Selection of Aflatoxin B1 Mimic Epitope Peptides by Phage Display

Ke Wu

, Xiaoyan Qiu

and Renrong Liu

School of Life Science, Jiangxi Science & Technology Normal University, Jiangxi, Nanchang, China

These authors contributed equally to this work

Keywords:

Aflatoxin B1, Phage Display Peptide Library, Mimic Epitope, ELISA.

Abstract:

Aflatoxin B1 (AFB1) is a natural pollutant with strong toxicity and carcinogenicity. It not only causes huge

economic losses, but also poses a serious threat to human, livestock and poultry health. To acquire the

mimic epitope peptide of aflatoxin B1 and establish a non-toxic detection system of aflatoxin, an anti-

afb13c7 monoclonal antibody was used as the target molecule, and the mimic epitopes of AFB1 were

screened from the phage random 7 peptide library. A total of 39 phage clones were selected for verification.

36 of them could specifically bind to the antibody, and 30 of them could be inhibited by AFB1, whose DNA

was extracted and sequenced. The results showed that 30 positive phage particles were actually 17 phage

clones. The common sequence was histidine (H) - proline (P) - tryptophan (W), abbreviated as xxxxhpw,

xxxhpwx, xxhpwxx, xhpwxxx (X was any amino acid). The linear range, detection limit and half inhibitory

concentration (IC50) of the 17 positive phage particles were similar. The linear range was 1-2000 pg / ml.

1 INTRODUCTION

Aflatoxin B1 (AFB1) is a secondary metabolite

produced by Aspergillus flavus, Aspergillus

parasiticus and Aspergillus wasabi, which has been

proved to be carcinogenic, teratogenic and

mutagenic (Riikka, 2017, Rushing, 2018). AFB1 is

easy to pollute wheat, rice and other agricultural

products, which poses a serious threat to human,

livestock and poultry health (Anja, 2016, Zhao,

2015). The method of monitoring and detecting

AFB1 is important to prevent its harm, which has

great research significance.

Domestic and foreign scholars have done a lot of

research on the detection methods of AFB1.

Since the immunoassay for AFB1 have to use the

toxin both in free and conjugated forms, it may pose

a toxicity risk to kit manufacturers and users, so it is

an urgent problem to find the substitute of AFB1

standard (Liu, 2016). The epitope peptide of AFB1

McAb was successfully screened by phage display

technology in this study.

https://orcid.org/0000-0002-6663-5928

https://orcid.org/0000-0003-2425-8316

https://orcid.org/0000-0001-7415-5169

2 MATERIALS AND METHODS

2.1 Reagents and Instruments

The anti-AFB1 monoclonal antibody was made in

Laboratory of Jiangxi Normal University of science

and technology. Aflatoxin B1 was purchased from

Solarbio. IPTG and X-gal were purchased from

Golden clone Biotechnology Co., Ltd. Random

phage 7 peptide library and E.coli ER2738 were

purchased from NEW ENGLAND BioLabs. Agar

Powder was purchased from Chembase.

Polyethylene glycol. PEG-8000 was purchased from

Shanghai Shenggong biology Co., Ltd. Mulitiskan

MK3 microplate reader (Thermo, USA).

Centrifuge5804r freezing centrifuge (Eppendorf,

USA). Washing machine (Thermo, USA). Pipette

gun (Thermo, USA). Polystyrene 96-well microtiter

plates (Costar).

2.2 Panning and Identification of

Positive Mimetic Peptides

The specific operation process is as follows (Wang,

2018, Shi, 2019)

The solid phase carrier (Polystyrene 96-well

microtiter plates) was coated with monoclonal

antibody and incubated with phage library for 60

Wu, K., Qiu, X. and Liu, R.

Selection of Aﬂatoxin B1 Mimic Epitope Peptides by Phage Display.

DOI: 10.5220/0011508600003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1279-1283

ISBN: 978-989-758-595-1

1279

minutes. The unconjugated phage was washed out,

and the specific binding phage was washed down to

amplify the phage. The above steps were repeated

for 3 times and the phage titer was determined. The

final product was confirmed by positive clone

sequencing, and the binding of the selected peptide

to the monoclonal antibody was detected by ELISA.

