Screening and Molecular Evolution of a Single Chain Variable

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Article

Screening and molecular evolution of a single chain variable fragment antibody (scFv) against Citreoviridin toxin Rongzhi Wang, Xiaosong Gu, Zhenghong Zhuang, Yanfang Zhong, Hang Yang, and Shihua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02637 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Journal of Agricultural and Food Chemistry

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Screening and molecular evolution of a single chain variable

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fragment antibody (scFv) against Citreoviridin toxin

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Rongzhi Wang#, Xiaosong Gu#, Zhenghong Zhuang#, Yanfang Zhong, Hang Yang,

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and Shihua Wang*

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Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key

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Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School

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of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.

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# These authors contribute equally to the work.

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*Corresponding author. Tel./Fax: +86 (591) 87984471.

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E-mail addresses: [email protected].

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ACS Paragon Plus Environment


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Abstract: Citreoviridin (CIT), a small food-borne mycotoxin produced by

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Penicillium citreonigrum, is generally distributed in various cereal grains and farm

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crop products around the world, and has caused cytotoxicity as an uncompetitive

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inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv)

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antibody that can detect the citreoviridin in samples is still not available, so it is very

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urgent to prepare an antibody for CIT detection and therapy. In this study, an

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amplified and assembled scFv from hybridoma was used to construct the mutant

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phage library by error-prone PCR, generating a capacity of 2×108 mutated phage

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display library. After six rounds of bio-panning, the selected scFv-5A10 displayed

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higher affinity and specificity to CIT antigen, with an increased affinity of 13.25-fold

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(Kaff =5.7×109 L/mol) compared to the original wild type scFv. Two critical amino

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acids (P100 and T151) distributed in H-CDR3 and L-FR regions that responsible for

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scFv-5A10 to CIT were found and verified by oligonucleotide-directed mutagenesis,

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and the resulted three mutants except of the mutant (P100K) lost binding activity

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significantly against CIT as predicated. Indirect competitive ELISA (ic-ELISA)

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indicated that the linear range to detect CIT was 25~562 ng/mL with IC50 of 120

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ng/mL. The limit of detection was 14.7 ng/mL, and the recovery average was

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(90.612±3.889)%. Hence, the expressed and purified anti-CIT MBP-linker-scFv can

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be used to detect CIT in corn and related samples.

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Keywords: citreoviridin (CIT), scFv, phage display, affinity, detection.

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Introduction

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Citreoviridin (CIT), a mainly toxic secondary metabolite secreted by Penicillium

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citreoviride and Penicillium ochrosalmoneum, spp, has been reported primarily in

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moldy corn, wheat and rice in parts of Asia and South America1-5. CIT is a small

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molecular weight toxin (about 402.5 dalton), and the basic structure was formed by a

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lactone ring conjugated to a furan ring6. It has been reported that CIT is toxic to

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human and animal with the typical symptoms of vomiting, convulsions, ascending

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paralysis, neurological symptoms, and depressed sensory responses5,7. CIT is a major

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food-borne toxin that causes Keshan disease in human through affecting the

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mitochondrial respiratory chain8. Meanwhile, CIT toxin can inhibit the beta-subunits

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of membrane to bind F1-ATPase as a noncompetitive inhibitor, resulting in the failure

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of the ATP synthesis9. Recently, researchers have indicated that CIT plays a key role

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in inducing DNA damage in HepG2 cells, most likely through oxidative stress

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mechanisms10, and also inhibits the synthesis of ectopic ATP by activating the

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unfolded protein response, further suppressing lung adenocarcinoma growth11.

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Disturbingly, the contamination levels of CIT in crop in the target areas of China

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reached 4.9~33.2 µg/kg, seriously endangering the health of human12. However, some

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related safety limits of CIT in crop samples have not been established by Food and

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Drug Administration in China as well as other countries until now. Hence, it is urgent

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to develop a fast, accurate and sensitive method to detect and quantitate the residual

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of CIT toxin in real crop samples.

