Chicken Single-Chain Antibody Fused to Alkaline Phosphatase

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Article

A chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich ELISA Sheng Xue, He–Ping Li, Jing–Bo Zhang, Jin–Long Liu, ZuQuan Hu, An-Dong Gong, Tao Huang, and Yu-Cai Liao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402608e • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 21, 2013

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Analytical Chemistry

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A chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus

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pathogens and their presence in natural samples by direct sandwich ELISA

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Sheng Xue†, He-Ping Li†,‡, Jing-Bo Zhang†,§, Jin-Long Liu†, Zu-Quan Hu†, An-Dong Gong†,

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Tao Huang†,‡, Yu-Cai Liao†,§,

⊥,*

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†

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Wuhan 430070, China ‡

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College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

§

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Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University,

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

⊥

National Center of Plant Gene Research (Wuhan), Wuhan 430070, China

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*Corresponding author: Tel./Fax: +86 27 8728 3008. E-mail: [email protected];

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ACS Paragon Plus Environment


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ABSTRACT

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A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus

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pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken

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single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from

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Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the

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highest activity towards both A. flavus and A. parasiticus and specifically recognized a

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surface target of their cell walls as revealed by immunofluorescence localization. Molecular

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modeling revealed a unique compact motif on the antibody surface mainly involving

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L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline

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phosphatase had a higher binding capability and six-fold higher affinity compared with

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AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to

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SCWP components from the two fungal species. Direct sandwich enzyme-linked

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immunosorbent assays with mouse anti-aspergillus monoclonal antibody mAb2A8 generated

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in parallel as a capture antibody revealed that the detection limit of the two fungi was as low

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as 10−3 µg/mL, 1000-fold more sensitive than that reported previously (1 µg/mL). The fusion

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protein was able to detect fungal concentrations below 1 µg/g of maize and peanut grains in

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both artificially and naturally contaminated samples, with at least 10-fold more sensitivity

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than that reported (10 µg/g) thus far. Thus, the fusion can be applied in rapid, simple, and

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specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

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Aspergillus flavus and A. parasiticus are the most ubiquitous aflatoxigenic Aspergillus fungi

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found to infect many field crops and to contaminate their products and other food/feed

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commodities during storage.1 These fungi can produce various types of aflatoxins and lead to

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huge economic losses. Additionally, A. flavus is second only to A. fumigatus as the cause of

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human invasive aspergillosis2 and the main Aspergillus species infecting insects.3 Naturally

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occurring aflatoxins have been classified as Group 1 human carcinogens by the International

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Agency for Research on Cancer, and they have demonstrated carcinogenicity in many animal

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species.4 Currently, more than 5 billion people worldwide suffer from uncontrolled exposure

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to aflatoxins.5 However, aflatoxins are difficult to detect owing to their complex structures

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and variable accumulation governed by their producers. Thus, rapid and accurate analytical

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tools for detecting aflatoxin producers are essential for predicting disease risk, preventing

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aflatoxins from entering food/feed chains, and making proper diagnoses in clinics.

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Conventional methods for detection of Aspergillus fungi usually rely on plate counting

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that is laborious and time-consuming, and the number of conidia may not reflect actual

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or potential mycotoxin production because aflatoxins are produced by mycelia. While conidia

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do undergo germination and growth to form mycelia, aflatoxin biosynthesis depends on

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mycelial growth.6 Therefore, direct detection of aflatoxin producers is more reliable than

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counting conidia. Enzyme-linked immunosorbent assay (ELISA) techniques generally have

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the advantages of speed, specificity, sensitivity, and ease-of-use in monitoring pathogens, and

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sandwich ELISA is especially advantageous when well-matched pairs of capture and

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detection antibodies are used.7 During the last three decades, immunoassays have been

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developed for detection of mold contamination by using polyclonal antisera8–11 and

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monoclonal12 or single-chain variable fragment (scFv) antibodies.13–16 For A. flavus and A.