The specific steps are as follows.

A. The anti-AFB1 monoclonal antibody was

dissolved in the PBS of 0.1M pH 7.4 (100, 75,

50μg/mL), and the 100μL/pore 4°C package was

spent overnight.

B. Discard the liquid in the hole, wash it 3 times

with TBST (including 0.1% Tween20), put the board

upside down on a clean tissue and pat it hard to

remove the residual solution. Add 3% BSA-PBS

300μL/hole 4°C to seal for 2 hours.

C. Wash it 3 times with TBST (including 0.1%-

0.05% Tween20), add the phage peptide bank

(Ph.D.-7 library) 100μL per hole (dillute it with

TBST at 1:10, containing phage about 2×1011pfu),

and shake gently at 16-22°C. It should be 1h.

D. Discard the liquid in the hole, wash it 10

times with TBST, and wash away the unbined

bacteriophages.

E. Add the lotion (Gly-HCL pH2.2) 100μL per

hole, shake it gently at 16-22°C for 8 minutes, suck

out the lotion, and add 15μL neutralizing buffer.

F. Except for 10μL for titer determination after

neutralization, the rest are added to 20mL

inoculation with the LB culture medium of E.coli

ER2738, which is in the pre- logarithmic growth

stage for amplification culture.

G. After 4.5 hours of oscillation culture of 37°C,

4°C 10000rpm centrifugal for 10 minutes, 1/6

volume of PEG/NaCl is added to the supernatant,

and 4°C precipitates overnight.

H.4°C centrifugal 15min (10000rpm), remove

the upper clearing, then use 1 mL TBST suspended

phages, and add 1/6 volume of PEG / NaCl ice to

incubate 60 minutes. 4°C centrifugal 15min

(10000rpm), de-clearing, precipitation with 200μL

TBST suspension and 10μL to measure titer.

A. Inoculated with E.coli ER2738

monobacterium colony in 5-10mL LB medium,

rocker cultured to logarithmic mid-term (OD600 at

about 0.5).

B. When cells grow, melt the upper agar and

divide it into 3mL equal parts in the sterilization test

tube, and dilute one tube for each phage. Store at

45°C for later use.

C.37°C warm up the LB/IPTG/Xgal plate, and

take one tablet for each phage dilution.

D. The bacteriophages to be tested are diluted

with a 10x series of LB medium.

E. When the bacterial culture reaches the middle

logarithmic, it is divided into 200μL equal parts in

the trace centrifugal tube, and each phage dilutes one

tube.

F. Add 10μL bacteriophages with different

dilution degrees to each tube, quickly oscillate and

mix well, and warm up 16-22oC for 1-5 min. Add

infected cells to the upper agar culture tube pre-

temperatured at 45°C, mix one tube quickly at a

time, and immediately pour them on the

LB/IPTG/Xgal plate pre-temperatured at 37°C. Tilt

the plate appropriately to spread the upper agar

evenly.

G. After the plate is cooled for 5 minutes, put it

upside down at 37°C for the night. Check the plate

and count the number of spots on the plate with

~102 phage plaques. Then multiply this number by

dilution factor to obtain the empty spot formation

unit (pfu) titer per 10μL phage. DNA of positive

phage clones was extracted and sequenced

A. Inoculate overnight cultures of E.coli ER2738

at 1: 100 dilution on LB medium and 1 mL into

culture tubes. One tube per clone to be identified.

B. Using a sterilized toothpick or tip, pick a blue

plaque into the 1 mL culture tube above. Note: The

plaque should be selected from a total of less than

100 plaque to ensure that each plaque selected

contains only one DNA sequence.

C. 37 °C shaker culture 4.5-5 h.

D. The cultures were transferred into

microcentrifuge tubes and centrifuged at 4C 30 sec

(10000 rpm). The supernatant is transferred into a

fresh tube and centrifuged again. 80% supernatant

was transferred to fresh centrifuge tube with pipette.

This was the amplified phage reservoir, stored at

4 °C or diluted with sterile glycerin 1: 1 and stored

at -20 °C.