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To date，some traditional techniques have been used for detection of CIT in

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corn and rice, including high-performance liquid chromatography (HPLC), gas

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chromatography copied with mass spectrometry (GC/MS), liquid chromatographye

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mass spectrometry (LC-MS), thin-layer chromatography (TLC) and monoclonal

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antibody technology 6, 13-15. Although these methods based on chromatography and

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spectrometry are effective and accurate for the detection of CIT, but they are often

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time-consuming, expensive, inconvenient and need expensive equipment16. Besides,

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the procedures for real pre-treated samples are complex, and required professional

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technicians for the detection of samples and data analysis17. Moreover, the

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monoclonal antibody has some disadvantages including high cost, failure in genetic

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engineering, and the gradual loss in antibody-secreting ability of selected hybridoma

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clones over time18. Single chain variable fragment (scFv) antibody, a special kind of

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engineering antibody that retains the original antigen specificity and affinity, and it

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has been widely used to detect toxins and pathogens in samples19-22. Compared to

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polyclonal antibodies or monoclonal antibodies, scFv antibody can be easily

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manipulated using molecular biology to improve their binding behavior, and to

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change the affinity and specificity20, 22, which has widely attracted the research

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interests18.

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Antibody affinity is a measurement of the binding capacity between an antibody

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and its corresponding antigen, which can be further improved in vitro by molecular

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evolution techniques such as error-prone PCR, DNA shuffling and DNA point

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mutation23. Error-prone PCR introduces random copying errors by addition of

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different concentration of Mg2+, Mn2+, dNTP, and low-fidelity DNA polymerase in the

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PCR system, creating a large number of gene variants24. It has been reported that the

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affinity of antibody could be significantly improved if the key hydrophobic amino

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acids of complementary determining region (CDR) in antibody were mutated into

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hydrophilic amino acids18. In the present study, a wild anti-CIT scFv antibody

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amplified from hybridoma cell has been first constructed and identified successfully,

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but the resulted scFv antibody has low affinity to CIT antigen, and the resulted

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affinity of the parental scFv was 4.3×108 L/mol, and can’t be used for the detection of

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CIT in real samples. To further improve the affinity of anti-CIT scFv, a mutant phage

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library with large capacity was constructed. After six rounds of bio-panning, a high

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affinity scFv antibody, named scFv-5A10 was isolated and identified successfully.

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The isolated scFv, with 13.25-fold higher affinity compared to the wild-type scFv, is

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specific to CIT antigen. At last, a competitive indirect ELISA was developed to detect

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CIT based on this scFv antibody, and this provides a necessary basis for the effective

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immunoassay for CIT.

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Materials and methods

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Reagents

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All the strains used in this study were from Fujian Agriculture and Forestry

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University (Fujian, China). PCR mixture and DNA restriction enzymes were 5



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purchased from Thermofisher Scientific (Massachusetts, USA). Taq DNA polymerase

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and T4 DNA ligase were purchased from Takara (Dalian, China). Anti 6×His tag

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monoclonal antibody was purchased from Abgent (San Diego, USA), and

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HRP-labeled goat anti-mouse IgG was from Boster Biological Technology Co.

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(Wuhan, China). All reagents used were of analytical reagent grade.

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ELISA assay for binding activity of wild type scFv

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To determine the binding activity of the purified wild type scFv antibody to CIT

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antigen, ELISA was performed as follows. The diluted antigen BSA-CIT conjugate

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(5.0 µg/mL) in coating buffer was coated in 96-well plate, and incubated at 37℃ for 2

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h. After blocking and washing, 100 µL/well of the diluted scFv antibody against CIT

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was added to the reaction wells (37℃ for 2 h) for antigen recognition, and washed

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three times with PBS and PBST, respectively. Subsequently, the binding activity of

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the purified MBP-Linker-scFv was detected by using an HRP-conjugated anti-His6

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tag antibody. The enzyme reaction was then performed using tetramethylbenzidine

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(TMB) as the substrate, and color development was stopped by adding 2 M H2SO4.

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The absorbance at 450nm was measured using a microplate reader25.

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Specificity analysis and western blot of scFv

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To identify the specificity of the purified wild type scFv, an indirect ELISA was

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performed. BSA, BSA-CIT, and CIT-correlated antigens such as FB1-BSA,

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AFB1-BSA, and CTN-BSA were diluted and coated in 96-well plate (2.5 µg/mL).

