Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody

Accepted Manuscript Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody

Chongxin Xu, Wenjie Miao, Yan He, Yao Zu, Xiaoqin Liu, Jianhong Li, Xianjin Liu PII: DOI: Reference:

S0141-8130(18)33373-7 https://doi.org/10.1016/j.ijbiomac.2018.11.122 BIOMAC 10999

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

6 July 2018 13 November 2018 13 November 2018

Please cite this article as: Chongxin Xu, Wenjie Miao, Yan He, Yao Zu, Xiaoqin Liu, Jianhong Li, Xianjin Liu , Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.122

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ACCEPTED MANUSCRIPT Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody

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Chongxin Xua,b, Wenjie Miaoa, Yan Hec, Yao Zua, Xiaoqin Liud, Jianhong Li a*, Xianjin Liub*

College of Life Sciences, Nanjing Normal University, Nanjing 210023, China

b

Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science

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a

and technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences,

Institute of food science and technology, Chinese Academy of Agricultural Sciences, Beijing

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c

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Nanjing 210014, China

100081, China

Huaihua Vocational and Technical College, Huaihua 418007, China

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*Corresponding author: Jianhong Li (E-mail: [email protected]), College of Life Sciences,

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Nanjing Normal University, Nanjing 210023, China; Xianjin Liu (E-mail: [email protected].),

China.

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Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014,

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Abstract: Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its

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capacity of 3.26×109 CFU/mL was constructed and used for anti-MC-LR phage scFv particles

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screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The

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most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7 μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6

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mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay

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(IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05 μg/L, and had strong cross-reactivity for

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MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of

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variation less than 6.8% for scFv-based IC-ELISA to detect MC-LR spiked in water samples, which met the requirement of indoor testing. The present results indicate that we have obtained a high

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sensitive anti-MC-LR scFv by the MC-LR-immunized phage library construction and screening, and

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the scFv-based IC-ELISA can be used for monitoring MC-LR in water samples. Keywords: Microcystin, Polyclonal antibodies, Phage display antibody library, ScFv, Immunoassay

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1.

Introduction Microcystins (MCs) are a class of small molecule polypeptide biotoxins produced with

cyanobacterial blooms, and they are the threats to ecological environmental safety and human health [1,

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2]. Until now, more than 100 MC isoforms are reported with molecular weights (M.W.) between 900

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and 1200 Da, which have strong biotoxicity to human and animals [3, 4]. Among them, MC-LR is the

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most common isoform and with the highest toxicity in waterbodies with cyanobacterial blooms [5-7], and its LD50 dose to mice is approximately 50 μg/kg body weight [8-12]. MC-LR has been classified as

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a possible human carcinogen for human health by International Agency for Research on Cancer, and its

Health Organization (WHO) [13-15].

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minimum residue limit (MRL) has been set a guideline value of 1.0 μg/L in drinking water from World

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With cyanobacterial blooms frequently erupting in rivers, lakes and oceans, MC-LR is

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accumulated largely in ecological environment and agricultural products through food chain [1]. Therefore, there is a great need for establishing a simple and practicable analytical method to detect

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MC-LR [16]. Enzyme-linked immunosorbent assay (ELISA) acts as the representative of immunoassay

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which is considered to be the most applicable method in detection and analytics, which is based on the principle of antibody specifically binding to antigen [17-19]. Liu et al. [3], Zeck et al. [20] and Sheng et al. [21] both have developed monoclonal antibodies (MAbs) based IC-ELISA for MC-LR, and their haif-maximum inhibition concentrations (IC50) were 0.27, 0.06 and 1.8 μg/L, respectively. Metcalf et al. [22] and Sheng et al. [23] have employed the PAbs-based ELISA to detect MC-LR, and the IC50 were 2.0 and 0.63 μg/L, respectively. All these reported ELISAs with the detection limits could satisfy the requirement of WHO about the MC-LR of MRL at 1.0 μg/L in drinking water. Even though using 3

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ELISA to detect MC-LR is a mature and widely used method, it cannot do without the toxin-specific antibody, as a result it has to suffer the burdensome programs of prepared traditional antibodies (PAbs and MAbs) from antigen-immunized animals [24, 25]. Hence, it is very necessary to develop a new technology with simplicity, rapidity and effectivity to prepare a novel antibody instead of the

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traditional antibodies for detecting MC-LR.

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Phage antibody library is an emerging antibody preparation technology by large-scale clone and

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display artificial antibody in phagemid vector, and it is extensively used for preparing antigen-specific artificial antibodies in biomedicine, environmental pollutants analysis and food safety detection fields

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[26, 27]. Wang et al. [25] and Zhang et al. [28] have obtained anti-Cry1Ac and Cry1B toxin scFvs from

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a naive human phage antibody library, respectively, and their IC 50 were 0.39 and 0.84 μg/mL. Zhao et al. [29] and Xu et al. [30] have obtained the broad-specificity domain antibodies for pyrethroid

reports

obtained

the

anti-zearalenone

scFv

[31]

and

polypeptide

antibody

[32],

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some

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pesticides and Bt Cry toxins by rounds of specific phage library biopanining, respectively. Furthermore,

anti-deoxynivalenol-mimic nanobody [33], anti-β-bungarotoxin scFv [34] and anti-tetrodotoxin scFv

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[35], which are all based on phage antibody library technology. Nevertheless, it is normally difficult to

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get the high sensitive antigen-specific antibodies from a native phage antibody library, unless to improve the screening strategy, construct antigen-immunized antibody library or further affinity maturation for antibody [36, 37]. To obtain a high sensitive anti-MC-LR scFv for detecting MC-LR, in this study, we constructed a large capacity and diversity of MC-LR-immunized rabbit phage display scFv library for anti-MC-LR phage scFv particles screening. Then we expressed and purified the most positive scFv in E. coli HB2151, and established a scFv-based IC-ELISA for MC-LR and its analogues. Finally, the IC-ELISA 4

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was used to detect MC-LR spiked in water samples for evaluating its accuracy, practicality and applicability. 2.

