation dramatically alters the chain structure, even for a good solvent backbone.

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

Science.gov (United States)

Crivianu-Gaita, Victor; Thompson, Michael

2016-11-15

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

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

Science.gov (United States)

Ferrari, A; Weill, F S; Paz, M L; Cela, E M; González Maglio, D H; Leoni, J

2012-03-01

Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

International Nuclear Information System (INIS)

X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

2005-01-01

The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO 2 2+ was used as a source of RNA for the development of a recombinant (Fab) 2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab) 2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab) 2 fragments that bound to the UO 2 2+ -DCP complex with an affinity indistinguishable from that of a (Fab) 2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea

Improving the accuracy of the structure prediction of the third hypervariable loop of the heavy chains of antibodies.

KAUST Repository

Messih, Mario Abdel; Lepore, Rosalba; Marcatili, Paolo; Tramontano, Anna

2014-01-01

MOTIVATION: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. RESULTS: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at http://www.biocomputing.it/H3Loopred/ .

Improving the accuracy of the structure prediction of the third hypervariable loop of the heavy chains of antibodies.

KAUST Repository

Messih, Mario Abdel

2014-06-13

MOTIVATION: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. RESULTS: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at http://www.biocomputing.it/H3Loopred/ .

Conductance of single microRNAs chains related to the autism spectrum disorder

Science.gov (United States)

Oliveira, J. I. N.; Albuquerque, E. L.; Fulco, U. L.; Mauriz, P. W.; Sarmento, R. G.; Caetano, E. W. S.; Freire, V. N.

2014-09-01

The charge transport properties of single-stranded microRNAs (miRNAs) chains associated to autism disorder were investigated. The computations were performed within a tight-binding model, together with a transfer matrix technique, with ionization energies and hopping parameters obtained by quantum chemistry method. Current-voltage (I× V) curves of twelve miRNA chains related to the autism spectrum disorders were calculated and analysed. We have obtained both semiconductor and insulator behavior, and a relationship between the current intensity and the autism-related miRNA bases sequencies, suggesting that a kind of electronic biosensor can be developed to distinguish different profiles of autism disorders.

Diversity and maturation in the anti-dansyl antibody response of the Balb/C mouse

International Nuclear Information System (INIS)

Burns, F.R.

1987-01-01

Ten hybridoma cell lines that produce antibodies with specificity for the 5-dimethylaminonaphthalene-1-sulfonyl(dansyl)-lysine hapten, were studied. Single stranded cDNAs were generated by reverse transcription of the immunoglobulin (Ig) mRNAs primed by 5' P-32 labeled oligonucleotides complementary to specific regions of the Ig message. Nucleic acid sequences of 4 mu heavy chains was sufficient to reveal that the early immune response involves members of at least three distinct heavy chain variable region (V/sub H/) gene families. Nucleic acid sequences and Southern blot data from 6 gamma heavy chains reveal that the gamma response is comprised of members of only a single V/sub H/ family although representation from that family derives from at least three distinguishable germline genes. The abrupt restriction of V/sub H/ family usage at the point of class switch can not be explained on the basis of antigen driven selection or of idio-type repression. The data indicate a mechanism for preferential class switch of a particular V/sub H/ family independent of affinity considerations or repression of other idiotypes

Diversity and maturation in the anti-dansyl antibody response of the Balb/C mouse

Energy Technology Data Exchange (ETDEWEB)

Burns, F.R.

1987-01-01

Ten hybridoma cell lines that produce antibodies with specificity for the 5-dimethylaminonaphthalene-1-sulfonyl(dansyl)-lysine hapten, were studied. Single stranded cDNAs were generated by reverse transcription of the immunoglobulin (Ig) mRNAs primed by 5' P-32 labeled oligonucleotides complementary to specific regions of the Ig message. Nucleic acid sequences of 4 mu heavy chains was sufficient to reveal that the early immune response involves members of at least three distinct heavy chain variable region (V/sub H/) gene families. Nucleic acid sequences and Southern blot data from 6 gamma heavy chains reveal that the gamma response is comprised of members of only a single V/sub H/ family although representation from that family derives from at least three distinguishable germline genes. The abrupt restriction of V/sub H/ family usage at the point of class switch can not be explained on the basis of antigen driven selection or of idio-type repression. The data indicate a mechanism for preferential class switch of a particular V/sub H/ family independent of affinity considerations or repression of other idiotypes.

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.

Preparation and characterization of chimeric CD19 monoclonal antibody

International Nuclear Information System (INIS)

Zola, H.; Macardle, P.J.; Bradford, T.; Weedon, H.; Yasui, H.; Kurosawa, Y.

1991-01-01

CD19 antibodies have been suggested as candidates for immunological attack on leukemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind a mouse-human chimera was produced in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed. 47 refs., 7 figs

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

Directory of Open Access Journals (Sweden)

Hust Michael

2008-09-01

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

Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

Energy Technology Data Exchange (ETDEWEB)

Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

1978-03-01

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

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

Science.gov (United States)

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

2016-07-01

Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

KAUST Repository

Hou, Zhipeng; Zhang, Qiang; Xu, Guizhou; Gong, Chen; Ding, Bei; Wang, Yue; Li, Hang; Liu, Enke; Xu, Feng; Zhang, Hongwei; Yao, Yuan; Wu, Guangheng; Zhang, Xixiang; Wang, Wenhong

2018-01-01

Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

KAUST Repository

Hou, Zhipeng

2018-01-04

Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

International Nuclear Information System (INIS)

Katus, H.A.; Hurrell, J.G.; Matsueda, G.R.; Ehrlich, P.; Zurawski, V.R. Jr.; Khaw, B.-A.; Haber, E.

1982-01-01

An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125 I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

Single-chain vascular endothelial growth factor variant with antagonist activity

DEFF Research Database (Denmark)

Boesen, Thomas P; Soni, Bobby; Schwartz, Thue W

2002-01-01

receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce...... dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing...

Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth disease

NARCIS (Netherlands)

Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Keulen, van L.; Rosalia, R.A.; Weerdmeester, K.; Cornelissen, A.H.M.; Bruin, de M.G.M.; Eble, P.L.; Dekker, A.

2007-01-01

Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody

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

DEFF Research Database (Denmark)

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

2009-01-01

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

Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

Science.gov (United States)

Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

2018-03-01

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 9 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

Baculovirus display of functional antibody Fab fragments.

Science.gov (United States)

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

2015-08-01

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

Surface water retardation around single-chain polymeric nanoparticles: critical for catalytic function?

Science.gov (United States)

Stals, Patrick J M; Cheng, Chi-Yuan; van Beek, Lotte; Wauters, Annelies C; Palmans, Anja R A; Han, Songi; Meijer, E W

2016-03-01

A library of water-soluble dynamic single-chain polymeric nanoparticles (SCPN) was prepared using a controlled radical polymerisation technique followed by the introduction of functional groups, including probes at targeted positions. The combined tools of electron paramagnetic resonance (EPR) and Overhauser dynamic nuclear polarization (ODNP) reveal that these SCPNs have structural and surface hydration properties resembling that of enzymes.

Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival.

Science.gov (United States)

Cilliers, Cornelius; Menezes, Bruna; Nessler, Ian; Linderman, Jennifer; Thurber, Greg M

2018-02-01

Current antibody-drug conjugates (ADC) have made advances in engineering the antibody, linker, conjugation site, small-molecule payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, coadministration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared with T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amount of payload delivered to each targeted cell but increasing the number of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist in vitro and has no single-agent efficacy in vivo , yet improves the effectiveness of T-DM1 in vivo Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metabolism and confirmed that the in vivo cellular dose of T-DM1 alone exceeded the minimum required for efficacy in this model. In addition, this technique characterized cellular pharmacokinetics with heterogeneous delivery after 1 day, degradation and payload release by 2 days, and in vitro cell killing and in vivo tumor shrinkage 2 to 3 days later. This work demonstrates that the intratumoral distribution of ADC, independent of payload dose or plasma clearance, plays a major role in ADC efficacy. Significance: This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoral distribution of a antibody-drug conjugate, with implications for improving the efficacy of this class of cancer drugs. Cancer Res; 78(3); 758-68. ©2017 AACR . ©2017 American Association for Cancer Research.