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The steps of ELISA were same as the above. To further investigate the interaction 6


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between wild type scFv and CIT-BSA, western blot was performed. The CIT-BSA

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antigen was transferred from SDS-PAGE gel onto a polyvinylidene difluoride

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membrane, and the membrane was incubated with the purified MBP-Linker-scFv.

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After wash and blocking, the membrane was subsequently incubated with HRP

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labeled anti-His6 tag IgG antibody, and signals were visualized by enhanced

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chemiluminescence (ECL).

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Construction of the mutational phage library against CIT

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To further enhance the binding activity of anti CIT scFv, a mutated phage library

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against CIT was constructed by error-prone PCR. The ratio of base mutation was

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induced and optimized by addition of different concentration of MnCl2 and MgSO4,

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and the mutated scFv was amplified using the wild anti-CIT scFv as template. After

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amplification and purification, the resulted PCR products was digested and inserted

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into the same enzyme digested phagemid pCANTAB-5E, and the ligated DNA

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mixture was transformed into the E. coli TG1 competent cells by calcium chloride

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transformation.

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Bio-panning of mutated scFv clones against CIT

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To further screen high affinity scFv clones against CIT from the constructed

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library, bio-panning was performed as described25. The eluted phage was used timely

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to infect the log phase TG1 cells, and the infected E. coli cells (100 µL) were plated

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onto SOB-AG plates through a continuous dilution method for the latter phage ELISA

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analysis. 7



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Screening of clones with high binding activity from enriched clones

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After six rounds of panning, 100 clones from different plates were picked

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randomly to culture individually with helper M13K07 for phage ELISA analysis. To

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detect the binding activities of enriched clones, the phage ELISA was carried out. In

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brief, the prepared phage was added into the pre-coated 96-well plate for incubation at

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37℃ for 2 h. After washing with PBST and PBS, the binding phage was tested with

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an HRP-labeled anti-M13 antibody at a 1:5000 dilution.

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Sequence of scFv gene and homology modeling

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The candidate clone was sequenced using the vector specific primer. The

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sequence of scFv gene was blasted with known murine genes for analysis from the

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IMGT/V-QUEST database (http://www.IMGT.org). The structure of wild type scFv

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and scFv-5A10 were generated using homology modeling method, and the 1DZB

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model was used as the template for homology modeling. The 3D structures of two

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scFv antibodies were analyzed using the software of Swiss-model and Pymol with the

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highest

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/SWISS-MODEL.html).

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Point mutation of key amino acids of scFv-5A10

level

of

optimization

setting

(http://www.expasy.ch/swissmod

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To determinate whether two mutated amino acids in the mutant scFv-5A10 play

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a key role in the interaction between scFv-5A10 and CIT, proline of the heavy-chain

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CDR3 (H-CDR3) and threonine of the light-chain FR (L-FR), were mutated to alanine.

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Meanwhile, each of these two amino acids was also mutated to lysine for further 8


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investigation of key binding sites. Oligonucleotide-directed mutagenesis was

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performed by Fast Mutagenesis Kit V2 with the mutant primers (Table S1), and the

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amplified PCR product was cloned into pBD-his6-mbp-linker vector. Protein

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purification and ELISA were carried out as the above described. The data are

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presented as means plus the SD for three separate experiments.

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Soluble expression and affinity determination

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Once obtained a high affinity scFv clone, the scFv fragment was amplified and

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inserted into the expression vector pBD-his6-mbp-linker for protein expression and

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purification as the former described, and the purified antibody was further analyzed

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by ELISA and SDS-PAGE. The affinity constant (Kaff) of the antibody against

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CIT-BSA was detected using the previous formula18.

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Establishment of ic-ELISA for CIT assay

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For indirect competitive ELISA (ic-ELISA), the CIT standard solution was used

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as the competing antigen and reacted to the purified MBP-Linker-scFv (1 µg/mL) at

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37℃ for 2 h, and other ELISA steps were corresponded to the steps described above.

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The data are presented as means and standard deviations (SD) for three separate

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experiments6.