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parasiticus pathogens, however, only polyclonal antisera have been used, with a detection

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limit in PBS of 1 µg/mL by ELISA17,18 and 10 µg/g in rice by a recently developed carbon

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nanotube field effect transistor device.19 Because of the disadvantages inherent in polyclonal

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antisera, such as heterogeneous population, limited quantities of antiserum, and variability

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from batch to batch, a commercial immunoassay usually requires the use of monoclonal

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antibodies (mAbs) that each contains a population of identical antibodies recognizing the

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same specific determinant on an antigen.20 Unlike polyclonal and monoclonal antibodies,

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scFv antibodies consisting of recombined variable domains of heavy and light chains can be

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isolated together with their coding sequence by phage display,15,21 expressed with a high yield

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with a bacterial expression system,22 and readily extracted from the periplasm space23 while

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still retaining their monoclonal properties. Various reports have shown the generation of

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highly reactive antibodies by phage display that would be difficult, if not impossible, to

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isolate by other methods.24

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Generation of recombinant antibodies from chickens is preferred because constructing a

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phage library is technically easier using chickens compared with other animals. Chickens

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carry only one functional V segment and one functional J segment in the immunoglobulin

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heavy and light chain loci and preserve antibody diversity by gene rearrangements and

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recombinations.25 This peculiar mechanism of immunoglobulin gene diversification leads to

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only one set of primers being required for each antibody chain, instead of the mixtures

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needed for amplification of the variable gene families from other animal.26 Phage display

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technology is also a powerful tool for screening high affinity antibodies from chicken

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antibody phage libraries.14,15,27

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Phage-displayed scFv antibodies are particularly apt for genetic manipulation such as

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construction of a fusion protein with an enzyme or protein to create bifunctional

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molecules.24,27 This unique feature allows an scFv antibody–alkaline phosphatase (AP) fusion

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to be easily generated for one-step ELISA detection, without the need for enzyme-labeled

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secondary antibodies. Such an analytical system is simple, rapid, and cost-effective, because 4


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AP or horseradish peroxidase (HRP) conjugation to antibodies by chemical cross-linking

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requires highly experienced technicians and is a random reaction that may lead to reduced or

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even lost activity and produces many unexpected conjugates.7 ScFv-AP fusions can be easily

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produced and purified in a bacterial expression system, and they accelerate analyte detection

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in antibody-based diagnostic tests.28–30

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In this work a highly reactive chicken scFv antibody, AfSA4, against A. flavus was isolated

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by phage display, and immunofluorescence assay localized the binding of the scFv antibody

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to a surface target of cell walls from both A. flavus and A. parasiticus. AfSA4 fused to AP

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showed a higher reactivity towards the two Aspergillus species in ELISA and had six-fold

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higher affinity compared with AfSA4 alone as revealed by surface plasmon resonance. Direct

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sandwich ELISAs based on a mouse anti-aspergillus mAb2A8 (capture antibody) and the

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AfSA4-AP fusion (detection antibody and enzyme) were developed. The fusion-based assay

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is sensitive, specific, accurate, and cost-effective, with limits of detection as low as 10−3

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µg/mL in PBS and 1 µg/g mycelia in maize or peanut, making it suitable for monitoring

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naturally contaminated samples.

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EXPERIMENTAL SECTION

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Two 4-month-old White Leghorn female chickens were immunized intramuscularly with 200

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µg of soluble cell wall proteins (SCWPs) from A. flavus strain wh-1 (Table S1) prepared as

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previously described31 for isolation of recombinant scFv antibodies and polyclonal IgY

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antisera.21,26 Five 6-week-old BALB/c female mice were immunized subcutaneously and

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intraperitoneally with 50 µg of the same SCWPs for generation of monoclonal antibodies.32

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Aflatoxins produced in maize and peanut by Aspergillus species (Table S1) were determined

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by liquid chromatography–tandem mass spectrometry (LC–MS/MS).33 RNA extracted from

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spleen cells of the immunized chickens was transcribed into first strand cDNA. Subsequently,

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a recombinant antibody library was constructed by cloning PCR amplified scFv genes into 5



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the pComb3X phagemid vector.34,35 Phage clones against A. flavus SCWPs were selected by

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panning of phage display library and soluble ELISAs.27 The selected scFv antibodies were

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expressed in bacteria and assayed for their binding to A. flavus and A. parasiticus (Table S1)

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by immunofluorescence labeling,15 and their three-dimensional structures were modeled.36,37

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The scFv antibody with the highest binding capacity was fused to a hyperactive mutant

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alkaline phosphatase (AP)28 through a flexible 218 linker15 with a His-tag at the 3′ end. The

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resulting fusion and its parent scFv antibody expressed in bacteria were analyzed by

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immunoblots and indirect ELISA for their binding activity and specificity with different

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fungal species (Table S1), and by surface plasmon resonance (SPR)38,39 for binding kinetics.