Somatic populations of PGT135-137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics

Directory of Open Access Journals (Sweden)

Jiang eZhu

2012-09-01

Full Text Available Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use deep sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family-specific primers were used to amplify heavy and light chain-variable domain sequences. 454 pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light chain sequences of ~90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations

Experimental studies of the dynamic mechanical response of a single polymer chain

DEFF Research Database (Denmark)

Thormann, Esben; Evans, Drew R.; Craig, Vincent S. J.

2006-01-01

The high-frequency and low-amplitude dynamic mechanical response from a single poly(vinyl alcohol) chain was investigated. Modification of a commercial atomic force microscope enabled high-frequency and low-amplitude periodic deformations of polymer chains during extension to be performed...... mechanical response from poly(vinyl alcohol) does not differ from its static response. This result is not unexpected as poly(vinyl alcohol) is a highly flexible polymer with intramolecular relaxation processes taking place on a short time scale. The choice of a polymer with a fast relaxation allows its...... static properties to be recovered from the dynamic measurements and enables the method suggested in this paper for decoupling the polymer response from the hydrodynamic response to be validated....

Single-Chain Antibody Fragment VEGF Inhibitor RTH258 for Neovascular Age-Related Macular Degeneration

DEFF Research Database (Denmark)

Holz, Frank G; Dugel, Pravin U.; Weissgerber, Georges

2016-01-01

Purpose To assess the safety and efficacy of different doses of RTH258 applied as single intravitreal administration compared with ranibizumab 0.5 mg in patients with neovascular age-related macular degeneration (AMD). Design Six-month, phase 1/2, prospective, multicenter, double-masked, randomized...

Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry.

Science.gov (United States)

Wang, Shunhai; Liu, Anita P; Yan, Yuetian; Daly, Thomas J; Li, Ning

2018-05-30

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers

Energy Technology Data Exchange (ETDEWEB)

Ebner, Andreas [Institute of Biophysics, University of Linz, 4040 Linz (Austria)], E-mail: andreas.ebner@jku.at; Hinterdorfer, Peter; Gruber, Hermann J. [Institute of Biophysics, University of Linz, 4040 Linz (Austria)

2007-10-15

Atomic force microscopy (AFM) has developed into a key technique for elucidation of biological systems on the single molecular level. In particular, molecular recognition force microscopy has proven to be a powerful tool for the investigation of biological interactions under near physiological conditions. For this purpose, ligands are tethered to AFM tips and the interaction forces with cognate receptors on the sample surface are measured with pico-Newton accuracy. In the first step of tip functionalization, amino groups are typically introduced on the initially inert AFM tip. Several methods have been developed to reproducibly adjust the desired low density of amino groups on the tip surface, i.e. esterification with ethanolamine, gas-phase silanization with aminopropyl-triethoxysilane (APTES), or treatment with aminophenyl-trimethoxysilane (APhS) in toluene solution. In the present study, the usefulness of these methods for attachments of antibodies to AFM tips was characterized by a standardized test system, in which biotinylated IgG was bound to the tip and a dense monolayer of avidin on mica served as test sample. All three methods of aminofunctionalization were found fully satisfactory for attachment of single antibodies to AFM tips, only in a parallel macroscopic assay on silicon nitride chips a minor difference was found in that APTES appeared to yield a slightly lower surface density of amino groups.

VHH Antibodies: Reagents for Mycotoxin Detection in Food Products

Directory of Open Access Journals (Sweden)

Jia Wang

2018-02-01

Full Text Available Mycotoxins are the toxic secondary metabolites produced by fungi and they are a worldwide public health concern. A VHH antibody (or nanobody is the smallest antigen binding entity and is produced by heavy chain only antibodies. Compared with conventional antibodies, VHH antibodies overcome many pitfalls typically encountered in clinical therapeutics and immunodiagnostics. Likewise, VHH antibodies are particularly useful for monitoring mycotoxins in food and feedstuffs, as they are easily genetic engineered and have superior stability. In this review, we summarize the efforts to produce anti-mycotoxins VHH antibodies and associated assays, presenting VHH as a potential tool in mycotoxin analysis.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

OpenAIRE

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies)...

Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

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Shradha Bagaria

Full Text Available Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

Direct observation of backbone planarization via side-chain alignment in single bulky-substituted polythiophenes

Science.gov (United States)

Raithel, Dominic; Simine, Lena; Pickel, Sebastian; Schötz, Konstantin; Panzer, Fabian; Baderschneider, Sebastian; Schiefer, Daniel; Lohwasser, Ruth; Köhler, Jürgen; Thelakkat, Mukundan; Sommer, Michael; Köhler, Anna; Rossky, Peter J.; Hildner, Richard

2018-03-01

The backbone conformation of conjugated polymers affects, to a large extent, their optical and electronic properties. The usually flexible substituents provide solubility and influence the packing behavior of conjugated polymers in films or in bad solvents. However, the role of the side chains in determining and potentially controlling the backbone conformation, and thus the optical and electronic properties on the single polymer level, is currently under debate. Here, we investigate directly the impact of the side chains by studying the bulky-substituted poly(3-(2,5-dioctylphenyl)thiophene) (PDOPT) and the common poly(3-hexylthiophene) (P3HT), both with a defined molecular weight and high regioregularity, using low-temperature single-chain photoluminescence (PL) spectroscopy and quantum-classical simulations. Surprisingly, the optical transition energy of PDOPT is significantly (˜2,000 cm‑1 or 0.25 eV) red-shifted relative to P3HT despite a higher static and dynamic disorder in the former. We ascribe this red shift to a side-chain induced backbone planarization in PDOPT, supported by temperature-dependent ensemble PL spectroscopy. Our atomistic simulations reveal that the bulkier 2,5-dioctylphenyl side chains of PDOPT adopt a clear secondary helical structural motif and thus protect conjugation, i.e., enforce backbone planarity, whereas, for P3HT, this is not the case. These different degrees of planarity in both thiophenes do not result in different conjugation lengths, which we found to be similar. It is rather the stronger electronic coupling between the repeating units in the more planar PDOPT which gives rise to the observed spectral red shift as well as to a reduced calculated electron‑hole polarization.

Development of Antibody-Coated Magnetite Nanoparticles for Biomarker Immobilization

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Christian Chapa Gonzalez

2014-01-01

Full Text Available Magnetic nanoparticles (MNPs have great potential in biomedical applications because of their magnetic response offers the possibility to direct them to specific areas and target biological entities. Magnetic separation of biomolecules is one of the most important applications of MNPs because their versatility in detecting cancer biomarkers. However, the effectiveness of this method depends on many factors, including the type of functionalization onto MNPs. Therefore, in this study, magnetite nanoparticles have been developed in order to separate the 5′-nucleotidase enzyme (5eNT. The 5eNT is used as a bio-indicator for diagnosing diseases such as hepatic ischaemia, liver tumor, and hepatotoxic drugs damage. Magnetic nanoparticles were covered in a core/shell type with silica, aminosilane, and a double shell of silica-aminosilane. A ScFv (fragment antibody and anti-CD73 antibody were attached to the coated nanoparticles in order to separate the enzyme. The magnetic separation of this enzyme with fragment antibody was found to be 28% higher than anti-CD73 antibody and the enzyme adsorption was improved with the double shell due to the increased length of the polymeric chain. Magnetite nanoparticles with a double shell (silica-aminosilane were also found to be more sensitive than magnetite with a single shell in the detection of biomarkers.

Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide γ-chain-specific monoclonal antibody

International Nuclear Information System (INIS)

Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

1987-01-01

The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the λ chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125 I-labeled denatured lysates of T3 + WT31 - lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of 125 I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3 + WT31 - cell populations are required to determine whether the TCR contains two λ polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa γ-chain polypeptide under reducing conditions expressed on purified L3T4 - Lyt2 - BALB/c mouse thymocytes. This γ-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions

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

NARCIS (Netherlands)

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

2002-01-01

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

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

Science.gov (United States)

McElhiney, J; Lawton, L A; Porter, A J

2000-12-01

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-08-07

Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on H_CA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

Single step, pH induced gold nanoparticle chain formation in lecithin/water system.

Science.gov (United States)

Sharma, Damyanti

2013-07-01

Gold nanoparticle (AuNP) chains have been formed by a single step method in a lecithin/water system where lecithin itself plays the role of a reductant and a template for AuNP chain formation. Two preparative strategies were explored: (1) evaporating lecithin solution with aqueous gold chloride (HAuCl4) at different pHs and (2) dispersing lecithin vesicles in aqueous HAuCl4 solutions of various pHs in the range of 2.5-11.3. In method 1, at initial pH 2.5, 20-50 nm AuNPs are found attached to lecithin vesicles. When pH is raised to 5.5 there are no vesicles present and 20 nm monodisperse particles are found aggregating. Chain formation of fine nanoparticles (3-5 nm) is observed from neutral to basic pH, between 6.5-10.3 The chains formed are hundreds of nanometers to micrometer long and are usually 2-3 nanoparticles wide. On further increasing pH to 11.3, particles form disk-like or raft-like structures. When method (ii) was used a little chain formation was observed. Most of the nanoparticles formed were found either sitting together as raft like structures or scattered on lecithin structures. Copyright © 2013 Elsevier B.V. All rights reserved.

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

Science.gov (United States)

Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

2017-10-01

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

DEFF Research Database (Denmark)

Skjødt, K; Schou, C; Koch, C

1984-01-01

A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

Extrasynaptic location of laminin beta 2 chain in developing and adult human skeletal muscle

DEFF Research Database (Denmark)

Wewer, U M; Thornell, L E; Loechel, F

1997-01-01

and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present...

Monoclonal antibody fragment removal mediated by mixed mode resins.

Science.gov (United States)

O'Connor, Ellen; Aspelund, Matthew; Bartnik, Frank; Berge, Mark; Coughlin, Kelly; Kambarami, Mutsa; Spencer, David; Yan, Huiming; Wang, William

2017-05-26

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities

Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition.

Science.gov (United States)

Umlauf, Benjamin J; Mix, Kalie A; Grosskopf, Vanessa A; Raines, Ronald T; Shusta, Eric V

2018-05-03

Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

Science.gov (United States)

Kawahara, Masahiro; Chen, Jianhong; Sogo, Takahiro; Teng, Jinying; Otsu, Makoto; Onodera, Masafumi; Nakauchi, Hiromitsu; Ueda, Hiroshi; Nagamune, Teruyuki

2011-09-01

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

Science.gov (United States)

Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel

2015-04-01

For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. © 2014 Society for Laboratory Automation and Screening.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Kaku Yoshihiro

2012-09-01

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

MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

Directory of Open Access Journals (Sweden)

Duarte Keila M.R.

2001-01-01

Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

International Nuclear Information System (INIS)

Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

1996-01-01

A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,κ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with 99m Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author)

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

Directory of Open Access Journals (Sweden)

Christiane Y Ozaki

Full Text Available Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT and heat-stable toxin (ST, from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

Science.gov (United States)

Matsushita, Takahiko; Arai, Hidenao; Koyama, Tetsuo; Hatano, Ken; Nemoto, Naoto; Matsuoka, Koji

2017-11-01

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efficient sampling of reversible cross-linking polymers: Self-assembly of single-chain polymeric nanoparticles

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Oyarzún, Bernardo; Mognetti, Bortolo Matteo

2018-03-01

We present a new simulation technique to study systems of polymers functionalized by reactive sites that bind/unbind forming reversible linkages. Functionalized polymers feature self-assembly and responsive properties that are unmatched by the systems lacking selective interactions. The scales at which the functional properties of these materials emerge are difficult to model, especially in the reversible regime where such properties result from many binding/unbinding events. This difficulty is related to large entropic barriers associated with the formation of intra-molecular loops. In this work, we present a simulation scheme that sidesteps configurational costs by dedicated Monte Carlo moves capable of binding/unbinding reactive sites in a single step. Cross-linking reactions are implemented by trial moves that reconstruct chain sections attempting, at the same time, a dimerization reaction between pairs of reactive sites. The model is parametrized by the reaction equilibrium constant of the reactive species free in solution. This quantity can be obtained by means of experiments or atomistic/quantum simulations. We use the proposed methodology to study the self-assembly of single-chain polymeric nanoparticles, starting from flexible precursors carrying regularly or randomly distributed reactive sites. We focus on understanding differences in the morphology of chain nanoparticles when linkages are reversible as compared to the well-studied case of irreversible reactions. Intriguingly, we find that the size of regularly functionalized chains, in good solvent conditions, is non-monotonous as a function of the degree of functionalization. We clarify how this result follows from excluded volume interactions and is peculiar of reversible linkages and regular functionalizations.

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

Science.gov (United States)

di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

2000-01-01

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

Construction and Self-Assembly of Single-Chain Polymer Nanoparticles via Coordination Association and Electrostatic Repulsion in Water.

Science.gov (United States)

Zhu, Zhengguang; Xu, Na; Yu, Qiuping; Guo, Lei; Cao, Hui; Lu, Xinhua; Cai, Yuanli

2015-08-01

Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly sensitive antibody-aptamer sensor for vascular endothelial growth factor based on hybridization chain reaction and pH meter/indicator.

Science.gov (United States)

Xu, Huifeng; Kou, Fangxia; Ye, Hongzhi; Wang, Zongwen; Huang, Suixin; Liu, Xianxiang; Zhu, Xi; Lin, Zhenyu; Chen, Guonan

2017-12-01

Vascular endothelial growth factor (VEGF) is a crucial signaling protein for the tumor growth and metastasis, which is also acted as the biomarkers for various diseases. In this research, we fabricate an aptamer-antibody sensor for point-of-care test of VEGF. Firstly, target VEGF is captured by antibody immobilized on the microplate, and then binds with aptamer to form the sandwich structure. Next, with the assist of glucose oxidase (GOx)-functionalized ssDNAs, hybridization chain reaction occurs using the aptamer as the primer. Thus, GOx are greatly gathered on the microplate, which catalyzes the oxidization of glucose, leading to the pH change. As a result, the detect limit at a signal-to-noise was estimated to be 0.5pg/mL of target by pH meter, and 1.6pg/mL of VEGF was able to be distinguished by naked eyes. Meanwhile, this method has been used assay VEGF in the serum with the satisfactory results. Copyright © 2017. Published by Elsevier B.V.

Mediating Dynamic Supply Chain Formation by Collaborative Single Machine Earliness/Tardiness Agents in Supply Mesh

Directory of Open Access Journals (Sweden)

Hang Yang

2014-01-01

Full Text Available Nowadays, a trend of forming dynamic supply chains with different trading partners over different e-marketplaces has emerged. These supply chains, which are called “supply mesh,” generally refer to heterogeneous electronic marketplaces in which dynamic supply chains, as per project (often make-to-order, are formed across different parties. Conceptually, in a supply mesh a dynamic supply chain is formed vertically, mediating several companies for a project. Companies that are on the same level horizontally are either competitors or cohorts. A complex scenario such as this makes it challenging to find the right group of members for a dynamic supply chain. Earlier on, a multiagent model called the collaborative single machine earliness/tardiness (CSET model was proposed for the optimal formation of make-to-order supply chains. This paper contributes the particular agent designs, for enabling the mediation of CSET in a supply mesh, and the possibilities are discussed. It is demonstrated via a computer simulation, based on samples from the U.S. textile industry, that by using intelligent agents under the CSET model it is possible to automatically find an ideal group of trading partners from a supply mesh.