Materials and methods

2.1 Materials and reagents

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MCs were purchased from ApexBio Company (USA). MC-LR-KLH (MC-LR conjugated to

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keyhole limpet haemocyanin (KLH)) and MC-LR-BSA (MC-LR conjugated to bovine serum albumin

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(BSA)) were previously prepared in our laboratory [38]. Freund’s adjuvants, DifcoTM skim milk, ovalbumin (OVA), Tween 20, trypsin and 3,3,5,5-tetramethylbenzidine (TMB) were purchased from

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Sigma-Aldrich (Beijing, China). New Zealand white rabbit was supplied by the National Research

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Center of Veterinary Biologicals engineering and technology (Nanjing, China), and all of the experimental protocols were approved by the animal experiment ethics committee of authors’ institute.

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Primers (Table 1) were synthesized from Sangon Bio.Co.Ltd. (Shanghai, China). pIT2 phagemid

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vector (Ampr and His tag), E.coli TG1, E.coli HB2151, KM13 helper phage (Kanar) were obtained from MRC HGMP Resource Centre (Cambridge, England). Goat anti-rabbit IgG-HRP, anti-M13-HRP

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and anti-His-HRP MAbs were purchased from GenScript Bio.Co.Ltd (Nanjing, China). PCR Mix,

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DNA and protein markers were purchased from ComWin Biotech Co.,Ltd (Beijing, China). NcoI and NotI restriction endonucleases and T4 DNA ligase were obtained from NEB (Beijing, China). Cell culture flask and 96-well plates were purchased from Corning (Beijing, China). 2.2 Rabbit immunization A healthy New Zealand white rabbit was immunized by five rounds of subcutaneous injection with MC-LR-KLH conjugates for inducing its spleen express anti-MC-LR PAbs. In brief, in the first round of immunization, 100 μg of MC-LR-KLH in sodium phosphate buffer (PBS) mixed with 5

ACCEPTED MANUSCRIPT Freund’s complete adjuvant was emulsified for rabbit subcutaneously multi-point injection. The second, third and fourth rounds were injected two, four and six weeks, respectively, the dose of MC-LR-KLH in PBS were all 125 μg that mixed with Freund’s incomplete adjuvant. A week after the fourth round of immunization, 150 μg of MC-LR-KLH in PBS was injected immediately (the booster immunization).

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Five days later, the blood and spleen were collected for obtaining the anti-MC-LR PAbs and

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constructing the phage display scFv library, respectively. Before each round of immunization, a little

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blood was collected to evaluate the anti-MC-LR response by ELISA [39]. 2.3 Polyclonal antibodies purification

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Total 80 mL blood was collected by heart blood sampling. The blood was standing at room

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temperature (RM) for 1 h, then transfered to 4 ℃ overnight, separating out the serum and then use it for purifying anti-MC-LR PAbs. Briefly, the serum mixed with 0.9% stroke-physiological saline

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solution (SPSS, contains 0.9% NaCl in double distilled water (ddH2O)) by ratio of 1:1 (v/v), then

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saturated ammonium sulfate solution (SASS) were added into drop by drop until its concentration reached to 20%. After standing at RM for 30 min, the mixture was centrifuged at 10000 g and 4 ℃ for

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20 min, and the supernatant were collected, adding the SASS until the concentration was 50%. After

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standing at RM for 30 min, the precipitated proteins were collected and dissolved in SPSS at RM for standing 30 min, then add the SASS until with a concentration of 33%, centrifuging at 10000 g and 4 ℃ for 20 min, and the precipitated proteins were the primarily purified PAbs. A high-purity anti-MC-LR PAbs proteins were obtained by using HiTrap Protein A HP columns (GE, USA) performed as described in the handbook (http://www.blossombio.com/pdf/products/UG_71700200AP.pdf). 2.4 Phage display scFv library construction Total RNA was extracted from the spleen B-cells by using TRIzol® reagent (Invitrogen, USA). 6

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The RNA integrity was assayed on 1% agarose gel, and its concentration was calculated by UV absorbance at OD260/OD280 from a Nanodrop 2000/2000C spectrophotometry (Thermo Fisher, USA). The RNA was used as the template for reverse transcribed to cDNA by using the PrimeScript TM II 1st strand cDNA synthesis kit (TaKaRa-Dalian, China) performed as described in the handbook

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(http://www.takara.co.kr/file/manual/pdf/6110_e.v0610-2.pdf). The cDNA was used to amplify the

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heavy chain variable (VH) and light chain variable (VL) genes based on the specific primers listed in

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Table 1, then spliced to a whole scFv gene by splicing overlap extension PCR (SOE-PCR). Briefly, the NcoI-VL genes were amplified by PCR as follow conditions in 50 μL system: contains 25 μL 2×Tap

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PCR master mix, 1 μL cDNA, 1 μL upstream primer mixtures (NcoI-RVL-F1, NcoI-RVL-F2,