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Sensitivity analysis and Real samples detection by scFv

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To further determinate the limit of detection, indirect competition ELISA was

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established by optimization condition according to the previous research with minor 9



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modification6. Different corn samples, purchased form the local farmer's market，were

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smashed through 100 mesh sieve, and the sample(1 g) was extracted with 5 mL of

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methanol/water(2:8, v/v) at room temperature. The samples were mixed well with

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ultrasonic treatment for 30 min. After centrifugation at 4000 g for 10 min, the

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supernatant was used in the latter ELISA detection. In the study, the mean recovery

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and coefficient of variation (CV%) values of spiked samples with different

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concentration of CIT (0.3, 1, 3, 10, 30 µg/mL) were detected at least three times.

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Practical samples were tested by the optimized ic-ELISA. Different corn samples

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purchased from the local farmer's market were smashed through 100 mesh sieve, and

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the sample (1 g) was extracted with 5 mL of methanol/water (2:8, v/v) at room

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temperature. After centrifugation at 4000 g for 10 min, the supernatant was used for

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latter ELISA detection.

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Results

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Binding activity and specificity of scFv by ELISA analysis

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To identify the binding activity and specificity of wild type scFv, correlative

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antigens such as CIT-BSA, FB1-BSA, AFB1-BSA, CTN-BSA, and BSA were used to

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test with the purified wild type scFv antibody, and each experiment was done in

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triplicate. As is shown in the Fig. 1A, the purified wild type scFv protein recognized

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CIT-BSA effectively, and showed higher titer significantly than the control group. The

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result from specificity analysis (Fig. 1B) indicated that this scFv is highly specific to

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CIT-BSA antigen. Western blot result showed that the wild type scFv could specificly 10


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binding to CIT-BSA (Fig. 1C). The result in Fig. 1D showed that the IC50 for

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detection CIT was 47.925 µg/mL, indicating that the affinity of the wild type scFv for

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CIT was not ideal for samples detection, and it is needed to further enhance the

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affinity by molecular evolution in vitro.

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Construction of mutant phage library and screening of specific scFvs.

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To screen high affinity scFv clones against CIT toxin, a mutational phage

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displayed library was constructed successfully. The mutant scFv genes were amplified

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by error-prone PCR with the original scFv as template, and the resulted products were

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digested with restriction endonuclease Bgl II and Not I and cloned into the phage

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plasmid vector pCANTAB-5E, generating a mutant scFv gene library comprised of

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2×108 independent clones. Then, six clones selected randomly were identified by

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bacterial PCR (Fig. 2A), and all the clones were positive in PCR. The diversity of

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resulted library was evaluated by sequencing clones that randomly selected (Fig. 2B),

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and each clone contains different DNA sequence, revealing that the diversity of

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mutated library is desirable. After six rounds panning, the input and output of the

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library were shown in Fig. 2C. The titer of the eluted phages after each round of

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panning was increased distinctly, and maintained a stable level at the fourth rounds,

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demonstrating that more enriched specific antibodies interacted with the antigen were

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happened in the process of panning. At last, five scFv antibody clones showing

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stronger binding activity to CIT-BSA were isolated from the library by phage ELISA,

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including scFv-5A10, scFv-5A12, scFv-5A15, scFv-5B17 and scFv-5B60 (Fig. 2D).

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Sequence of scFv gene and homology modeling

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The result from DNA sequence displayed that scFv-5A10, scFv-5A12 and

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scFv-5B17 have 100% identity in sequence, but they have two changes of amino acids

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that distributed in H-CDR3 and L-FR1 to the original wild type scFv, and the

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corresponding amino acids were T100P and M151T, respectively. The remaining

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group contained two clones scFv-5A15 and scFv-5B60 with only one mutation

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(M151T) in L-FR1 to the original wild type scFv (Table 1). Interestingly, five clones

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have the same mutation (M151T) in L-FR1, and the above results demonstrated that

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this site might be hot site for mutation and play a key role in the interaction between

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antibody and antigen. In view of the affinity of scFv, scFv-5A10 has the highest

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binding activity to the CIT antigen, and this clone was chosen for the further study.