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Energy Technology Data Exchange (ETDEWEB)

Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

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

Directory of Open Access Journals (Sweden)

Guowei Wei

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

Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

Science.gov (United States)

Yuasa, Noriyuki; Iida, Noriko; Sakaue, Hiroyuki; Zhang, Wei; Wilczynski, Sharon; Fujita-Yamaguchi, Yoko

2007-10-01

We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

First-order reversal curves of single domain particles: diluted random assemblages and chains

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Egli, R.

2009-04-01

Exact magnetic models can be used to calculate first-order reversal curves (FORC) of single domain (SD) particle assemblages, as shown by Newell [2005] for the case of isolated Stoner-Wohlfarth particles. After overcoming experimental difficulties, a FORC diagram sharing many similarities to Newell's model has been measured on a lake sediment sample (see A.P. Chen et al., "Quantification of magnetofossils using first-order reversal curves", EGU General Assembly 2009, Abstracts Vol. 11, EGU2009-10719). This sample contains abundant magnetofossils, as shown by coercivity analysis and electron microscopy, therefore suggesting that well dispersed, intact magnetosome chains are the main SD carriers. Subtle differences between the reversible and the irreversible contributions of the measured FORC distribution suggest that magnetosome chains might not be correctly described by the Stoner-Wohlfarth model. To better understand the hysteresis properties of such chains, a simple magnetic model has been implemented, taking dipole-dipole interactions between particles within the same chain into account. The model results depend on the magnetosome elongation, the number of magnetosomes in a chain, and the gap between them. If the chain axis is subparallel to the applied field, the magnetic moment reverses by a pseudo-fanning mode, which is replaced by a pseudo-coherent rotation mode at greater angles. These reversal modes are intrinsically different from coherent rotation assumed Stoner-Wohlfarth model, resulting in FORC diagrams with a smaller reversible component. On the other hand, isolated authigenic SD particles can precipitate in the sediment matrix, as it might occur for pedogenic magnetite. In this case, an assembly of randomly located particles provides a possible model for the resulting FORC diagram. If the concentration of the particles is small, each particle is affected by a random interaction field whose statistical distribution can be calculated from first

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

Science.gov (United States)

Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

2017-08-08

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

Science.gov (United States)

Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

2016-01-01

Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

Differential neutralizing activities of a single domain camelid antibody (VHH specific for ricin toxin's binding subunit (RTB.

Directory of Open Access Journals (Sweden)

Cristina Herrera

Full Text Available Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody" specific for ricin's enzymatic (RTA and binding (RTB subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50 that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Quantum phase transitions driven by rhombic-type single-ion anisotropy in the S =1 Haldane chain

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Tzeng, Yu-Chin; Onishi, Hiroaki; Okubo, Tsuyoshi; Kao, Ying-Jer

2017-08-01

The spin-1 Haldane chain is an example of the symmetry-protected-topological (SPT) phase in one dimension. Experimental realization of the spin chain materials usually involves both the uniaxial-type, D (Sz)2 , and the rhombic-type, E [(Sx)2-(Sy)2] , single-ion anisotropies. Here, we provide a precise ground-state phase diagram for a spin-1 Haldane chain with these single-ion anisotropies. Using quantum numbers, we find that the Z2 symmetry breaking phase can be characterized by double degeneracy in the entanglement spectrum. Topological quantum phase transitions take place on particular paths in the phase diagram, from the Haldane phase to the large-Ex, large-Ey, or large-D phases. The topological critical points are determined by the level spectroscopy method with a newly developed parity technique in the density matrix renormalization group [Phys. Rev. B 86, 024403 (2012), 10.1103/PhysRevB.86.024403], and the Haldane-large-D critical point is obtained with an unprecedented precision, (D/J ) c=0.9684713 (1 ) . Close to this critical point, a small rhombic single-ion anisotropy |E |/J ≪1 can destroy the Haldane phase and bring the system into a y -Néel phase. We propose that the compound [Ni (HF2) (3-Clpy ) 4] BF4 is a candidate system to search for the y -Néel phase.

Magnetic properties of CsCrCl/sub 3/, an antiferromagnetic chain compound with single-ion anisotropy

Energy Technology Data Exchange (ETDEWEB)

Day, P; Gregson, A K; Leech, D H [Oxford Univ. (UK). Inorganic Chemistry Lab.; Hutchings, M T [UKAEA Atomic Energy Research Establishment, Harwell. Materials Physics Div.; Rainford, B D [Imperial Coll. of Science and Technology, London (UK). Dept. of Physics

1979-01-01

The magnetic structure and excitations of the linear chain hexagonal perovskite salt CsCrCl/sub 3/ have been studied by susceptibility, powder and single crystal neutron diffraction, and coherent inelastic neutron scattering. Below the Neel temperature, Tsub(N) = 16 K, the spins lie in the basal plane with antiferromagnetic ordering along the c-axis chains. At 4.5 K there is strong dispersion of the spin-wave energy along c but no measurable dispersion perpendicular to c.

Safety, efficacy and pharmacokinetics of rVIII-SingleChain in children with severe hemophilia A: results of a multicenter clinical trial.

Science.gov (United States)

Stasyshyn, O; Djambas Khayat, C; Iosava, G; Ong, J; Abdul Karim, F; Fischer, K; Veldman, A; Blackman, N; St Ledger, K; Pabinger, I

2017-04-01

Essentials rVIII-SingleChain is a novel recombinant factor VIII with covalently bonded heavy and light chains. Efficacy, safety and pharmacokinetics were studied in pediatric patients with severe hemophilia A. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00. rVIII-SingleChain showed excellent hemostatic efficacy and a favorable safety profile. Background rVIII-SingleChain is a novel B-domain truncated recombinant factor VIII (rFVIII) comprised of covalently bonded FVIII heavy and light chains, demonstrating a high binding affinity to von Willebrand factor. Objectives This phase III study investigated the safety, efficacy and pharmacokinetics of rVIII-SingleChain in previously treated pediatric patients hemophilia A. Patients/Methods Patients could be assigned to prophylaxis or on-demand therapy by the investigator. For patients assigned to prophylaxis, the treatment regimen and dose were based on the bleeding phenotype. For patients receiving on-demand therapy, dosing was guided by World Federation of Hemophilia recommendations. The primary endpoint was treatment success, defined as a rating of 'excellent' or 'good' on the investigator's clinical assessment of hemostatic efficacy for all treated bleeding events. Results The study enrolled 84 patients (0 to 50 EDs. In the 347 bleeds treated and evaluated by the investigator, hemostatic efficacy was rated as excellent or good in 96.3%. The median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.00, 2.20), and the median annualized bleeding rate was 3.69 (Q1, Q3: 0.00, 7.20) across all prophylaxis regimens. No participant developed an inhibitor. Conclusions rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy and a favorable safety profile in a clinical study in children hemophilia A. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and

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

NARCIS (Netherlands)

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

2005-01-01

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

Impact of Age and Antibody Type on Progression From Single to Multiple Autoantibodies in Type 1 Diabetes Relatives.