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NcoI-RVL-F3 and NcoI-RVL-F4 were 0.25 μL respectively), 1 μL downstream primer mixtures (RVL-B1, RVL-B2, RVL-B3 and RVL-B4 were 0.25 μL respectively) and 22 μL ddH2O. The system

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was at 95 ℃ for 10 min, then cycled 35 times at 94 ℃ for 60 s, 56 ℃ for 60 s and 72 ℃ for 60 s,

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and finally extended at 72 ℃ for 10 min. The VH-NotI genes were amplified by PCR as follow conditions in 50 μL system: contains 25 μL 2×Tap PCR master mix, 1 μL cDNA, 1 μL upstream primer

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mixtures (RVH-F1, RVH-F2, RVH-F3 and RVH-F4 were 0.25 μL respectively), 0.5 μL downstream

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primer (RVH-B-NotI) and 22.5 μL ddH2O. The system was at 95 ℃ for 10 min, then cycled 35 times at 94 ℃ for 60 s, 58 ℃ for 60 s and 72 ℃ for 60 s, and finally extended at 72 ℃ for 10 min. The NcoI-VL and VH-NotI genes were used as the templates for amplifying NcoI-VL-Linker and Linker-VH-NotI genes. NcoI-VL-Linker genes amplification conditions in 50 μL system: contains 25 μL 2×Tap PCR master mix, 0.5 μL NcoI-VL, 1 μL upstream primer mixtures (NcoI-RVL-F1, NcoI-RVL-F2, NcoI-RVL-F3 and NcoI-RVL-F4 were 0.25 μL respectively), 0.5 μL downstream primer (RV L-Linker) and 23 μL ddH2O. The system was at 95 ℃ for 10 min, then cycled 30 times at 95 ℃ for 60 s, 56 ℃ 7

ACCEPTED MANUSCRIPT for 60 s and 72 ℃ for 60 s, and finally extended at 72 ℃ for 10 min. Linker-VH-NotI genes amplification conditions in 50 μL system: contains 25 μL 2×Tap PCR master mix, 0.5 μL VH-NotI, 0.8 μL upstream primer mixtures (RVH-F1, RVH-F2, RVH-F3 and RVH-F4 were 0.2 μL respectively), 0.5 μL downstream primer (RVL-Linker) and 23.2 μL ddH2O. The system was at 95 ℃ for 10 min, then

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cycled 30 times at 95 ℃ for 60 s, 57 ℃ for 60 s and 72 ℃ for 60 s, and finally extended at 72 ℃

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for 10 min. The NcoI-VL-Linker and Linker-VH-NotI genes were spliced to NcoI-VL-Linker-VH-NotI

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(scFv) genes by SOE-PCR. 50 μL system: contains 25 μL 2×Fast Pfu PCR Super Mix, 0.5 μL NcoI-VL-Linker, 0.5 μL Linker-VH-NotI, 1 μL upstream primer mixtures (NcoI-RVL-F1, NcoI-RVL-F2,

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NcoI-RVL-F3 and NcoI-RVL-F4 were 0.25 μL respectively), 1 μL downstream primer (RVH-B-NotI)

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and 22 μL ddH2O. The system was at 95 ℃ for 10 min, then cycled 30 times at 95 ℃ for 90 s, 58 ℃ for 60 s and 72 ℃ for 60 s, and finally extended at 72 ℃ for 10 min. The scFv genes ligated into

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pIT2 phagemid vectors, and then transformed into E.coli TG1 electro-competent cells as described by

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Xu et al. [39], which constructed MC-LR-immunized rabbit phage display scFv library. The quality and diversity of the library were evaluated by randomly single-colony PCR (primers were LMB3 and

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PHEN) and their DNA sequence analysis. All of the primers and PCR products in the experiments were

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dissolved in ddH2O with a concentration of μM range. 2.5 Phage display scFv library amplification 500 μL phage scFv library in E.coli TG1 was added into 500 mL 2×TY-AG medium (contains 100 μg/mL Amp and 1% Glu) which was grown at 37 ℃ and 250 rpm until the OD600 reached 0.6, then adding 500 μL KM13 helper phage (1010 CFU/mL in PBS) and growing for 1.5 h at 30 ℃ and 250 rpm. The cultures centrifuged at 1800 g and 30 ℃ for 30 min, and the cell pellets were resuspended with 500 mL 2×TY-AK medium (contains 100 μg/mL Amp and 50 μg/mL Kana) for growing overnight 8

ACCEPTED MANUSCRIPT at 30 ℃ and 250 rpm. Next day, 500 mL supernatant was collected by centrifuged at 3300 g and 4 ℃ for 30 min, then mixed with 125 mL PEG/NaCl for keeping ice-bath for 2 h. The mixture was centrifuged at 3300 g and 4 ℃ for 30 min, and the precipitate was resuspended with 5 mL sterile PBS and then centrifuged at 11600 g and 4 ℃ for 10 min. The finally collected supernatant which

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contained phage particles were the amplified library, and its concentration was adjusted to 109 CFU/mL

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2.6 Anti-MC-LR phage scFv particles enrichment and screening

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for follow-up experiment.