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The nucleotide of scFv-5A10 was sequenced, and the scFv has 738 nucleotides

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encoding 246 amino acids. The complementary determining regions (CDRs) of the VH

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and VL domains were determined according to the IMGT/V-QUEST database (Fig.

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3A). Three dimensional models of wild type scFv and scFv-5A10 were constructed by

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SWISS-MODEL and PYMOL software. As is shown in Fig. 3A and Fig. 3B, the 3D

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structures of wild type scFv and mutated scFv-5A10 were different obviously.

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Compared to the wild type scFv, the structure of three CDR loops in heavy chain of

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scFv-5A10 were changed, and the loop of H-CDR3 was more nearly to the loop of

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L-CDR3, while the hole formed by VH and VL became more smaller. The above

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result indicated that this change might be more favorable for the antigen-antibody

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interaction. 12


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Alanine mutation of two amino acids

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To understand the interaction between H-CDR3/L-FR1 and CIT-BSA antigen,

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proline of the H-CDR3 and threonine of the L-FR1, were mutated to alanine by

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site-directed mutagenesis to scan the key amino acids responsible for binding (Fig.

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4A). As is shown in Fig. 4B, the mutated proteins were purified and identified by

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SDS-PAGE and used for iELISA analysis. Compared to the scFv-5A10, the binding

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activities of the three mutated proteins (P100A, T151A, and P100A/T151A)

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decreased greatly (Fig. 4C), showing low binding activity to CIT-BSA. This result

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demonstrated that threonine of L-FR1 and proline of H-CDR3 was critical for the

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binding.

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Lysine mutation of two amino acids

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To further improve the affinity of the scFv, proline of the H-CDR3 and threonine

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of the L-FR1, were mutated to lysine by site-directed mutagenesis (Fig. 5A). The

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purified mutated proteins were analyzed by SDS-PAGE (Fig. 5B) and used for

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iELISA analysis. Compared to the scFv-5A10, the binding activity of the mutated

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proteins (P100K) was not changed obviously, but the other two mutated proteins

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(T151K, P100K/T151K) showed low binding activities to CIT-BSA antigen (Fig. 5C),

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which further demonstrated that threonine of L-FR1 and proline of H-CDR3 were

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critical for the binding. Interestingly, both of two mutations (T151A and T151K) in

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L-FR1 can lead to a decline of the binding activity. Combined with the previous

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sequencing results, we further speculated that threonine of the light-chain FR1 is 13



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foremost in the binding activity in this study, and two key sites are very critical for the

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interaction of scFv-5A10 to CIT antigen.

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Soluble expression and affinity determination

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On the basis of the above results, the scFv-5A10 with the highest binding activity

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was selected for affinity and ELISA analysis. The MBP-linker-scFv-5A10 fusion

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protein (defined as scFv-5A10) was expressed and purified by immobilized metal

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affinity chromatography. As shown in Fig. 6A and 6B, wild type scFv and scFv-5A10

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were expressed and purified successfully, and the scFv-5A10 has higher binding

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activity to CIT antigen than the wild-type. These results indicated that the binding

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activity of the scFv-5A10 antibody was improved. Meanwhile, the purified

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scFv-5A10 is also specific to CIT antigen and no cross-reaction was observed to other

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antigens (Fig. 6C). The affinity constant of scFv-5A10 was 5.7×109 L/moL(Fig. 6D),

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with an increase of 13.25-fold to the wild-type scFv. Therefore, the scFv-5A10 fusion

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protein was used for sensitively determination and samples detection.