Science.gov (United States)

Bosi, Emanuele; Boulware, David C; Becker, Dorothy J; Buckner, Jane H; Geyer, Susan; Gottlieb, Peter A; Henderson, Courtney; Kinderman, Amanda; Sosenko, Jay M; Steck, Andrea K; Bingley, Polly J

2017-08-01

Islet autoantibodies are markers of type 1 diabetes, and an increase in number of autoantibodies detected during the preclinical phase predicts progression to overt disease. To refine the effect of age in relation to islet antibody type on progression from single to multiple autoantibodies in relatives of people with type 1 diabetes. We examined 994 relatives with normal glucose tolerance who were positive for a single autoantibody, followed prospectively in the TrialNet Pathway to Prevention. Antibodies to glutamic acid decarboxylase (GADA), insulin (IAA), insulinoma-associated antigen 2, and zinc transporter 8 and islet cell antibodies were tested every 6 to 12 months. The primary outcome was confirmed development of multiple autoantibodies. Age was categorized as <8 years, 8 to 11 years, 12 to 17 years, and ≥18 years, and optimal age breakpoints were identified by recursive partitioning analysis. After median follow-up of 2 years, 141 relatives had developed at least one additional autoantibodies. Five-year risk was inversely related to age, but the pattern differed by antibody type: Relatives with GADA showed a gradual decrease in risk over the four age groups, whereas relatives with IAA showed a sharp decrease above age 8 years. Recursive partitioning analysis identified age breakpoints at 14 years in relatives with GADA and at 4 years in relatives with IAA. In relatives with IAA, spread of islet autoimmunity is largely limited to early childhood, whereas immune responses initially directed at glutamic acid decarboxylase can mature over a longer period. These differences have important implications for monitoring these patients and for designing prevention trials. Copyright © 2017 Endocrine Society

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

Energy Technology Data Exchange (ETDEWEB)

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

2007-07-13

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-05-20

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

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

International Nuclear Information System (INIS)

Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo; Marco, Ario de

2011-01-01

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

Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

NARCIS (Netherlands)

Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

2005-01-01

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

Science.gov (United States)

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

Science.gov (United States)

Collazo, M. Thelma; Garza, John A.; Hayhurst, Andrew

2010-01-01

Background There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype. Methods and Findings A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism. Conclusions Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively. PMID:20098614

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Science.gov (United States)

Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

2013-01-01

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

Radiolabelled antibodies in imaging

International Nuclear Information System (INIS)

Khaw, B.A.; Haber, E.

1982-01-01

Recent technological advances make it possible to produce pure (monoclonal) antibodies in unlimited quantities without the need for continuous immunization of animals and to label these antibodies with a variety of radionuclides which can be traced by single-photon computed tomography. An outline review of the state of the art is presented, with particular reference to the imaging of myocardial infarcts and to tumour imaging studies using labelled monoclonal antibodies (sup(99m)Tc and 125 I). Lengthy bibliography. (U.K.)

Monte Carlo simulated dynamical magnetization of single-chain magnets

Energy Technology Data Exchange (ETDEWEB)

Li, Jun; Liu, Bang-Gui, E-mail: bgliu@iphy.ac.cn

2015-03-15

Here, a dynamical Monte-Carlo (DMC) method is used to study temperature-dependent dynamical magnetization of famous Mn{sub 2}Ni system as typical example of single-chain magnets with strong magnetic anisotropy. Simulated magnetization curves are in good agreement with experimental results under typical temperatures and sweeping rates, and simulated coercive fields as functions of temperature are also consistent with experimental curves. Further analysis indicates that the magnetization reversal is determined by both thermal-activated effects and quantum spin tunnelings. These can help explore basic properties and applications of such important magnetic systems. - Highlights: • Monte Carlo simulated magnetization curves are in good agreement with experimental results. • Simulated coercive fields as functions of temperature are consistent with experimental results. • The magnetization reversal is understood in terms of the Monte Carlo simulations.

Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

Directory of Open Access Journals (Sweden)

Bogen Bjarne

2010-02-01

Full Text Available Abstract Background Efficient expression systems exist for antibody (Ab molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs. Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc TCRs. Results The effect of 1 over-expression of the periplasmic chaperon FkpA, 2 culture conditions and 3 molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1 high yield recovery sufficient for biophysical characterization and 2 high throughput screening of such molecules following molecular engineering.

A novel affinity purification method to isolate peptide specific antibodies

DEFF Research Database (Denmark)

Karlsen, Alan E; Lernmark, A; Kofod, Hans

1990-01-01

Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin

NARCIS (Netherlands)

Harmsen, M.M.; Fijten, H.P.D.; Dekker, A.; Eble, P.L.

2008-01-01

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-hoofed animals that occasionally causes outbreaks in Europe. We aim to develop an immunotherapy that confers rapid protection against FMD in outbreak situations. For this purpose, we previously isolated llama single-domain antibody

The exact solution of the Ising quantum chain with alternating single and sector defects

International Nuclear Information System (INIS)

Zhang Degang; Li Bozang; Li Yun

1992-10-01

The Ising quantum chain with alternating single and sector defects is solved exactly by using the technique of Lieb, Schultz and Mattis. The energy spectrum of this model is shown to have a tower structure if and only if these defects constitute a commensurate configuration. This means that conformal invariance is preserved under these circumstances. (author). 13 refs

Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

Directory of Open Access Journals (Sweden)

Phelps Amanda L

2009-11-01

Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

Science.gov (United States)

Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes

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Consuelo Garcia-Rodriguez

2018-03-01

Full Text Available Human botulism is most commonly caused by botulinum neurotoxin (BoNT serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS. A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD. By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633 is in pre-clinical development with an investigational new drug (IND application filing expected in 2018.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

Science.gov (United States)

Garcia-Rodriguez, Consuelo; Razai, Ali; Geren, Isin N; Lou, Jianlong; Conrad, Fraser; Wen, Wei-Hua; Farr-Jones, Shauna; Smith, Theresa J; Brown, Jennifer L; Skerry, Janet C; Smith, Leonard A; Marks, James D

2018-03-01

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (K D ). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had K D values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

International Nuclear Information System (INIS)

Saga, T.; Endo, K.; Koizumi, M.; Kawamura, Y.; Watanabe, Y.; Konishi, J.; Ueda, R.; Nishimura, Y.; Yokoyama, M.; Watanabe, T.

1990-01-01

A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111 In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111 In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

International Nuclear Information System (INIS)

Wang Yuzhen; Zhang Xiaohua; Yuan Li; Xu Tao; Rao Yu; Li Jia; Dai Heping

2008-01-01

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV

Targeting Antibodies to Carbon Nanotube Field Effect Transistors by Pyrene Hydrazide Modification of Heavy Chain Carbohydrates

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Steingrimur Stefansson

2012-01-01

Full Text Available Many carbon nanotube field-effect transistor (CNT-FET studies have used immobilized antibodies as the ligand binding moiety. However, antibodies are not optimal for CNT-FET detection due to their large size and charge. Their size can prevent ligands from reaching within the Debye length of the CNTs and a layer of charged antibodies on the circuits can drown out any ligand signal. In an attempt to minimize the antibody footprint on CNT-FETs, we examined whether pyrene hydrazide modification of antibody carbohydrates could reduce the concentration required to functionalize CNT circuits. The carbohydrates are almost exclusively on the antibody Fc region and this site-specific modification could mediate uniform antibody orientation on the CNTs. We compared the hydrazide modification of anti-E. coli O157:H7 polyclonal antibodies to pyrenebutanoic acid succinimidyl ester-coated CNTs and carbodiimide-mediated antibody CNT attachment. Our results show that the pyrene hydrazide modification was superior to those methods with respect to bacteria detection and less than 1 nM labeled antibody was required to functionalize the circuits.

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

Science.gov (United States)

Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

International Nuclear Information System (INIS)

Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

2014-01-01

Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp"*_2Co] (Cp"*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K.

An indirect antibody assay using haptenated antigen and 125I-labelled anti-hapten antibody

International Nuclear Information System (INIS)

Aalberse, R.C.; Amsterdam Univ.

1978-01-01

Hapten (trinitrophenyl) was coupled to antigen (ovalbumin). The haptenated antigen was bound by anti-ovalbumin antibody and binding was quantitated with 125 I-labelled anti-hapten antibodies. Thus, with a single radioactive reagent, antibodies against a variety of antigens can be detected while the problems inherent in a labelled antiglobulin binding test are avoided. In the ovalbumin system, the haptenated antigen binding test proved to be approximately 20 times as sensitive as the iodinated ovalbumin binding test

Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors.