The amplified library was gone through four rounds of biopanning with coat antigen MC-LR-BSA

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for anti-MC-LR phage scFv particles enrichment. Briefly, a sterile cell culture flask was coated with 3

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mL MC-LR-BSA (the first round was 50 μg/mL, and the remaining three rounds were 25, 12 and 6 μg/mL, respectively) in PBS solution at 4 ℃ for standing overnight. After being washed five times

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with sterile PBST solution (contains 0.1% Tween in PBS), the flask was blocked at RM for 2 h with 5

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mL MPBS solution (contains 5% DifcoTM skim milk in PBS). After being washed with PBST solution, 1 mL library phage particles (109 CFU/mL) was added into the flask for shaking 1 h with 150 rpm at

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RM, then standing for 1 h. After being washed with PBST solution, the phage scFv particles binding to

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MC-LR-BSA were eluted by 1 mL trypsin solution (1mg/mL in PBS). The eluent was the first round of enrichment library, and the phage scFv particles were amplified for the next round of enrichment and total four rounds were performed. Each round of input and output library phage particles were quantified to 109 CFU/mL, and then used for MC-LR determined by polyclonal phage ELISA as described by Xu et al. [38]. The fourth round of enriched anti-MC-LR phage particles were infected to E.coli TG1 and spread on TYE-AG medium (contains 100 μg/mL Amp and 1% Glu) for culturing at 37 ℃ overnight. 9

ACCEPTED MANUSCRIPT Individual colonies were randomly picked into 96-well plate with 100 μL per well of 2×TY-AG for growing overnight at 37 ℃ and 250 rpm. Next day, transfering 10 μL cultures per well

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into another fresh 96-well plate for culturing 2 h at 37 ℃ and 250 rpm, then adding KM13 helper phages for rescuing 2 h at 37 ℃ and 250 rpm. The plate was centrifuged at 37 ℃ and 3300 g for 15

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min, and the pellets were resuspended with 200 μL per well of 2×TY-AK medium, then cultured

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overnight at 30 ℃ and 250 rpm, and the phage scFv particles were proliferated. The plate was

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centrifuged at 4 ℃ and 3300 g for 30 min, then the supernatants were used to monoclonal phage ELISA for MC-LR as described by Xu et al. [38]. The positive monoclonal phage scFv for MC-LR was

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from the ratio of P/N>3.0. “P” was coated MC-LR-BSA (1.5 μL/mL in PBS), and “N” was BSA (1.5

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μL/mL in PBS).

2.7 Phage scFv colony PCR and its DNA sequencing

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The positive anti-MC-LR phage scFv colonies were chosen and cultured in 2×TY-AG medium

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until logarithmic phase for colony PCR, and the PCR products were examined by 1% agarose gel electrophoresis. After being purified, the single-stranded DNAs of scFvs were sequenced by Invitrogen

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(Shanghai, China). Gene Construction Kit software was used for translating the scFv nucleotide

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sequence into amino acid sequence, and Vector NTI advance software was used for aligning its protein sequence.

2.8 Anti-MC-LR scFv expression and purification The most positive anti-MC-LR phage scFv particles were infected to E.coli HB2151, then spread on TYE-AG, and the individual colonies were randomly picked into 2×TY-AG medium for growing until reached to logarithmic phase and used for identifying by colony PCR and DNA sequencing. 500 μL positive inoculum was transfered into 500 mL 2×TY-AG medium for growing at 37 ℃ and 250 10

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rpm until OD600 reached 0.8, then IPTG was added with a concentration of 0.8 mM to induce the scFv proteins expressing at 30 ℃ and 250 rpm for overnight. The supernate was collected and concentrated to 100-fold by using MWCO: 8000-14000 Da of dialysis bag in PEG/NaCl. The collected pellets were lysed by TES buffer (contains 0.2 mM Tris-HCl, 0.5 mM EDTA and 0.5 M sucrose in 1 L ddH 2O) for

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obtaining the periplasmic lysate and lysed by sonication for obtaining the whole cell lysate,

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respectively, as described by Zhang et al. [28]. The supernate, periplasmic lysate and whole cell lysate

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were loaded a small samples into 12% SDS-PAGE for analyzing the scFv proteins expression index. Finally, the anti-MC-LR scFv proteins were purified by using His-Trap HP columns.

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2.9 Anti-MC-LR PAbs and scFv based IC-ELISAs for MC-LR and its analogues

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The purified anti-MC-LR PAbs and scFv were used to establish IC-ELISAs for MC-LR. Briefly, 100 μL per well of MC-LR-BSA solution (1.5 μL/mL in PBS) were coated into 96-well plates

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overnight at 4 ℃, then the plates were washed with PBST solution and blocked with 250 μL per well

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of OPBS solution (contains 5% OVA in PBS) at 37 ℃ for 2 h. After being washed with PBST, 100 μL mixture per well of MC-LR at various concentrations (contains 50 μL of 0.01, 0.05, 0.1, 0.5, 1.0,

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2.0, 5.0, 10 and 100 μg/L in PBS, respectively) and purified anti-MC-LR PAbs/scFv were added into

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the plates and incubated at 37 ℃ for 2 h. After being washed with PBST, 100 μL per well of HRP-conjugated goat anti-rabbit IgG-HRP/anti-His-HRP MAbs was added into the plates and incubated at 37 ℃ for 2 h. Follow being washed with PBST, 100 μL per well of TMB substrate was added and incubated at RT for 15 min, then the color development reaction was stopped with 50 μL per well of 2 M H2SO4 solution. Absorbance was measured at OD450 by an automatic microplate reader (Thermo, USA). The inhibition ratio of anti-MC-LR PAbs and scFv, IC10, IC20, IC50 and IC80 were calculated by the 11

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formula of [(P-S-N)]/(P-N)]×100%. The CRs of anti-MC-LR PAbs and scFv for MC-LR analogues, MC-RR, MC-YR, MC-WR, MC-LW, MC-LY and MC-LF were calculated by the formula of [CR (%) = IC50 (MC-LR)/IC50 (MC-LR analogue)] ×100%. “P” was the OD450 value of positive (50 μL anti-MC-LR PAbs/scFv mixed with 50 μL CBS), “S” was the OD450 value of standard (50 μL

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PAbs/scFv mixed with 50 μL serial concentration of MC-LR), “N” was the OD450 value of negative

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control (100 μL CBS).