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The fusion was finally used for direct sandwich ELISA for quantification of

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Aspergillus-contaminated maize and peanut samples (Tables S2 and S3). For a more detailed

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experimental description, see the Supporting Information.

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RESULTS AND DISCUSSION

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Antigen preparation and scFv antibody library. To generate antibodies against

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aflatoxigenic A. flavus, SCWPs from a representative strain, wh-1 isolated from diseased

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peanut in Wuhan, China, were prepared as antigens for immunization of chickens. The strain

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wh-1 was morphologically identified as A. flavus, the predominant species causing aflaroot in

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peanut or rot of matured peanuts and ear rot of maize in China. It was chemically shown to

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produce a large quantity of aflatoxins in maize (775.39 µg/kg AFB1, 26 µg/kg AFB2 and 2.31

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µg/kg AFG2) and peanut (1056.8 µg/kg AFB1 and 55.04 µg/kg AFB2) cultured for 8 days at

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28°C as determined by LC–MS/MS analysis (Table S1). After the fourth immunization, blood

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and pAIgY antisera were analyzed by indirect ELISA. The results showed a clear robust

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humoral response up to 1:204,800. Spleen cells from the immunized chickens were prepared

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for RNA isolation, and purified mRNA was transcribed into cDNA for construction of an

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scFv antibody library. A phagemid library with a size of 1.2 × 107 clones and 22 clones 6


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analyzed by PCR amplification all contained the expected inserts, which displayed a good

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diversity as revealed by BstNI digestion (data not shown), and was subsequently used for

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panning.

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Biopanning and ELISA analysis. The constructed phage library containing approximately

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1012 phage particles was screened against SCWPs of A. flavus through three rounds of

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panning. To select scFv antibodies with high affinity, washing with PBST was increased five

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times in each round (10, 15, and 20 times in the first, second, and third rounds, respectively),

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while amount of antigens were fixed. Under these stringent conditions, the ratios of output

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and input phages increased steadily (Table S4), with about 102-fold increased phage recovery

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after the third round of panning compared with the first one, demonstrating an efficient

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enrichment of specific antibodies. Subsequently, the final panned library served as a valuable

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pool for further selection of highly reactive antibodies.

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To isolate soluble scFv antibodies with high affinity, 35 clones randomly selected from the

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panned library were directly used for IPTG-induced expression. The supernatants from

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bacterial cultures were analyzed by soluble ELISA. All 35 clones gave a reactive signal

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against SCWPs of A. flavus, with varied intensities (Fig. S1). Among them, 10 clones that

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exhibited absorbance values at 450 nm (OD450nm) higher than 1.8 were selected for further

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analyses (marked in Fig. S1).

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Sequence analysis of scFv antibodies. Sequencing analyses of the selected 10 clones (Fig.

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S1) showed four groups of antibodies: clones 2 and 4 contained identical sequences

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(designated as AfSA4); clones 3, 11, and 24 carried the same sequences (AfSA5); four clones,

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5, 12, 26, and 31, belonged to one group (AfSA8); and clone 35 contained a sequence that

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was different from others (AfSD10). Figure 1 illustrates the alignments of the deduced amino

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acid sequences encoded by these four groups of scFv antibody genes, which were confirmed

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by NCBI BlastN search to contain functionally rearranged chicken VH and VL regions. All 7



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four phage-derived scFv antibodies contained conserved sequences in the framework regions

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(FRs) and variable sequences in the complementary determining regions (CDRs) compared

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with the chicken germline, demonstrating that the selected antibodies were indeed derived

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from the immune response rather than the naïve antibody repertoire. Among them, AfSA4

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showed the highest sequence variation in its CDRs compared with the germline. Furthermore,

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pair-wise comparison also showed that AfSA4 had the lowest sequence similarity to any of

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the other three antibodies, whereas AfSA8 and AfSD10 displayed up to 95.1% identity (Table

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S5). In addition, amino acid variations in the four antibodies were widely distributed in all six

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CDRs (Fig. 1).

