Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

An immunized mouse phage display scFv library with a capacity of 3.34 × 109 CFU/mL was constructed and used for screening of recombinant anti-ciprofloxacin single-chain antibody for the detection of ciprofloxacin (CIP) in animal-derived food. After four rounds of bio-panning, 25 positives were isolated and identified successfully. The highest positive scFv-22 was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residue Val160 was the key residue for the binding of scFv to CIP. Based on the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of contact amino acid residue Val160 to Ser. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant scFv was established for CIP, respectively. The IC50 value of the assay established with the ScFv mutant was 1.58 ng/mL, while the parental scFv was 26.23 ng/mL; this result showed highly increased affinity, with up to 16.6-fold improved sensitivity. The mean recovery for CIP ranged from 73.80% to 123.35%, with 10.46% relative standard deviation between the intra-assay and the inter-assay. The RSD values ranged between 1.49% and 9.81%. The results indicate that we obtained a highly sensitive anti-CIP scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of CIP residue in animal-derived edible tissues.

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Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

10.21203/rs.3.rs-310107/v2 ◽

2021 ◽

Author(s):

Fangyu Wang ◽

Ning Li ◽

Yunshang Zhang ◽

Xuxefeng Sun ◽

Yali Zhao ◽

...

Keyword(s):

Amino Acid ◽

Scfv Antibody ◽

Phage Library ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Single Chain ◽

Single Chain Antibody ◽

Relative Standard ◽

Directional Evolution ◽

Scfv Library

Abstract A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

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Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

10.21203/rs.3.rs-310107/v1 ◽

2021 ◽

Author(s):

Guangxu Xing ◽

Yunshang Zhang ◽

Fangyu Wang ◽

Liuding Wen ◽

Gaiping Zhang

Keyword(s):

Amino Acid ◽

Scfv Antibody ◽

Phage Library ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Single Chain ◽

Single Chain Antibody ◽

Relative Standard ◽

Directional Evolution ◽

Scfv Library

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Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Toxins ◽

10.3390/toxins12040273 ◽

2020 ◽

Vol 12 (4) ◽

pp. 273

Author(s):

Caixia Zhang ◽

Weiqi Zhang ◽

Xiaoqian Tang ◽

Qi Zhang ◽

Wen Zhang ◽

...

Keyword(s):

Amino Acid ◽

Ochratoxin A ◽

Limit Of Detection ◽

Basic Knowledge ◽

Affinity Constant ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Assay Sensitivity ◽

Coating Antigen ◽

Relative Standard

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

10.1101/2020.07.27.224089 ◽

2020 ◽

Author(s):

Matthew D. Beasley ◽

Sanja Aracic ◽

Fiona M. Gracey ◽

Ruban Kannan ◽

Avisa Masarati ◽

...

Keyword(s):

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Single Chain ◽

Single Chain Antibody ◽

High Affinity ◽

Scfv Library ◽

Antibody Libraries ◽

Prophylactic Use

AbstractAntibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display™ (ReD) platform employing a 1 × 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.

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Dissolution of arterial platelet thrombi in vivo with a bifunctional platelet GPIIIa49-66 ligand which specifically targets the platelet thrombus

Blood ◽

10.1182/blood-2010-01-264358 ◽

2010 ◽

Vol 116 (13) ◽

pp. 2336-2344 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Yong-Sheng Li ◽

Michael A. Nardi ◽

Suying Dang ◽

Jing Yang ◽

...

Keyword(s):

Blood Flow ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Single Chain ◽

Single Chain Antibody ◽

Antithrombotic Agent ◽

Scfv Library ◽

Platelet Thrombus ◽

12 Lipoxygenase ◽

Carotid Vessels

Abstract Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance.

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Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

Current Biotechnology ◽

10.2174/2211550108666190417113342 ◽

2019 ◽

Vol 8 (1) ◽

pp. 24-31

Author(s):

Chol-Jin Kim ◽

Sunll Choe ◽

Kum-Chol Ri ◽

Chol-Ho Kim ◽

Hyon-Gwang Li ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Phage Display ◽

Affinity Purification ◽

Phage Library ◽

Amino Acid Residues ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Selection Of

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

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Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Journal of Biological Chemistry ◽

10.1074/jbc.m110.116699 ◽

2010 ◽

Vol 285 (25) ◽

pp. 19116-19124 ◽

Cited By ~ 30

Author(s):

Willie W. L. Koh ◽

Soren Steffensen ◽

Maria Gonzalez-Pajuelo ◽

Bart Hoorelbeke ◽

Andrea Gorlani ◽

...

Keyword(s):

Antibody Fragments ◽

Phage Library ◽

Single Chain ◽

Single Chain Antibody ◽

Specific Phage ◽

Hiv 1

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Fully “Recombinant Enzyme-Linked Immunosorbent Assays” Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus

Phytopathology ◽

10.1094/phyto.2000.90.12.1337 ◽

2000 ◽

Vol 90 (12) ◽

pp. 1337-1344 ◽

Cited By ~ 20

Author(s):

Estela Terrada ◽

Randolf J. Kerschbaumer ◽

Giuseppe Giunta ◽

Patrizia Galeffi ◽

Gottfried Himmler ◽

...

Keyword(s):

Fusion Proteins ◽

Citrus Tristeza Virus ◽

Sandwich Elisa ◽

Viral Disease ◽

Genetically Engineered ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

Tristeza Virus ◽

Citrus Tristeza

Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.68.11.5288-5295.2002 ◽

2002 ◽

Vol 68 (11) ◽

pp. 5288-5295 ◽

Cited By ~ 42

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.

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Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108330113 ◽

2009 ◽

Vol 14 (3) ◽

pp. 282-293 ◽

Cited By ~ 38

Author(s):

Laura Turunen ◽

Kristiina Takkinen ◽

Hans Söderlund ◽

Timo Pulli

Keyword(s):

Phage Display ◽

High Efficiency ◽

Magnetic Bead ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Technology ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. ( Journal of Biomolecular Screening 2009:282-293)

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