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2.10 Assessment of the scFv based IC-ELISA for MC-LR by spiked samples

Tap water from our laboratory, drinking bottled water from supermarket and lake water from

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Nanjing Dongwazi were spiked with series gradient concentration of MC-LR for assessing the

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practicability and applicability of the scFv-based IC-ELISA, respectively. Briefly, the water samples were filtered through with 0.45 μm nitrocellulose membrane, then spiked with MC-LR at 0, 0.2, 1.0

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and 2.0 μg/L concentration levels, respectively. The MC-LR spiked water samples were used for

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detecting directly by the scFv-based IC-ELISA as described above, and the recoveries, SDs and CVs of MC-LR in the water samples from intra-assay and inter-assay were calculated as described by Zhang et

Results and discussion

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3.

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al. [40].

3.1 MC-LR-KLH based rabbit immunization Mainly because MC-LR is a high toxic biotoxin for animals [8, 41], in order to avoid rabbit death during immunization, in this study, we used the method of low dose injection, long time interval and more rounds of immunizations. In this way, the injected MC-LR-KLH antigen could slowly stimulate the rabbit spleen B-cells to produce and accumulate the anti-MC-LR polyclonal antibodies and genes. The results as shown in Fig. 1, after five rounds of immunization, the anti-MC-LR polyclonal 12

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antibodies were gradually produced and accumulated. Finally, total 80 mL anti-MC-LR serum was obtained by heart blood collection and its titer reached to 1: 2500000, and the rabbit spleen was obvious swelling (picture not shown) after dissected, these results indicated that this study have achieved a good immune effectiveness. In other words, the immunized rabbit have expressed lots of

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high activity of anti-MC-LR PAbs and genes, and its spleen B-cells could be used to extract total RNA

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for constructing the phage display scFv library.

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3.2 MC-LR-KLH immunized rabbit phage display scFv library construction

As shown in Fig. 2A, the extracted total RNA of rabbit existed three sharp and clear eukaryotes

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characteristic of 28s, 18s and 5s nucleotide bands on a gel, and its concentration was 2.98 μg/μL with a

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OD260/OD280 ratio of 1.99, which indicated that the total RNA had a high concentration and purity, and it could be used for follow-up experiments. As shown in Fig. 2B, the NcoI-VL and VH-NotI genes were

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amplified based on the reverse transcriptional cDNA template, then the long complementary linker

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sequence were added respectively, finally, the amplified and purified NcoI-VL-Linker and Linker-VH-NotI were spliced to the whole scFv genes by SOE-PCR, successfully. In this study, we have

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optimized the PCR primers design, and the NcoI and NotI restriction site sequences were directly

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synthesized into the VL and VH genes of upstream and downstream primers. As a result, the spliced and amplified whole scFv genes had the NcoI and NotI double restriction sites that could be used for constructing pIT2-scFv recombinant plasmid immediately, thereby avoiding the step of final PCR amplification for adding restriction sites that compared with some previously reports [39, 42, 43]. The capacity of final successfully constructed MC-LR-KLH immunized rabbit phage antibody library was 3.26×109 CFU/mL, and all the randomly picked single-colony phage particles had the corresponding size of scFv gene by colony PCR on a gel (Fig. 2C). After sequencing and blast, all those determined 13

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scFv genes were from rabbit antibody and with the complete scFv characteristic fragments, which had the same VL-FR, linker and VH-FR regions and an abundant diversity at V L-CDR1-3 and VH-CDR1-3 (Fig. 3). The capacity, completeness and diversity were the most important indicators to evaluate a phage display antibody library [44, 45]. Based on these indicators, the present experimental results

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showed that we had successfully constructed a high quality of rabbit immunized phage antibody library,

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and it could be used for anti-MC-LR phage display scFv particles screening.

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3.3 Enrichment and screening of anti-MC-LR phage display scFv particles

As showed in Fig. 4A, after four rounds of biopanning, the anti-MC-LR phage display scFv

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particles were enriched obviously, and the OD450 value of the fourth round enriched library (0.812) was

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increased more than 3-fold which compared with the original (0.25) by polyclonal phage ELISA for MC-LR. Total 18 positive anti-MC-LR phage scFvs were obtained, and the most positive (RscFv3)

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OD450 value was 1.52 (control was 0.124) and the lowest (RscFv18) was 0.601 (control was 0.135) as

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shown in Fig. 4B. All these selected positives had a real size of scFv gene fragments which were the same as showed in Fig. 2C by colony PCR on a gel, and they completed scFv gene sequences by DNA

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sequencing, which were more or less different in VL-CDR1-3 and VH-CDR1-3. Among them, the most

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two positive anti-MC-LR phage scFvs (RscFv3 and RscFv11) of amino acid sequences were chosen and shown in Fig. 4C. Even though it had the more possibility to obtain antigen-specific and high sensitive phage antibody particles from an immunized library compared with the naive library, it was very necessary to optimize the method of screening [46-48]. In above experiments, the coated antigen used in anti-MC-LR phage particles enrichment, screening and identification was MC-LR-BSA not MC-LR-KLH, the main aim was to avoid the interference from KLH carrier protein because the immunogen was MC-LR-KLH, and thereby improving the efficiency and probability for obtaining the 14

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high sensitive anti-MC-LR phage particles. Furthermore, we have designed a method of alternately using different blocked proteins in experiments, which was blocked skim milk in library enrichment and blocked OVA in ELISA identification to reduce the probability of obtained false positives. The present experimental results indicated that we had successfully obtained multiple positive anti-MC-LR

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3.4 Preparation of pure anti-MC-LR PAbs and RscFv3-scFv proteins

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phage scFvs by the optimized conditions.