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Moreover, the lengths in the L-CDR1, L-CDR3, and H-CDR3 were clearly variable,

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especially for the AfSA4 antibody (Fig. 1). For instance, AfSA4 contained one amino acid

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more in L-CDR1 compared with AfSA5 and AfSA8 and also in L-CDR3 compared with

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AfSA8 and AfSD10; and more substantially, this antibody had five and six amino acids more

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in H-CDR3 (19 aa in total) than AfSA5 (14 aa), and AfSA8 and AfSD10 (13 aa), respectively.

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Since residues in CDRs directly interact with antigen determinants, variations in CDRs

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generally result in a conformational change responsible for the binding capacity.40–42 The

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AfSA4 antibody displayed the highest reactivity to SCWPs from A. flavus in the first

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screening by soluble ELISA (Fig. S1). To further reveal their binding activity towards A.

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flavus wh-1 and A. parasiticus isolated from a diseased peanut in China that also produced

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aflatoxins in peanut (478.31 µg/kg) and maize (43.75 µg/kg) (Table S1), soluble ELISAs with

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four scFv antibodies, AfSA4 (clone 2), AfSA5 (clone 3), AfSA8 (clone 5), and AfSD10

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(clone 35), purified from large-scale expression were carried out. The results confirmed that

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AfSA4 still had the highest reactivity to both fungi, with OD450nm values being 2.26 (A. flavus)

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and 2.19 (A. parasiticus), whereas the lowest reactive antibody was AfSA5 whose OD450nm

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values were only 1.28 (A. flavus) and 0.13 (A. parasiticus), respectively. Comparable OD450nm 8


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values for AfSA8 (1.91 and 1.66) and AfSD10 (1.93 and 1.66) were observed (Fig. S2),

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consistent with the soluble ELISA screening (Fig. S1). These results suggested that CDRs in

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the scFv AfSA4, with variation in both the sequence and length, may play an important role

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in its interaction with antigens resulting in the highest binding activity, and thus subsequent

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modeling may provide more insights into molecular structure and mechanisms associated

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with the high binding capacity.

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Structural modeling of scFv antibodies. The iterative threading assembly refinement

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(I-TASSER) server was used to generate three-dimensional (3D) atomic models of the four

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selected scFv antibodies. I-TASSER is an integrated platform for automated protein structure

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and function prediction based on the sequence-to-structure-to-function paradigm recently

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developed.36,37 Based on amino acid sequences the I-TASSER server revealed that the four

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antibodies had C-scores (confidence score)43 ranging from 1.08 to 1.35 and TM-scores

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(template modeling) from 0.86 ± 0.07 to 0.90 ± 0.06, respectively. The C-scores are usually

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in the ranges of −5 to 2 and a higher C-score indicates higher confidence,36 while a TM-score

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larger than 0.5 indicates correct topology of the model.36,43 Thus, the models built by this

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system are satisfactory for further analysis.

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The generated 3D structures of the four scFv antibodies displayed varied conformations,

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mainly in the L-CDR2 and H-CDR3 regions (Fig. 2). For instance, the distance between the

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Cα of the residue proline in L-CDR2 (Pro51) and the first glycine in H-CDR3 (Gly225) of

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the antibody AfSA4 was calculated to be 4.6 Å (Fig. 2a), substantially smaller than that in the

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antibodies AfSA5 (12.0 Å), AfSA8 (9.6 Å) and AfSD10 (10.9 Å) (Fig. 2b-d). Thus, the loops

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of L-CDR2 and H-CDR3 of AfSA4 compared with the other three antibodies were closer to

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each other, forming a unique compact motif on the surface of the antibody (Fig. 2a). The

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other three antibodies (Fig. 2b–d) had a looser structure, in which L-CDR2 and H-CDR3