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Sensitivity determination and standard curve

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To further determinate the sensitivity of detection by this scFv-5A10, ic-ELISA

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was performed to develop a standard curve for CIT detection. The relationship

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between concentration of CIT and inhibition value was analyzed using Microcal

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Originpro 8.1. The half inhibitory concentration (IC50) of CIT binding to scFv-5A10

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was 120 ng/mL, where the linear range to detect CIT was 25~562 ng/mL, which

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defined as the concentration of CIT toward from 20% to 80% inhibition, and the limit 14


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of detection (LOD) was 14.7 ng/mL (Fig. 7A). The linear equation is y =

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-0.4428x+1.4221, with a correlation coefficient (R2) of 0.98706 (Fig. 7B)

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Real samples detection

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In this study, the recovery of detection was analyzed based on the standard curve

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by ic-ELISA, and corn samples without any contamination were spiked with different

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concentrations of CIT. The recovery of detection was ranged from (86.401±5.204)%

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to (94.312±1.192)% with the average of (90.612±3.889)%, and the variation

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coefficient is 1.263%~8.058% (average 4.329%) in intra-assay. While the recovery

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rate was ranged from (76.401±0.502)% to (91.570±1.561)% with the average of

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(83.991±3.613)% and the variation coefficient is 0.657%~10.792% (average 4.278%)

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in inter-assay (Table 2). The above results demonstrated that this assay had high

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repeatability and accuracy, and it could be used for quantitative detection of CIT in

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real samples. At last, four different non-spiked corn samples with CIT (corn flour,

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corn steamed bread, niblet, and maize) were collected randomly from farmer's

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markets for quantitate detection of CIT toxin. As shown in Table 3, CIT was not

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detected in these samples.

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Discussion

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At present, many different methods have been used for detecting CIT toxin. The

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most commonly used methods are mass spectrometry and chromatography analysis,

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but these methods are unfixable for the actual samples detection as the large

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equipment and professional staff6, 17. Immunoassay based on monoclonal antibody was 15



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also used widely in immunological detection of small molecules/toxins and pathogens

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in samples6,

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time-consuming and can’t be manipulated genetically18. These disadvantages severely

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limited its application in biological toxin detection. Genetic engineering antibody

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(such as scFv) is a new format antibody with high affinity and specificity, and it has

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been widely used in pathogenic microorganism detection18, 20, 25. In view of the harm

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of CIT toxin, it is urgent to develop a fast, accurate and sensitive method based on

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scFv for CIT detection in real samples. In this study, we developed a feasible

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immunoassay based on scFv antibody for the first time to detect CIT toxin in crop

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samples, and the MBP tag was used for improving the solubility of scFv via inserting

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a flexible DNA linker. The resulted fusion protein have good solubility and binding

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activity, and this phenomenon indicated that this format for protein solubility

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expression is effective and viable, and the insertion of Linker DNA may reduce

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effectively the interference of folding between MBP and scFv on the space.

26-28

, however, the traditional monoclonal antibody are often cost,

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How to improve effectively the affinity of scFv in vitro is still a problem faced in

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the present study. Currently, several strategies have been applied to enhance the

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affinity of recombinant antibodies, such as using specific host cell, error-prone PCR,

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site-directed mutagenesis and chain shuffling29-31. In this study, we constructed a

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capacity of 2×108 mutant phage library by error-prone PCR to screen a high affinity

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scFv antibody against CIT. Exhilaratingly, the isolated scFv-5A10 has high affinity

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and specificity, with a increased affinity of 13.25 fold to the original scFv. The

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sequences analysis showed that H-CDR3 played an important role for binding affinity, 16


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and some studies have been shown that mutations in CDRs generally resulted in a

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change of the binding capacity because of the direct contact of loop residues with

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antigen determinants32. Compared to the wild type scFv, two changes of amino acids

335

were found in protein sequence, and they are proline (P100) in H-CDR3 and threonine

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(T151) in L-FR region, respectively. This change in amino acids may cause a

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rearrangement of the electric field nearby, which further leads to the peptide chain

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exploring the lowest energy conformation and space structure changes of H-CDR3

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and L-FR1. To further verify the critical role of mutated amino acids for

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antibody-antigen interaction, oligonucleotide-directed mutagenesis was performed.

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Three alanine mutants (P100A, T151A, and P100A/T151A) lost binding activity

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significantly against CIT antigen as predicated, and the result from lysine mutants

343

also showed that the mutant (P100K) has similar binding activity to CIT, while the

344

binding activities of two mutants (T151K and P100K/T151K) had decreased greatly.

345

All the above results further confirmed that these two amino acids (P100 and T151)

346

were very critical for the interaction of scFv-5A10 to CIT antigen.