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Fig. 5A showed that the anti-MC-LR PAbs were efficiently purified by using HiTrap Protein A HP columns, which existed in a sharp and clear protein band and released VH and VL characteristic

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fragments after dealing with β-mercaptoethanol denaturing buffer on SDS-PAGE gel. The collected

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pure anti-MC-LR PAbs with an initial concentration of 3.6 mg/mL that completely satisfied follow-up application. Phage displayed scFv was not suitable for accurate analysis and further application for

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antigen directly because of the tandem super large phage capsid protein, therefore it need to soluble

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express for preparing purify scFv proteins. E.coli HB2151 is the recommended host bacteria for the soluble expression of pIT2 phagemid vector display antibody library, which the selected phage

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antibody could be directly expressed without reconstructing the recombinant plasmid by host

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replacement [28, 39]. In this study, the RscFv3-scFv was expressed in E.coli HB2151, and its proteins purified from periplasmic lysate, which existed in a sharp and clear scFv size of protein band (approximately 30 kDa) on SDS-PAGE gel as showed in Fig. 5B. The initial concentration of purified RscFv3-scFv was 796.7 μg/mL, and it switched back to the original culture system was 3.98 mg/L. Although the expression levels of RscFv3-scFv in E.coli HB2151 were better than some previously reports which were at 0.2-1.0 mg/L [25, 48, 49], there still existed a big gap compared with the report by Moricoli et al. [50] and Qiu et al. [33] that reached up to 35 and 40 mg/L, respectively. Usually, it is 15

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important for an exogenous protein high-expressing levels in prokaryotic expression systems under suitable culture conditions, which include expression vector and host bacteria types, IPTG concentration, culturing time and temperature, and shaking speed of incubator [51-54]. In this study, the expression level of RscFv3-scFv fully met the demand of qualitative analysis and preliminary

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application for MC-LR indoor. Of course, to rapidly and efficiently prepare lots of RscFv3-scFv for

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subsequent commercial applications, it is still necessary to further optimize the expression conditions

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as listed above.

3.5 IC-ELISAs for MC-LR and its analogues based on anti-MC-LR PAbs and RscFv3-scFv

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To evaluate the activity and specificity of anti-MC-LR antibodies for MC-LR and its analogues,

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IC-ELISAs were established based on the purified anti-MC-LR PAbs and RscFv3-scFv, respectively. As showed in Fig. 6, according to the standard curves of anti-MC-LR PAbs and RscFv3-scFv based

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IC-ELISAs, the IC10 were 0.03 and 0.05 μg/L, the IC50 were 0.6 and 0.76 μg/L, and IC20-IC80 were

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0.13-2.68 and 0.15-3.98 μg/L, respectively. The CRs of anti-MC-LR antibodies for MC-LR analogues were showed in Table 2. The anti-MC-LR PAbs had strong CRs of 92-130% for MC-RR, MC-WR and

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MC-YR, and weak CRs for MC-LW (6.5%), MC-LY (0.7%) and MC-LF (<0.1%). The anti-MC-LR

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RscFv3-scFv also had strong CRs for MC-RR (97%), MC-WR (85%) and MC-YR (56%), and weak CRs for MC-LW (0.9%), but had no detection for MC-LY and MC-LF. These results showed that the RscFv3-scFv was of similar activity and specificity compared with the anti-MC-LR PAbs for MC-LR and its analogues, which further confirmed that the constructed MC-LR-immunized phage library and the matched screening strategy in above presented studies were successful and feasible. The IC50 of RscFv3-scFv for MC-LR was below to the MRL of 1.0 μg/L in drinking water by WHO, and the IC10 reached to 0.05 μg/L which fully met the requirements of MC-LR detection. The IC10 of RscFv3-scFv 16

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for MC-LR was better than the reports by Murphy et al. [55], Akter et al. [56] and Zhang et al. [57] were 0.19, 0.2 and 0.13 μg/L from scFv-based ELISAs, and the reports by Pirez-Schirmer et al. [58] and Xu et al. [38] were 0.4 and 0.12 μg/L from VHH-based ELISAs, respectively. Some reports demonstrated that it was possible to improve the affinity and sensitivity of scFv by using antibody

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affinity maturation in vitro or label-fluorophore [16, 59-63], which could be used for further improving

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3.6 RscFv3-scFv based IC-ELISA for MC-LR by spiked samples

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the application value of RscFv3-scFv for MC-LR.

It is an effective method to evaluate the suitability of a new established detection method before

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practical application by spiked samples indoor [16]. In this study, the MC-LR was spiked in tap water,

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drinking bottled water and lake water samples at the concentration of 0.2, 1.0 and 2.0 μg/L respectively, which according to the linear working range of 0.15-3.98 μg/L from RscFv3-scFv based IC-ELISA

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showed in Fig. 6. The analytical results shown in Table 3. The recovery rates in tap water samples

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were 88.3-97.5% with CVs of 5.9-10.4% and 91.6-100.5% with CVs of 4.4-10.3%, the drinking bottled water samples were 95.3-106% with CVs of 1.8-5.4% and 93.1-102.2% with CVs of 2.5-11.5%, and

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the lake water samples were 83-93.9% with CVs of 2.7-10.8% and 86.8-95.1% with CVs of 4.2-6.0%

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at intra-assay and inter-assay, respectively. These results converted to the average recovery rate was 91.9% with CV of 6.8%, and it indicated that the RscFv3-scFv based IC-ELISA has a good accuracy, stability and reproducibility for detecting MC-LR in present water samples. 4.