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were further apart from each other. H-CDR3 of AfSA4 was especially exposed, bringing 9



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L-CDR2, H-CDR1, and H-CDR3 into a similarly high level to the surface, with H-CDR3

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being substantially prominent (Fig. 2a). For the other antibodies, H-CDR3 was located below

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either H-CDR1 in AfSA5 (Fig. 2b) or both L-CDR2 and H-CDR1 in the AfSA8 and AfSD10

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antibodies (Figs. 2c–d). The longer H-CDR3 in AfSA4 gave rise to this conformational

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change on the antibody surface. Furthermore, residues in H-CDR3 and L-FR2 of AfSA4 may

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have chemically interacted to strengthen the association between H-CDR3 with L-CDR2. As

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shown in Fig. 2a, one hydrogen bond is predicted to form between Thr42 of L-FR2 and

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Asp239 of H-CDR3, and a similar interaction is also present between Tyr45 of L-FR2 and

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Ile238 of H-CDR3. Moreover, parallel π-stacking is predicted to result from the interaction

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between the aromatic side chains of Try45 of L-FR2 and Trp232 of H-CDR3. However, no

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such interactions were present in the remaining three antibodies. H-CDR3 in an antibody has

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been shown to play a critical role in the establishment of the antigen-binding surface, broad

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neutralization, and a high binding ability.40–42 Therefore, the unique amino acid sequence in

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AfSA4 apparently contributes a proper structural conformation and thus the high binding

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capacity towards the antigens from different Aspergillus species.

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Immunofluorescence localization of scFv antibodies. The fundamental objective of this

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study was to generate antibodies specific to the mycelial surface of A. flavus for a sensitive

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immunoassay. To ascertain whether the selected scFv antibodies recognize surface antigens in

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mycelia and spores, immunofluorescence labeling was used to identify the site of antibody

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binding and to select the antibody with the best surface binding capacity. As shown in Fig. 3,

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the binding of AfSA4 localized with the brightest fluorescence intensity to the cell walls of

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mycelia (Fig. 3b) and conidiospores (Fig. 3f) of A. flavus, confirming the specificity of this

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antibody for a cell surface target. Moreover, a similar immunofluorescence labeling pattern

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was evident for A. parasiticus (Fig. 3j for mycelia and Fig. 3n for conidia). On the other hand,

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the remaining three antibodies, AfSA5, AfSA8, and AfSD10, showed no clear fluorescence

10


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signal on the cell walls of mycelia and spores of A. flavus under the same conditions (Fig. S3),

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indicating no or very weak binding to the surface. However, there was no fluorescence signal

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detected in the controls (Figs. 3d, h, l, p) by adding a non–Aspergillus-specific PIPP scFv

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antibody that is specific to human HCG.44 These results indicated that the AfSA4 is the most

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reactive antibody specific to the surface antigen that is commonly and constitutively present

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on fungal conidiospores and mycelia of both A. flavus and A. parasiticus, although the precise

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component recognized by the scFv antibody is unknown. Thus, the AfSA4 may be a proper

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candidate for further genetic manipulation for wide use in immunoassays.

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Activity analysis of AfSA4-AP fusion and its comparison with AfSA4 antibody. The

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AfSA4 scFv antibody was chosen to construct the AfSA4-AP fusion (Fig. 4a) whose soluble

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form expressed in E. coli was confirmed (Fig. 4b). A flexible 218 linker was used to link the

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scFv antibody and AP enzyme because it had been shown to promote the correct folding of

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the fusion protein for proper function.45 The purified AfSA4 antibody and AfSA4-AP fusion

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protein displayed a single band with an expected molecular size on SDS-PAGE gel. The

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activity assay of the fusion protein showed that both the AP enzyme activity and the scFv

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antibody affinity were retained in the AfSA4-AP fusion. For analyses of antigen-binding

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properties of scFv and its fusion, the SCWPs from A. flavus wh-1 and A. parasiticus were

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detected by immunoblots, and the results showed that AfSA4 and its fusion could bind to the

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same antigens in cell wall preparations of two Aspergillus fungi (Fig. 5). To compare the

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affinity of the AfSA4 antibody and its fusion, the detection limits of the two antibodies were

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measured by indirect ELISA with different fungal concentrations. As shown in Figure 6, the

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detection limits were below 10−1 µg/mL of A. flavus and A. parasiticus for both the AfSA4

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antibody and the AfSA4-AP fusion, and the latter was superior in antigen-binding capacity.