347

Until now, immunoassay based on scFv antibody for CIT detection in samples

348

has not been reported. In this study, the positive scFv-5A10 against CIT was obtained

349

by phage display for the first time, and the MBP-Linker-scFv fusion protein was

350

expressed and purified successfully by Ni2+ affinity chromatography. The affinity of

351

scFv antibody in this study was 5.7×109 L/moL, showing a high affinity scFv antibody.

352

Under optimal condition, the linear range to detect CIT was 25-562 ng/mL with a

353

recovery average of (90.612±3.889)%, and the limit of detection (LOD) of the 17



354

ic-ELISA was 14.7 ng/mL, which keep similar level with the LOD reported

355

previously6. At last, no CIT was found in four different kinds of real samples that

356

non-spiked samples with CIT (Table 3). This is a similar result to our group's earlier

357

findings with the mAb6. In conclusion, ELISA detection based on this selected mutant

358

scFv has high accuracy and stability, and this laid the foundation for the detection of

359

CIT in real samples, and might used for the development of specific drug candidate

360

for diseases caused by CIT.

361

362

Funding

363

This study was supported by Science and Technology Project in Fujian Province

364

(2016Y0001; 2014YZ0001; 2013Y0004), Science and Technology Project of Fujian

365

Education Department (K8015025A), the Program for New Century Excellent Talents

366

in Fujian Province University (JA13087), and Agricultural Five-new Engineering

367

Projects of Fujian Development and Reform Commission.

368 369

18


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370

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the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin. Appl Environ

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26. Kato, Y.; Fujinaka, R.; Juri, M.; Yoshiki, Y.; Ishisaka, A.; Kitamoto, N.; Nitta, Y.;

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Method for Monitoring T-2 Toxin in Rice. J Agric Food Chem 2014, 62, (7),

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maturation of natural antibody using a chain shuffling technique and the expression of

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Antibody-Based


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32. Juarez-Gonzalez,

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antibody specific to scorpion toxin Cn2. J Mol Biol 2005, 346, (5), 1287-97.

V.

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Quintero-Hernandez,

479

480

24


V.;

Page 25 of 35


481

Table 1 Clones, mutated amino acids, and their respective regions and positions of the

482

selected scFv antibodies

Clone

scFv-5A10

scFv-5A15

Number of changes

2

1

Residue and position

Region

T100P

H-CDR3

M151T

L-FR1

M151T

L-FR1

483

25



Page 26 of 35

484

Table 2 The recovery and coefficient of variation detection in spiked corn samples

485

with CIT.

Intra-assaya

Spiked

level

Inter-assayb

Measured

Recovery

CV

(µg/mL)

(%)

(%)

Measured

Recovery

CV

n

(µg/mL)

(%)

(%)

(µg/mL)

n

0.3

3

0.273±0.022

91.001±7.333

8.058

4

0.242±0.012

80.667±4.012

4.973

1

3

0.864±0.052

86.401±5.204

6.023

4

0.764±0.005

76.401±0.502

0.657

3

3

2.732±0.089

91.066±2.966

3.256

4

2.549±0.084

86.333±2.820

3.266

10

3

9.431±0.119

94.312±1.192

1.263

4

9.157±0.156

91.570±1.561

1.704

30

3

27.084±0.825

90.281±2.753

3.049

4

25.496±2.751

84.986±9.172

10.792

90.612±3.889

4.329

83.991±3.613

4.278

Average

486

a

Intra-assay variation was determined by 3 replicates of each spiked level on 1th day.

487

b

Inter-assay variation was determined by 3 replicates of each spiked level on 4th day. Data was

488

given as the mean value.

489

c

490

the recovery test.

The coefficient of variation (CV) was defined as the ratio of the standard deviation to the mean in

491

26


Page 27 of 35


492

493

Table 3 The detection results of Citreoviridin in real samples (n=4).

Sample

OD450 Value

Detection results

PBS

1.253±0.105

-

niblet

1.246±0.126

-

maize

1.252±0.056

-

corn Flour

1.275±0.084

-

corn steamed bread

1.322±0.112

-

- means no Citreoviridin was detected out in real samples.