Conclusion In this study, we have successfully constructed a large capacity and diversity of MC-LR

immunized phage display scFv library that could be used for anti-MC-LR phage scFv particles screening. The most positive anti-MC-LR RscFv3-scFv was expressed in E.coli HB2151, and it 17

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showed high sensitively and broad recognizably for MC-LR and its analogues, MC-RR, MC-WR and MC-YR. The RscFv3-scFv based IC-ELISA showed a good accuracy, stability and reproducibility for detecting MC-LR in water samples, which indicated a wide application prospect for rapidly and sensitively monitoring MC-LR and it analogues in environmental samples.

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Acknowledgments

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This study was supported by the National Natural Science Foundation of China (31701724,

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31630061 and 31370217), and the Social Development Projects of Jiangsu Province in China

Conflict of interest

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The authors declare no conflict of interest.

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(BE2017706).

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Fig. 1. The immune efficacy analysis of MC-LR-KLH to rabbit by serum based ELISA for MC-LR, and the coated antigen were MC-LR-BSA. The presented OD450 values were the means ± SDs from triplicate measurements.

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Fig. 2. (A) The total RNA from the MC-LR-KLH immunized rabbit spleen B-cells. M: DNA marker;

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Lane 1: Total RNA. (B) The PCR amplified products of during the processes of rabbit phage display

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scFv library construction. M: DNA marker; Lanes 1-3: NcoI-VL, approximately 350 bp; Lanes 4-6: VH-NotI, approximately 365 bp; Lanes 7-9: NcoI-VL-Linker, approximately 390 bp; Lanes 10-12:

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Linker-VH-NotI, approximately 410 bp. Lanes 13-17: NcoI-VL-Linker-VH-NotI (scFv), approximately

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800 bp; ck1-5: Without templates. (C1-2) The scFv amplified products by random colony PCR from the constructed MC-LR-KLH immunized rabbit phage display scFv library. M: DNA marker; Lanes 1-42:

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random colonies, their PCR products were all approximately 950 bp (contains 800 bp of scFv and 150

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bp of pIT2 phagemid vector nucleotide sequences). ck: Without templates. Fig. 3. Diversity analysis of the constructed MC-LR-KLH immunized rabbit phage display scFv library

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by randomly pick multiple single-colony for the inserted scFv genes sequencing. The present graphics

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were made by using of WebLogo website (http://weblogo.berkeley.edu/logo.cgi), and the relative nucleotide bias at each position of the R-VL-CDR1-3 and R-VH-CDR1-3 sequence motifs were from the randomly picked 42 single-colony scFv genes. Fig. 4. (A) Polyclonal phage ELISA that based on the enrichment of library (contains anti-MC-LR phage scFv particles) for MC-LR. The coated antigen used in the enrichment of library were MC-LR-BSA, and in the polyclonal phage ELISA were MC-LR-KLH and the negative controls were KLH. The presented OD450 values were the means ± SDs from 27

ACCEPTED MANUSCRIPT triplicate measurements. (B) Monoclonal phage ELISA that based on the selected anti-MC-LR phage scFv particles for MC-LR. The coated antigen used in the monoclonal phage ELISA were MC-LR-KLH and the negative controls were KLH. The presented OD450 values were the means from triplicate measurements. (C) Amino acid sequences of the most two positive

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anti-MC-LR phage scFv genes (named RscFv3 and RscFv11, respectively) from above

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present selected clones.

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Fig. 5. (A) SDS-PAGE of anti-MC-LR PAbs proteins from the booster immunized rabbit serum and purified by using HiTrap Protein A HP columns. M: Protein marker; Lane 1: The primary purified

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PAbs proteins by SASS from serum; Lanes 2, 3: The obtained purified PAbs proteins (approximately

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150 kDa) by using HiTrap Protein A HP columns. Lanes 4-7: The purified PAbs proteins with β -mercaptoethanol denaturing buffer, and the two heavy chain (approximately 50 kDa) and the two light

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chain (approximately 25 kDa) were released, respectively. (B) SDS-PAGE of anti-MC-LR scFv

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(RscFv3) proteins expressed in E.coli HB2151 and purified by using His-Trap HP columns. M: Protein marker; Lane 1: The obtained purified scFv proteins (approximately 30 kDa) from the periplasmic

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lysate by using His-Trap HP columns; Lane 2: Periplasmic lysate; Lane 3: Expressed of supernatant;

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Lane 4: Whole of cell lysate.

Fig. 6. The standard curves of IC-ELISAs based on anti-MC-LR PAbs and scFv (RscFv3) for MC-LR, respectively. The presented inhibition ratio values were the means ± SDs from triplicate measurements.

