Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1

1996 ◽  
Vol 59 (11) ◽  
pp. 1208-1212 ◽  
Author(s):  
HUI-REN ZHOU ◽  
JAMES J. PESTKA ◽  
L. PATRICK HART
Keyword(s):  
Polymerase Chain Reaction Amplification ◽  
Recombinant Antibody ◽  
Fumonisin B1 ◽  
Cloning And Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Recombinant Phage ◽  
Recombinant Scfv ◽  
Food Borne ◽  
Variable Heavy

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B1 (FB1)-specific antibodies, composed of a single chain containing a variable heavy (VH) and light (VL) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB1-BSA conjugate or from an existing hybridoma cell line that produces anti-FB1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of VH and VL cDNAs, (iii) ligation of VL and VH chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB1, the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB2 or FB3. The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other food borne disease hazards.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals 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

2002 ◽  
Vol 68 (11) ◽  
pp. 5288-5295 ◽  
Author(s):  
Jacqui McElhiney ◽  
Mathew Drever ◽  
Linda A. Lawton ◽  
Andy J. Porter
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Phage Display ◽  
High Performance ◽  
Recombinant Antibody ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Cyanobacterial Toxin

ABSTRACT 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.

scholarly journals Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA

2017 ◽  
Vol 11 (03) ◽  
pp. 234-241 ◽  
Author(s):  
Sara Chandy ◽  
Lokeshwaran Kirubanandhan ◽  
Priya Hemavathy ◽  
Anees Mohammad Khadeeja ◽  
Siby Jacob Kurian ◽  
...  
Keyword(s):  
Tamil Nadu ◽  
Polymerase Chain Reaction Amplification ◽  
Public Health Problem ◽  
Cloning And Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Public Health Problem ◽  
Diagnostic Systems ◽  
Serum Samples ◽  
Specificity And Sensitivity ◽  
Leptospira Serovars

Introduction: Leptospirosis is a major public health problem in India. However, it has been underreported and under-diagnosed due to a lack of awareness of the disease, a functional surveillance system, and appropriate laboratory diagnostic facilities. Methodology: This multicenter study aimed to understand the Leptospira serovars causing leptospirosis in seven secondary-level hospitals in six states in India. Since early and accurate diagnosis of leptospirosis is one of the challenges faced by clinicians in India due to the poor specificity and sensitivity of commercially available diagnostic systems, an in-house indirect enzyme-linked immunosorbent assay (ELISA) was developed. Genomic DNA from L. interrogans serovar Canicola was used for polymerase chain reaction amplification, cloning, and expression of the lipL32 gene in E. coli to amplify, clone, and express the lipL32 gene. Results: Australis was the common serovar seen at all the study centers. Serovar Icterohaemorrhagiae was seen in samples from Tamil Nadu and Assam. In-house ELISA was standardized using the purified recombinant LipL32 polypeptide and was used to evaluate serum. Subsequently, acute serum samples from leptospirosis patients (n = 60) were screened. Compared to the gold standard, the microscopic agglutination test, sensitivity and specificity of the in-house ELISA was 95% and 90%, respectively. Conclusions: Understanding Leptospira serovars circulating in leptospirosis-endemic areas will help to formulate better vaccines. LipL32-based ELISA may serve as a valuable tool for early diagnosis of leptospirosis.

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.

Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis

2014 ◽  
Vol 90 (1) ◽  
pp. 74-80 ◽  
Author(s):  
R. Kamatchi ◽  
J. Charumathi ◽  
R. Ravishankaran ◽  
P. Kaliraj ◽  
S. Meenakshisundaram
Keyword(s):  
Monoclonal Antibody ◽  
Lymphatic Filariasis ◽  
Antibody Fragment ◽  
Wuchereria Bancrofti ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Recombinant Scfv ◽  
Overlap Extension ◽  
Field Samples ◽  
Filarial Antigen

AbstractGlobal programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ–Linker–VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

scholarly journals Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

2019 ◽  
Vol 41 (1) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Expression Vector ◽  
Recombinant Antibody ◽  
Soluble Form ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Insoluble Form ◽  
Single Chain Variable Fragments

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Generation of recombinant scFv antibody against Ochratoxin A (OTA)

2018 ◽  
Vol 22 (2) ◽  
pp. 107 ◽  
Author(s):  
Ranya Pranomphon ◽  
Witsanu Srila ◽  
Montarop Yamabhai
Keyword(s):  
Ochratoxin A ◽  
Recombinant Antibody ◽  
Cross Reactivity ◽  
Agricultural Product ◽  
Binding Property ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Recombinant Scfv ◽  
Contaminated Food ◽  
Soluble Scfv

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

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.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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