Construction and Expression of a Bifunctional Single-Chain Antibody against Bacillus cereusSpores

1998 ◽  
Vol 64 (7) ◽  
pp. 2490-2496 ◽  
Author(s):  
Kai Koo ◽  
Peggy M. Foegeding ◽  
Harold E. Swaisgood
Keyword(s):  
Monoclonal Antibody ◽  
Recombinant Antibody ◽  
Expression System ◽  
Variable Region ◽  
Hybridoma Cells ◽  
Antigen Specificity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Western Blots ◽  
Variable Region Genes

ABSTRACT The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

scholarly journals Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

2016 ◽  
Vol 64 (1) ◽  
Author(s):  
Róza Sawicka ◽  
Paweł Siedlecki ◽  
Barbara Kalenik ◽  
Jan P Radomski ◽  
Violetta Sączyńska ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Structural Model ◽  
Expression System ◽  
Antigenic Specificity ◽  
Hybridoma Cells ◽  
Single Chain Variable Fragment ◽  
Single Chain

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitated epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope has been located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibition activity and its antigenic specificity. Additionally, total RNA from hybridoma cells secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed slight differences in specificity are discussed.

Polymemse Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes from Single Hybridoma Cells

1989 ◽  
Vol 7 (9) ◽  
pp. 934-938 ◽  
Author(s):  
James W. Larrick ◽  
Lena Danielsson ◽  
Carol A. Brenner ◽  
Ellen F. Wallace ◽  
Magnus Abrahamson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Monoclonal Antibody ◽  
Variable Region ◽  
Hybridoma Cells ◽  
Chain Reaction ◽  
Variable Region Genes

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereusSpores

1998 ◽  
Vol 64 (7) ◽  
pp. 2497-2502 ◽  
Author(s):  
Kai Koo ◽  
Peggy M. Foegeding ◽  
Harold E. Swaisgood
Keyword(s):  
Fusion Protein ◽  
Magnetic Beads ◽  
Detection System ◽  
Expression System ◽  
Recombinant Antibodies ◽  
Antigen Specificity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
T7 Promoter ◽  
Enzyme Detection

ABSTRACT Control of microorganisms such as Bacillus cereusspores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encodeB. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay ofB. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 104 B. cereusspores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.

scholarly journals Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3343-3349 ◽  
Author(s):  
William J. J. Finlay ◽  
Iain Shaw ◽  
Joanna P. Reilly ◽  
Marian Kane
Keyword(s):  
Domoic Acid ◽  
Recombinant Antibody ◽  
Variable Region ◽  
Antibody Fragments ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Single Chain Fv ◽  
Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

scholarly journals Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

2003 ◽  
Vol 10 (4) ◽  
pp. 587-595 ◽  
Author(s):  
Xiao K. Deng ◽  
Lance A. Nesbit ◽  
K. John Morrow
Keyword(s):  
Monoclonal Antibody ◽  
Clostridium Difficile ◽  
Phage Display ◽  
Recombinant Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Display System ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Toxin B ◽  
Single Chain Antibodies

ABSTRACT Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10−8 M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

scholarly journals Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

2002 ◽  
Vol 68 (7) ◽  
pp. 3261-3269 ◽  
Author(s):  
Sau-Ching Wu ◽  
Jonathan C. Yeung ◽  
Yanjun Duan ◽  
Ruiqiong Ye ◽  
Steven J. Szarka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bacillus Subtilis ◽  
Molecular Chaperones ◽  
Blood Clot ◽  
Expression System ◽  
Antibody Fragment ◽  
High Sensitivity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Major Factors

ABSTRACT To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2133-2133
Author(s):  
Jingyu Zhang ◽  
Zhenni Ma ◽  
Ningzheng Dong ◽  
Jian Su ◽  
Anyou Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Proteolytic Activity ◽  
Recombinant Proteins ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Proteolytic Cleavage ◽  
Terminal Region ◽  
Full Length ◽  
Western Blots ◽  
A2 Domain

Abstract Abstract 2133 Poster Board II-110 Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Primary Structure and Functional Expression of Heavy- and Light-Chain Variable Region Genes of a Monoclonal Antibody Specific for Human Fibrin

Hybridoma ◽  
1997 ◽  
Vol 16 (3) ◽  
pp. 235-241 ◽  
Author(s):  
ZENGXUAN SONG ◽  
YINGLIN CAI ◽  
DANYING SONG ◽  
JING XU ◽  
HONGWEI YUAN ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Primary Structure ◽  
Variable Region ◽  
Functional Expression ◽  
Variable Region Genes

scholarly journals Identification of a Novel Marker for Primordial Smooth Muscle and Its Differential Expression Pattern in Contractile vs Noncontractile Cells

1997 ◽  
Vol 137 (4) ◽  
pp. 925-937 ◽  
Author(s):  
Jill E. Hungerford ◽  
James P. Hoeffler ◽  
Chauncey W. Bowers ◽  
Lisa M. Dahm ◽  
Rocco Falchetto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vessel Wall ◽  
Recombinant Antibody ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Differential Expression Pattern ◽  
Extracellular Matrix Components

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375–392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle α-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle α-actinin. Our results suggest that the 1E12 antigen is a member of the α-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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