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Table 1. The PCR primers used for construction of the MC-LR-KLH immunized rabbit phage display scFv library (W=A or T, S=G or C, M= A or C, R=G or A) Nucleotide sequences (5’→3’)

Primer names NcoI-RVL-F1

CATGCCATGGGGCCCAGCCGGCCGAGCTCGTGMTGACCCAGACTCCA

NcoI-RVL-F2

CATGCCATGGGGCCCAGCCGGCCGAGCTCGATMTGACCCAGACTCCA

NcoI-RVL-F3

CATGCCATGGGGCCCAGCCGGCCGAGCTCGTGATGACCCAGACTGAA

NcoI-RVL-F4

CATGCCATGGGGCCCAGCCGGCCGAGCTCGTGCTGACTCAGTCGCCCTC

RVL-B1

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATAGGATCTCCAGCTCGGTCCC

RVL-B2

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATTTGACSACCACCTCGGTCCC

RVL-B3

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATTTGATTTCCAGATTGGTGCC

RVL-B4

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGAGCCTGTGACGGTCAGGGTCCC

RVH-F1

GGTGGTTCCTCTAGATCTTCCCAGTCGGTGGAGGAGTCCRGG

RVH-F2

GGTGGTTCCTCTAGATCTTCCCAGTCGGTGAAGGAGTCCGAG

RVH-F3

GGTGGTTCCTCTAGATCTTCCCAGTCGYTGGAGGAGTCCGGG

RVH-F4

GGTGGTTCCTCTAGATCTTCCCAGSAGCAGCTGGWGGAGTCC

RVH-B-NotI

ATAAGAATGCGGCCGCCCTGGCCCCCGAGGCCACAGTGACTGAYGGAGCCTTAGGTTGCCC

scFv

RVL-Linker

GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGGGAAGATCTAGAGGAACCACCCCCACC

(VL-Linker-VH )

Linker-RVH

CCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACCGGTGGTTCCTCTAGATCTTCCCAG

LMB3

CAGGAAACAGCTATGAC

PHEN

CTATGCGGCCCCATTCA

RI

SC

NU

MA

RVH

PT

RVL

AC

CE

PT E

D

pIT2

29

ACCEPTED MANUSCRIPT

Table 2. The CRs of anti-MC-LR PAbs and scFv (RscFv3) by IC-ELISAs for MC-LR analogues (the presented values were the means from triplicate measurements) MC-LR analogues

Antibo

0.60

100

MC-RR

1037

0.44

136

MC-WR

1068

0.50

119

MC-YR

1044

0.65

92

MC-LW

1024

9.2

MC-LY

1001

86

MC-LF

985

878

MC-LR

994

0.76

MC-RR

1037

MC-WR

1068

3

MC-YR

1044

MC-LW

1024

MC-LY

1001

MC-LF

985

AC

CE

PT E

D

“nd”: not detected.

PT

994

6.5

0.7

RI

MC-LR

<0.1 100

SC

RscFv

CRs (%)

M.W.(Da)

0.78

97

0.89

85

NU

PAbs

IC50 (μg/L)

Competitors

MA

dies

30

1.36

56

66.7

0.9

nd

nd

nd

nd

ACCEPTED MANUSCRIPT

Table 3. Anti-MC-LR scFv (RscFv3) based IC-ELISA for MC-LR in spiked water samples (the presented values were the means ± SDs from triplicate measurements) Intra-assay

Inter-assay

MC-LR Measured Water

Recov

C

Measured

Recov

samples

values ± SDs

eries ± SDs

(μg/L)

(%)

Vs

concentrations (μg/L)

(

values ± SDs

eries ± SDs

(μg/L)

(%)

0

nd

%)

nd

nd

nd

water 94.5±7.5

7.9

0.201±0.012

1.0

0.975±0.058

97.5±5.8

5.9

0.933±0.096

2.0

1.766±0.183

88.3±9 .2

Average

.4 93.4±7

0

nd

0 nd

bottled 0.2 1.0

0.212±0.011 0.992±0.018

2.0

1.905±0.102

106.0±5.5

MA

water

nd

99.2±1.8

D

Average

0

water 0.2

nd

AC

2.0

6.0

93.3±9.6

10.3

1.832±0.080

91.6±4 .0

4 .4

95.1±6 .5

nd

6 .8

nd

n d

94.5±7.0

7.4

1.8

1.022±0.026

102.2±2.6

2.5

5.

1.861±0.214

4

93.1±1 0.7

4.

96.6±6

1 nd

.8 nd

1 1.5

nd

7 .0

nd

n d

0.166±0.018

83.0±9.0

10.8

0.178±0.010

89.0±5.0

0.905±0.024

90.5±2.4

2.7

0.868±0.038

86.8±3.8

CE

1.0

PT E

Lake

d

0.189±0.014

101.2±

4.1

n

5.2

95.3±5

.1

nd

100.5±6.0

8.

NU

.5 Drinki

10

RI

0.189±0.015

SC

0.2

PT

Tap

Vs (

%)

ng

C

spiked

5.6 4 .2

1.878±0.156

93.9±7 .8

Average

8. 3

89.1±6 .4

31

95.1±5 .8

7. 2

“nd”: not detected.

1.902±0.116

6 .0

90.3±4 .7

5 .3

ACCEPTED MANUSCRIPT Highlights Constructed a large capacity and diversity of MC-LR immunized phage scFv library.

2.

Successfully isolated 18 positive anti-MC-LR phage scFv particles by biopanning.

3.

Prepared high purified and concentrative anti-MC-LR PAbs and scFv proteins.

4.

The anti-MC-LR PAbs and scFv were high-sensitive and broad-recognizable for MCs.

5.

The scFv based IC-ELISA can be used for determination of MC-LR in water samples.

AC

CE

PT E

D

MA

NU

SC

RI

PT

1.

32

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6