E. coli ER2738 was inoculated in 20 mL LB

medium and cultured at 37 °C to prologarithmic

stage. Alternatively, the overnight culture of E.coli

ER2738 was diluted in a 1: 100 ratio in 20 mL LB

medium.

F. Adding 5L phage reservoir to each tube of

Ecoli ER 2738 culture medium, aerated at 37 °C for

4.5 h.

G. The above cultures were transferred into

centrifuge tubes and centrifuged at 10,000 rpm for

10 min. The supernatant is moved into a fresh

centrifuge tube and centrifuged again.

H. Take 80% supernatant in fresh centrifuge tube,

add 1 / 6 volume of PEG / NaCl. Precipitation at

4 °C overnight.

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I.4℃ 10000rpm 15 min Centrifugally precipitate

and discard the supernatant.

J. Precipitate was resuspended in 1 mL TBS. The

suspension was transferred to a microcentrifuge tube

and centrifuged at 4 °C for 5 min (10000 rpm) to

remove the residual cells.

K. supernatant was transferred into a fresh

microcentrifuge tube and added 1 / 6 volume of PEG

/ NaCl to precipitate. Ice action 15-60 min.

Centrifuge 10 min (10000 rpm) at 4 °C, discard the

supernatant, and centrifuge briefly to remove the

residual supernatants. The precipitate was

resuspended in 50 L TBS and the titer of phage was

determined. 4 °C storage.

A. Monoclonal antibody against AFB1 was

dissolved in 0.1 M pH 7.4PBS (10 μg / mL) and

coated overnight with 100 μL / well 4 °C.

B. PBST washed plate three times, 3% skim milk

300μL / hole 4 °C closed 2h.

C. Addition of 1 μg / mL of AFB1 (soluble in 20%

methanol PBS) 50 μL and PBST diluted 50 μL.

(Dilution ratio was twice the maximum dilution for

identification of specifically bound positive phage

clones with absorbance values of 1.0-1.5), 37 °C 1 h.

D. PBST washed 6 times and added horseradish

peroxidase labeled anti-M13 monoclonal antibody

(100 μL / well); 37 °C acts for 1h. Wash 6 times.

Benzidine (OPD) color, 2M H2SO4 terminated

reaction, OD value was measured. The original

peptide library coated as positive control, McAb

coated with original peptide as negative control,

monoclonal antibody coated with PBST as blank

control. According to the ratio of OD value (S) of

test sample to OD (N) of negative control, the S / N

was more than 2.1. The phage which can be blocked

by AFB1 molecule is the mimotope of AFB1.

500μl of the phage-containing supernatant was

transferred to a fresh microfuge tube and added with

200μl PEG/NaCl. The mix was let stand at room

temperature for 10 minutes, then it was centrifuged

for 10 minutes, supernatant was discarded. Pellet

was suspended thoroughly in 100μl Iodide Buffer

and added with 250μl ethanol. The resuspended

solution was incubated for 10 minutes at room

temperature and spun for 10 minutes, supernatant

was discarded, the pellet was washed in 70% ethanol

and dried briefly under vacuum. The pellet was

suspended in 30μl TE buffer. Take 5ul of the above

solution for nucleic acid electrophoresis, and send

the rest to the Shanghai Shenggong biology Co., Ltd

for sequencing.

2.3 Establishment of Competitive

ELISA Standard Curve with AFB1

Mimic Epitope

Dilute AFB1 standard stock solution into a series of

concentration standard solutions. According to the

competitive steps under the optimized conditions,

the luminescence value was determined through

experiments, and the standard curve was established

(the logarithm of the toxin standard solution

concentration was the abscissa and the binding rate

was the ordinate). Specific steps are as follows

A. After amplification and purification of

positive phage particles, the optimal antibody

coating concentration and phage concentration were

determined by matrix titration.

B. anti-AFB1 monoclonal antibody soluble in 0.1

M pH7.4PBS, 100 μ L / pore 4°C was coated

overnight. The original peptide library was coated as

positive control, the monoclonal antibody coated

with primitive peptide library as negative control

and the mAb coated with PBST as blank control.

C. PBST washed plate three times, 3% skim milk

300μL / hole 4°C closed 2h.