Science.gov (United States)

Taniguchi, K; Yoshihara, S; Maruya, E; Ikegame, K; Kaida, K; Hayashi, K; Kato, R; Inoue, T; Fujioka, T; Tamaki, H; Okada, M; Onuma, T; Fujii, N; Kusunoki, Y; Soma, T; Saji, H; Ogawa, H

2012-10-01

Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

Probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies.

Science.gov (United States)

Niikura, Takahito; Yamamoto, Izumi; Nakada, Yasuyuki; Kamejima, Sahoko; Katsumata, Haruki; Yamakawa, Takafumi; Furuya, Maiko; Mafune, Aki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

2015-07-01

We report a case of probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies. A 44 year-old male was admitted to our hospital for a kidney transplant. The donor, his wife, was an ABO minor mismatch (blood type O to A) and had Gitelman syndrome. Graft function was delayed; his serum creatinine level was 10.1 mg/dL at 3 days after transplantation. Open biopsy was performed immediately; no venous thrombosis was observed during surgery. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Flow cytometric crossmatching was positive, but no panel-reactive antibodies against HLA or donor-specific antibodies (DSAbs) to major histocompatibility complex class I-related chain A (MICA) were detected. Taken together, we diagnosed him with probable C4d-negative accelerated antibody-mediated rejection due to non-HLA, non-MICA antibodies, the patient was treated with steroid pulse therapy (methylprednisolone 500 mg/day for 3 days), plasma exchange, intravenous immunoglobulin (40 g/body), and rituximab (200 mg/body) were performed. Biopsy at 58 days after transplantation, at which time S-Cr levels were 1.56 mg/dL, found no evidence of rejection. This case, presented with a review of relevant literature, demonstrates that probable C4d-negative accelerated acute AMR can result from non-HLA antibodies. © 2015 Asian Pacific Society of Nephrology.

Radioimmunoassay of single-stranded DNA antibodies for control of diagnosis and therapy

Energy Technology Data Exchange (ETDEWEB)

Meffert, H; Boehm, F; Soennichsen, N; Gens, J [Humboldt-Universitaet, Berlin (German Democratic Republic). Bereich Medizin (Charite)

1980-10-01

Several years experience in quantitative determination of single-stranded DNA antibodies is reported and the normal range as well as the diagnostic hit rate of the method is outlined. In the controls the mean DNA attachment rate was 1.5% and the upper normal range limit was 12.8%, the risk of erroneous rejection being 1%. The DNA binding rate was greater than 12.8% in 74.7% of untreated patients suffering from lupus erythematodes visceralis, in 47.6% of patients with circumscribed sclerodermia, in 14.4% of patients with progressive sclerodermia, and in 10.3% of those suffering from lupus erythematodes chronicus. The findings emphasize the importance of regulatory mechanisms of the immune system to the process of autosensitization.

Radioimmunoassay of single-stranded DNA antibodies for control of diagnosis and therapy

International Nuclear Information System (INIS)

Meffert, H.; Boehm, F.; Soennichsen, N.; Gens, J.

1980-01-01

Several years experience in quantitative determination of single-stranded DNA antibodies is reported and the normal range as well as the diagnostic hit rate of the method is outlined. In the controls the mean DNA attachment rate was 1.5% and the upper normal range limit was 12.8%, the risk of erroneous rejection being 1%. The DNA binding rate was greater than 12.8% in 74.7% of untreated patients suffering from lupus erythematodes visceralis, in 47.6% of patients with circumscribed sclerodermia, in 14.4% of patients with progressive sclerodermia, and in 10.3% of those suffering from lupus erythematodes chronicus. The findings emphasize the importance of regulatory mechanisms of the immune system to the process of autosensitization

Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

Science.gov (United States)

Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

2015-01-01

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

Pharmacokinetic Effects of Antidrug Antibodies Occurring in Healthy Subjects After a Single Dose of Intravenous Infliximab.

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Ehrenpreis, Eli D

2017-12-01

Infliximab pharmacokinetic studies have been performed in patients receiving chronic infliximab therapy. In these patients, infliximab antidrug antibodies (ADAs) increase infliximab clearance and decrease serum levels and drug efficacy. This study analyzed the pharmacokinetic effect of infliximab ADAs in healthy subjects receiving a single dose of intravenous infliximab. Data were obtained from a single-blind, parallel-group, single-dose study of healthy subjects receiving 5 mg/kg of intravenous SB2 (infliximab biosimilar), EU-sourced Remicade (EU-IFX) or US-sourced Remicade (US-IFX). Serum infliximab was measured at 1, 2, 3, 6, 12, 24, 48, and 72 h and at 5, 7, 14, 21, 28, 42, 56, and 70 days after administration. ADAs were measured pre-dose and at 29 and 71 days. Data from the first ten subjects randomized to each treatment arm were utilized for this study. A two-compartment model of the serum infliximab vs. time curve was developed using nonlinear regression. At 10 weeks, 11 subjects (37%) developed ADAs. ADAs were detected in four subjects after SB2, one subject after EU-IFX, and six subjects after US-IFX infusion. Of these, neutralizing antibodies occurred in one subject after SB2, in no subjects after EU-IFX, and in three subjects after US-IFX infusion. Infliximab clearance was increased in subjects with ADAs vs. those without ADAs (12.89 ± 2.69 vs. 9.90 ± 1.74 ml/h; p ADAs (282.4 ± 56.4 vs. 343.3 ± 61.9 h; p ADAs are common in healthy subjects after a single intravenous dose of infliximab and result in faster infliximab clearance, shorter elimination time, and lower serum infliximab levels. These data confirm that ADAs are common with biologic therapy and significantly impact the efficacy of these drugs.

The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1.

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Liu, Lihong; Wang, Weiming; Matz, Julie; Ye, Chaobaihui; Bracq, Lucie; Delon, Jerome; Kimata, Jason T; Chen, Zhiwei; Benichou, Serge; Zhou, Paul

2016-12-01

The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4 + cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI

A monoclonal antibody distinguishes between two IgM heavy chain isotypes in Atlantic salmon and brown trout: protein characterization, 3D modeling and epitope mapping.

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Kamil, Atif; Falk, Knut; Sharma, Animesh; Raae, Arnt; Berven, Frode; Koppang, Erling Olaf; Hordvik, Ivar

2011-09-01

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of an EGFRvIII specific recombinant antibody

Directory of Open Access Journals (Sweden)

Li Gordon

2010-10-01

Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

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Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

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Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

2015-06-01

The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies 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, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bubble dynamics and bubble-induced turbulence of a single-bubble chain

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Lee, Joohyoung; Park, Hyungmin

2016-11-01

In the present study, the bubble dynamics and liquid-phase turbulence induced by a chain of bubbles injected from a single nozzle have been experimentally investigated. Using a high-speed two-phase particle image velociemtry, measurements on the bubbles and liquid-phase velocity field are conducted in a transparent tank filled with water, while varying the bubble release frequency from 0.1 to 35 Hz. The tested bubble size ranges between 2.0-3.2 mm, and the corresponding bubble Reynolds number is 590-1100, indicating that it belongs to the regime of path instability. As the release frequency increases, it is found that the global shape of bubble dispersion can be classified into two regimes: from asymmetric (regular) to axisymmetric (irregular). In particular, at higher frequency, the wake vortices of leading bubbles cause an irregular behaviour of the following bubble. For the liquid phase, it is found that a specific trend on the bubble-induced turbulence appears in a strong relation to the above bubble dynamics. Considering this, we try to provide a theoretical model to estimate the liquid-phase turbulence induced by a chain of bubbles. Supported by a Grant funded by Samsung Electronics, Korea.

A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

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Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

2017-01-01

To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

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Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

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Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

2018-02-16

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Monoclonal antibodies: potential role in radiation therapy and oncology

International Nuclear Information System (INIS)

Order, S.E.