27



Fig. 1 Binding activity and specificity analysis of wild type scFv. A: The activity analysis of wild type scFv was carried out by iELISA. CIT-BSA and BSA were coated at the same concentration, and (-) means no coating antigen as a black control. B: Similarly, the different antigens such as CIT-BSA, FB1-BSA, AFB1-BSA, CTNBSA and BSA, were used to test specificity of purified scFv. C: Western blotting results. Lane M：Protein markers；Lane 1: control; Lane 2-3: the detected bands for wild type scFv. D: The ic-ELISA was carried out for sensitivity detection of wild type scFv. The data obtained in the presence of various inhibitor concentrations and without inhibitor are referred to as "B" and "B0", respectively. 98x85mm (300 x 300 DPI)


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Fig. 2 Phage panning and phage ELISA. A: The bacterial PCR was carried out by selecting six clones randomly. Lane M: DL-2000 DNA Markers; Lane 1-6: the amplified products of six clones; Lane 7-8: the negative control and positive control; B: The sequencing results of randomly selecting clones. C: the in-put and out-put of recombinant phage. In each panning round, the number of input phage was kept constant at 2×108CFU/mL and the phage that did not bind to CIT-BSA was removed by washing with phosphate buffer saline. D: the ELISA result of the selected scFv clones. Here, the results showed that the five scFv antibody clones have strong binding activity to the CIT-BSA antigen. 93x66mm (300 x 300 DPI)



Fig. 3 Bioinformatics analysis of scFv-5A10. A: The analysis of scFv-5A10 sequence. B: The threedimensional structure modeling of wild type scFv (left) and scFv-5A10 (right). The VH and VL domains are shown with their names adjacent to domains (different colors) as follows: HCDR1 (red), HCDR2 (darksalmon), HCDR3 (yellow), LCDR1 (magenta), LCDR2 (cyans), and LCDR3 (hotpink). Residues mutated in scFv-5A10 and the corresponding ones in the parent scFv are indicated with amino acid together with its number. 149x199mm (300 x 300 DPI)


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Fig. 4 Alanine mutation of HCDR3/LFR1. A: The construction diagram of alanine mutation vectors. B: The SDS-PAGE analysis of purified mutated proteins. Lane M: Blu Plus II Protein Marker, Lane 1: scFv-5A10, Lane 2: P100A, Lane 3: T151A, Lane 4: P100A/T151A. C: The ELISA analysis of alanine variants. The asterisks (*) represent significant difference. 63x35mm (300 x 300 DPI)



Fig. 5 Lysine mutation of HCDR3/LFR1. A: The construction diagram of lysine mutation vectors. B: The SDS-PAGE analysis of purified mutated proteins. Lane M: Blu Plus II Protein Marker, Lane 1: scFv-5A10, Lane 2: P100K, Lane 3: T151K, Lane 4: P100K/T151K. C: The ELISA analysis of lysine variants. The asterisks (*) represent significant difference. 58x29mm (300 x 300 DPI)


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Fig. 6 The characteristic analysis of purified antibodies. A: SDS-PAGE analysis of purified antibodies. Lane M: standard protein marker, Lane 1: wild type scFv, Lane 2: scFv-5A10. B: The affinity analysis of two purified antibodies. Values represent a mean of three parallel replicates. C: Cross reactivity of scFv-5A10 to other toxins was tested by iELISA. D: The affinity constant of scFv-5A10 was carried out with different concentrations (0.43, 0.87 and 1.75 µg/ml) of coating antigen by iELISA. 96x86mm (300 x 300 DPI)



Fig. 7 Sensitivity analysis of scFv-5A10 antibody. A: Standard curve was carried out by ic-ELISA. The data obtained in the presence of various inhibitor concentrations and without inhibitor are referred to as “B” and “B0”, respectively. Standard curves were generated by plotting the inhibition percentage (B/B0) versus the log of free inhibitor concentrations. B: The linear equation is y= -0.4428x+1.4221, with a correlation coefficient (R2) of 0.98706. 57x26mm (300 x 300 DPI)


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48x27mm (300 x 300 DPI)


Screening and Molecular Evolution of a Single Chain Variable

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