D. Adding concentrations of 200, 100, 50, 25,

12.5, 6.25, 4, 2, 1, 0.5, 0.25, 0.125, 0.10, 0.05, 0 ng /

mL AFB1 standard (dissolved in 20% methanol PBS)

50 μ L and positive phage 50 μL, shake and mix,

keep moisture at 37°C for 1 hour.

E. PBST washed 6 times, added horseradish

peroxidase labeled anti-M13 monoclonal antibody

(100 μL / pore), 37°C for 1 h.

F. PBST washed 6 times. The reaction was

terminated by 2M H2SO4. The optical density (OD

value) at 450 nm was determined. The binding rate

(%) = B / B0 × 100% (B0 is the OD value without

AFB1 and B is the value of OD with AFB1).

Drawing Competition Inhibition Curve.

3 RESULTS

3.1 Analysis of DNA Sequence and

Polypeptide Core Series

After DNA sequencing, the amino acid sequence

was translated by the DNAMAN software. The 30

phage particles are actually 17 phage clones., No.

A1-17 The common sequence of aflatoxin mimic

epitope peptide is histidine (H) - proline (P) -

tryptophan (W), abbreviated as xxxhpwx, xhpwxxx,

xxxxhpw, xxhpwxx. X is any amino acid. The DNA

and amino acid sequences are shown in Table 1.

Selection of Aﬂatoxin B1 Mimic Epitope Peptides by Phage Display

1281

Table. 1 The inserted DNA sequences of positive phages

age

mber

DNA sequence

Petide

sequence

A1 1

AGATACAAAGTAG

GAACCAGC

SMFHPW

A2 2

CCACGCACCGTAG

GAACCAGA

GAWHP

A3 1

ATAGTAGGAACCT

CAACCACC

YHPWSW

A4 1

GTCGTAAACACCG

TAGGCACC

QHLWHP

A5 6

ACCGGACACAAAG

TAGGAACC

WPVFHP

A6 1

CTCCAAAAAGTAG

GAACCAGC

EVFHPW

A7 2

ATAGGCCGAACCG

TAGGAACC

YPAWHP

A8 1

CTAAAAGGCACCG

TAGGCACC

DFPWHP

A9 2

CTAGTAGGCACCC

ACATAAGC

DHPWVY

CGCGTAGGCACCC

CAGGATAA

AHPWGP

AGCCAAACCGTAG

GAACCAGA

SVWHPW

ATAGTAGGAACCA

GACACTGC

YHPWSV

AAACACGTAGGCA

CCTAAACC

FVHPWI

TTATGCGTAGGAA

CCAAAACC

NTHPWF

CAATTACAAATAG

TAGGAACC

VNVYHP

TGAAACGTAGGCA

CCGTAACC

TLHPWH

CACGTCGGAATAG

TAGGCACC

VQPYHP

3.2 Establishment of Standard Curve

of Non-toxic ELISA with AFB1

Mimic Epitope

The standard curve of ELISA was established by

coating 96 well plate with anti AFB1 monoclonal

antibody (3c7) and incubating with phage particles

a1-a17 with mimic epitope of AFB1. The results

showed that the standard curve of ELISA based on

these phage particles showed a good linear

relationship. The linear range, detection limit and

half inhibitory concentration (IC50) were basically

similar, and the linear range was 1-2000 pg / ml (as

shown in Fig1).

Figure 1 The standard curves of competitive ELISA of

A1-A17

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4 CONCLUSION

Appropriate concentration of target protein is

beneficial to the screening of mimic peptides.

Reducing target protein concentration may improve

the specificity of washing. In order to screen antigen

mimic epitopes with high affinity, the detergent

concentration of the first round of elutriation was

0.1%, and that of the second round of elutriation was

0.5%. The number of ligands that have affinity with

the target protein at the beginning of panning is very

small. If the detergent concentration is too high, the

ligands with weak binding force may be lost. In the

second round, due to the amplification of ligands, the

concentration of detergent can be increased, and high

affinity ligands are more easily elutriated.

ACKNOWLEDGMENTS

This work was financially supported by grants from

Natural Science Foundation of China (No.31960501)

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