1982-01-01

Specificity, which is a hallmark of the immune system, will be used in radiation oncology in both diagnosis and therapy through the application of radiolabelled monoclonal and polyclonal antibodies. Antigenic specificities, antibody preparations, and the tumor as a target for radiolabelled antibody is reviewed. Several clinical situations, i.e. single tumor cell suspensions, intraperitoneal single cells and masses, and solid tumors are reviewed in regard to both immune antibody targeting and specific differences between tumors in these regions. The concentration of tumor associated antigens is introductory to radiolabelled antibodies in diagnosis. In the radiation therapy of solid tumors, data regarding tumor dose, tumor effective half-life, varied antibody preparations, and the use of radiolabelled antibody as a method of tumor implantation is discussed using antiferritin 131 I-IgG as a model in hepatoma. The theoretical applications of monoclonal antibody integrated in cancer therapy are then presented as a new goal for future development

X-ray computed tomography imaging of a tumor with high sensitivity using gold nanoparticles conjugated to a cancer-specific antibody via polyethylene glycol chains on their surface

Science.gov (United States)

Nakagawa, Tomohiko; Gonda, Kohsuke; Kamei, Takashi; Cong, Liman; Hamada, Yoh; Kitamura, Narufumi; Tada, Hiroshi; Ishida, Takanori; Aimiya, Takuji; Furusawa, Naoko; Nakano, Yasushi; Ohuchi, Noriaki

2016-01-01

Contrast agents are often used to enhance the contrast of X-ray computed tomography (CT) imaging of tumors to improve diagnostic accuracy. However, because the iodine-based contrast agents currently used in hospitals are of low molecular weight, the agent is rapidly excreted from the kidney or moves to extravascular tissues through the capillary vessels, depending on its concentration gradient. This leads to nonspecific enhancement of contrast images for tissues. Here, we created gold (Au) nanoparticles as a new contrast agent to specifically image tumors with CT using an enhanced permeability and retention (EPR) effect. Au has a higher X-ray absorption coefficient than does iodine. Au nanoparticles were supported with polyethylene glycol (PEG) chains on their surface to increase the blood retention and were conjugated with a cancer-specific antibody via terminal PEG chains. The developed Au nanoparticles were injected into tumor-bearing mice, and the distribution of Au was examined with CT imaging, transmission electron microscopy, and elemental analysis using inductively coupled plasma optical emission spectrometry. The results show that specific localization of the developed Au nanoparticles in the tumor is affected by a slight difference in particle size and enhanced by the conjugation of a specific antibody against the tumor.

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

Directory of Open Access Journals (Sweden)

Cesar Andres Diaz Arias

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

A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

International Nuclear Information System (INIS)

Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

2014-01-01

Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp*_2Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134 ±0.8 K (93 ±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T <6 K, with an impressive coercive field of 3.4 T at 2 K. (Copyright copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

International Nuclear Information System (INIS)

Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

2007-01-01

PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

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

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

2012-03-01

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

Efficacy of feline anti-parvovirus antibodies in the treatment of canine parvovirus infection.

Science.gov (United States)

Gerlach, M; Proksch, A L; Unterer, S; Speck, S; Truyen, U; Hartmann, K

2017-07-01

This prospective, randomised, placebo-controlled, double-blinded study aimed to evaluate efficacy of commercially available feline anti-parvovirus antibodies in dogs with canine parvovirus infection. First, cross-protection of feline panleukopenia virus antibodies against canine parvovirus was evaluated in vitro. In the subsequent prospective clinical trial, 31 dogs with clinical signs of canine parvovirus infection and a positive faecal canine parvovirus polymerase chain reaction were randomly assigned to a group receiving feline panleukopenia virus antibodies (n=15) or placebo (n=16). All dogs received additional routine treatment. Clinical signs, blood parameters, time to clinical recovery and mortality were compared between the groups. Serum antibody titres and quantitative faecal polymerase chain reaction were compared on days 0, 3, 7, and 14. In vitro, canine parvovirus was fully neutralised by feline panleukopenia virus antibodies. There were no detected significant differences in clinical signs, time to clinical recovery, blood parameters, mortality, faecal virus load, or viral shedding between groups. Dogs in the placebo group showed a significant increase of serum antibody titres and a significant decrease of faecal virus load between day 14 and day 0, which was not detectable in dogs treated with feline panleukopenia virus antibodies. No significant beneficial effect of passively transferred feline anti-parvovirus antibodies in the used dosage regimen on the treatment of canine parvovirus infection was demonstrated. © 2017 British Small Animal Veterinary Association.

In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences.

Directory of Open Access Journals (Sweden)

Angel M Cuesta

Full Text Available There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed "trimerbody", comprises a single-chain antibody (scFv fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA, a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.

Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes

DEFF Research Database (Denmark)

Atar, D; Dyrberg, T; Michelsen, Birgitte

1989-01-01

The HLA-DQ beta-chain gene shows a close association with susceptibility or resistance to autoimmune insulin-dependent diabetes mellitus (IDDM) and it has been suggested that the amino acid in position 57 may be of pathogenetic importance. To study the expression of the IDDM associated HLA-DQ beta......-chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera...

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

Science.gov (United States)

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

International Nuclear Information System (INIS)

Jeong, Jong-Geun; Kim, Dong-Sik; Kim, Yong-Sung; Kwon, Myung-Hee

2011-01-01

Highlights: → We generate ' H3 Tat-3D8' by grafting Tat 48-60 peptide to VH CDR of 3D8 scFv antibody. → H3 Tat-3D8 antibody retains nucleic acid binding and hydrolyzing activities. → H3 Tat-3D8 acquires a preference for TAR RNA structure. → Properties of Tat 48-60 is transferred to an antibody via Tat-grafting into a CDR. -- Abstract: The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ( H3 Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat 48-60 peptide (GRKKRRQRRRPPQ). H3 Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

DEFF Research Database (Denmark)

Dziegiel, M; Borre, Mette; Petersen, E

1992-01-01

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

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

Science.gov (United States)

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

2015-06-17

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

Utility of HLA Antibody Testing in Kidney Transplantation

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Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

Growth control of genetically modified cells using an antibody/c-Kit chimera.

Science.gov (United States)

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Adsorption of a single polymer chain on a surface: effects of the potential range.

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Klushin, Leonid I; Polotsky, Alexey A; Hsu, Hsiao-Ping; Markelov, Denis A; Binder, Kurt; Skvortsov, Alexander M

2013-02-01

We investigate the effects of the range of adsorption potential on the equilibrium behavior of a single polymer chain end-attached to a solid surface. The exact analytical theory for ideal lattice chains interacting with a planar surface via a box potential of depth U and width W is presented and compared to continuum model results and to Monte Carlo (MC) simulations using the pruned-enriched Rosenbluth method for self-avoiding chains on a simple cubic lattice. We show that the critical value U(c) corresponding to the adsorption transition scales as W(-1/ν), where the exponent ν=1/2 for ideal chains and ν≈3/5 for self-avoiding walks. Lattice corrections for finite W are incorporated in the analytical prediction of the ideal chain theory U(c)≈(π(2)/24)(W+1/2)(-2) and in the best-fit equation for the MC simulation data U(c)=0.585(W+1/2)(-5/3). Tail, loop, and train distributions at the critical point are evaluated by MC simulations for 1≤W≤10 and compared to analytical results for ideal chains and with scaling theory predictions. The behavior of a self-avoiding chain is remarkably close to that of an ideal chain in several aspects. We demonstrate that the bound fraction θ and the related properties of finite ideal and self-avoiding chains can be presented in a universal reduced form: θ(N,U,W)=θ(NU(c),U/U(c)). By utilizing precise estimations of the critical points we investigate the chain length dependence of the ratio of the normal and lateral components of the gyration radius. Contrary to common expectations this ratio attains a limiting universal value /=0.320±0.003 only at N~5000. Finite-N corrections for this ratio turn out to be of the opposite sign for W=1 and for W≥2. We also study the N dependence of the apparent crossover exponent φ(eff)(N). Strong corrections to scaling of order N(-0.5) are observed, and the extrapolated value φ=0.483±0.003 is found for all values of W. The strong correction to scaling effects found here explain why

Glycosylation profiles of therapeutic antibody pharmaceuticals.

Science.gov (United States)

Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

2011-11-01

Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

Antibody proteases: induction of catalytic response.

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Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

2002-10-01

Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

Science.gov (United States)

Pan, Lucy Yan; Salas-Solano, Oscar; Valliere-Douglass, John F

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C H 2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

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Imperiale Valentina

2007-07-01

Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

Antibody structural modeling with prediction of immunoglobulin structure (PIGS)

DEFF Research Database (Denmark)

Marcatili, Paolo; Olimpieri, Pier Paolo; Chailyan, Anna

2014-01-01

Antibodies (or immunoglobulins) are crucial for defending organisms from pathogens, but they are also key players in many medical, diagnostic and biotechnological applications. The ability to predict their structure and the specific residues involved in antigen recognition has several useful...... applications in all of these areas. Over the years, we have developed or collaborated in developing a strategy that enables researchers to predict the 3D structure of antibodies with a very satisfactory accuracy. The strategy is completely automated and extremely fast, requiring only a few minutes (∼10 min...... on average) to build a structural model of an antibody. It is based on the concept of canonical structures of antibody loops and on our understanding of the way light and heavy chains pack together....

Antibody-Based Strategies to Prevent and Treat Influenza

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Ram eSasisekharan

2015-07-01

Full Text Available Passive immunization using antibodies has been suggested to offer several benefits in comparison to other antiviral treatment options. The potential for seasonal protection arising from a single injection of antibodies is appealing and has been pursued for a number of infectious agents. However, until recently, antibody-based strategies to combat infectious agents has been hampered due to the fact that typical antibodies have been found to be strain-specific, with the virus evolving resistance in many cases. The discovery of broadly neutralizing antibodies (bNAbs in, for example, influenza, dengue virus, and HIV, which bind to multiple, structurally-diverse strains has provided renewed interest in this area. This review will focus on new technologies that enable the discovery of bNAbs, the challenges and opportunities of immunotherapies as an important addition to existing antiviral therapy, and the role of antibody discovery in informing rational vaccine discovery – with agents targeting influenza specifically addressed. Multiple agents have entered the clinic and raise the possibility that a single antibody or small combination of antibodies can effectively neutralize a wide variety of strains. However, challenges remain - including combating escape variants, pharmacodynamics of antibody distribution, and development of efficacy biomarkers beyond virologic endpoints.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

Science.gov (United States)

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

2013-01-01

Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

OpenAIRE

Narat, Mojca; Biček, Ajda; Vadnjal, Robert; Benčina, Dušan

2004-01-01

Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs) that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY) shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of...

Antibodies against Venom of the Snake Deinagkistrodon acutus.

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Lee, Chi-Hsin; Lee, Yu-Ching; Liang, Meng-Huei; Leu, Sy-Jye; Lin, Liang-Tzung; Chiang, Jen-Ron; Yang, Yi-Yuan

2016-01-01

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Clonal relationships between thyroid-stimulating hormone receptor-stimulating antibodies illustrate the effect of hypermutation on antibody function

DEFF Research Database (Denmark)

Padoa, Carolyn J; Larsen, Sanne L; Hampe, Christiane S

2009-01-01

relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity......, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease....

Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

Science.gov (United States)

Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

2012-10-01

We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

Early recognition is important when multiple magnets masquerade as a single chain after foreign body ingestion

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Auriel August

2016-10-01

Full Text Available Ingestions of multiple magnets can lead to serious damage to the gastrointestinal tract. Moreover, these foreign bodies can take deceptive shapes such as single chains which may mislead clinicians. We report the case of a ten-year-old boy who swallowed 33 magnets, the most yet reported, which took on the appearance of a single loop in the stomach, while actually being located in the stomach, small bowel, and colon. Early recognition and prompt intervention are necessary to avoid complications of this foreign body misadventure.

Phase transitions of single polymer chains and of polymer solutions: insights from Monte Carlo simulations

International Nuclear Information System (INIS)

Binder, K; Paul, W; Strauch, T; Rampf, F; Ivanov, V; Luettmer-Strathmann, J

2008-01-01

The statistical mechanics of flexible and semiflexible macromolecules is distinct from that of small molecule systems, since the thermodynamic limit can also be approached when the number of (effective) monomers of a single chain (realizable by a polymer solution in the dilute limit) is approaching infinity. One can introduce effective attractive interactions into a simulation model for a single chain such that a swollen coil contracts when the temperature is reduced, until excluded volume interactions are effectively canceled by attractive forces, and the chain conformation becomes almost Gaussian at the theta point. This state corresponds to a tricritical point, as the renormalization group theory shows. Below the theta temperature a fluid globule is predicted (at nonzero concentration then phase separation between dilute and semidilute solutions occurs), while at still lower temperature a transition to a solid phase (crystal or glass) occurs. Monte Carlo simulations have shown, however, that the fluid globule phase may become suppressed, when the range of the effective attractive forces becomes too short, with the result that a direct (ultimately first-order) transition from the swollen coil to the solid occurs. This behavior is analogous to the behavior of colloidal particles with a very short range of attractive forces, where liquid-vapor-type phase separation may be suppressed. Analogous first-order transitions from swollen coils to dense rodlike or toroidal structures occur for semiflexible polymers. Finally, the modifications of the behavior discussed when the polymers are adsorbed at surfaces are also mentioned, and possible relations to wetting behavior of polymer solutions are addressed.

The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag

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Selma Djender

2014-04-01

Full Text Available We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.

Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.

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Ran Taube

Full Text Available BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

First principles studies of the electronic properties and catalytic activity of single-walled carbon nanotube doped with Pt clusters and chains

International Nuclear Information System (INIS)

Hayes, Kayla E.; Lee, Hee-Seung

2012-01-01

Highlights: ► Electronic and magnetic properties of (5, 5)-SWNT doped with Pt clusters and chains. ► Pt-doping can change metallic (5, 5)-SWNT to semiconducting CNT. ► Oxygen adsorption on Pt-doped (5, 5)-SWNT is barrierless process. ► Pt-doping reduces the activation barrier of oxygen dissociation reaction. ► Adsorbed oxygen has 2 O 2 - – character. - Abstract: We report the results of density functional theory calculations on the electronic structures, geometrical parameters, and magnetic properties of a wide variety of Pt clusters/chains adsorbed on metallic (5,5) single-walled carbon nanotube (SWNT). It was found that the electronic band structures of Pt/CNT systems are very sensitive to the small changes in the geometries of Pt clusters and chains. In some cases, metallic (5, 5)-SWNT becomes a small-gap semiconducting nanotube with adsorbed Pt clusters and chains. We also investigated the dissociation of molecular oxygen on the (5, 5)-SWNT doped with a single Pt atom via the nudged elastic band (NEB) method. The NEB results showed that the activation barrier is lowered even with a single Pt atom compared to that of pristine SWNT. It was found that the electronic structure of molecular oxygen adsorbed on Pt-doped CNT resembles that of 2 O 2 - , which should facilitate the dissociation process.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Science.gov (United States)

Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

2013-01-01

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

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

Science.gov (United States)

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

2015-01-01

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

{sup 68}Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours

Energy Technology Data Exchange (ETDEWEB)

Eder, Matthias; Eisenhut, Michael [German Cancer Research Center, Radiopharmaceutical Chemistry, Heidelberg (Germany); Knackmuss, Stefan; Gall, Fabrice Le; Reusch, Uwe; Little, Melvyn [Affimed Therapeutics AG, Heidelberg (Germany); Rybin, Vladimir [European Molecular Biology Laboratory, Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)

2010-07-15

Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope {sup 68}Ga. In order to combine the promising tumour targeting properties of antibodies with {sup 68}Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naive antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N{sup '}-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N{sup '}-diacetic acid (HBED-CC) to enable {sup 68}Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments. The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to {sup 68}Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation. The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the {sup 68}Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the