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Therefore, AfSA4-AP fusion expressed in E. coli can be better used for detecting

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aflatoxigenic Aspergillus pathogens directly and simply.

11



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To further evaluate the specificity of the AfSA4 scFv antibody and the AfSA4-AP fusion,

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six isolates of Aspergillus and nine fungal species from six other genera (Table S1) that may

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be co-present in their hosts with Aspergillus species46 were used for indirect ELISA analysis.

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The results indicated that both the AfSA4 antibody and its fusion had a high affinity for

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Aspergillus, especially for A. flavus and A. parasiticus, but did not cross-react with other

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fungal species (Table S1). These results congruously suggested that the AfSA4 antibody is

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Aspergillus genus specific, and the AFSA4-AP fusion had antigen-binding characteristics

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similar to its parental scFv antibody and was able to specifically detect Aspergillus among

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mixtures of different fungal pathogens.

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Binding kinetics of AfSA4 and AfSA4-AP fusion. To assess the ability of the AfSA4

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antibody and its fusion to bind to the SCWPs of A. flavus, binding kinetics were determined

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using SPR measurements. As shown in Table 1, both the AfSA4 antibody and the AfSA4-AP

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fusion displayed a high affinity for SCWPs of A. flavus. The equilibrium association constant

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(KA) of AfSA4-AP fusion (KA ~ 1.20 × 108) was approximately six-fold higher than that of

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the AfSA4 antibody (KA ~ 2.18 × 107), owing to the increase of the kinetic association rate of

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the fusion. SPR sensorgrams and kinetic constant determination are presented in Fig. S4. AP

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is known to spontaneously dimerize in solution, imparting avidity to the fused scFv antibody

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partner and resulting in effective binding to antigen.47,48 Thus, the dimerization of the

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AfSA4-AP fusion apparently contributed the higher binding affinity.

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Ds-ELISA for rapid detection of Aspergillus contamination in field and storage. A

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sandwich ELISA procedure requires testing match pair antibodies, capture and detection

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antibodies that detect different epitopes on the target antigen so that they do not interfere with

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the other antibody binding. Here ds-ELISA refers to direct use of the AfSA4-AP fusion for

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two functions, detection and enzymatic reaction, thus omitting the secondary antibody

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conjugated with enzyme. To optimize ds-ELISA detection through the AfSA4-AP fusion, 12


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298

three different capture antibodies, the scFv antibody AfSA4 used for construction of the

299

AfSA4-AP, a chicken polyclonal pAIgY antiserum and a mouse monoclonal antibody

300

mAb2A8 derived from a hybridoma cell line generated in parallel (See Supporting

301

Information), were compared for their applicability and sensitivity. The results showed that

302

the detection limit for the AfSA4 and pAIgY was approximately 1 µg/mL, the same

303

magnitude as reported by others,17 whereas the limit for the mAb2A8 was as low as 10−3

304

µg/mL, with 1000 times more sensitivity (Fig. 7; Fig. S5). The lower sensitivity for the

305

pAIgY may result from a common epitope shared by the pAIgY and AfSA4 antibodies

306

because they were derived from the same immunized chickens, or lower capture capability

307

due to the heterogeneity of the polyclonal pAIgY. A similarly lower sensitivity for the AfSA4

308

was likely due to a competitive binding to a common epitope by the capture antibody AfSA4

309

and the detection antibody motif in the AfSA4-AP. Under the mAb2A8 condition, a good

310

correlation was observed between logarithmic concentration of mycelia and OD405nm values

311

(R2 = 0.9992 for both fungi). Therefore, the mAb2A8 is an ideal capture antibody, and the

312

matched pair mAb2A8 and AfSA4-AP fusion are an optimized combination for ds-ELISA

313

detection of Aspergillus pathogens.

314

To determine the limit of quantification, different concentrations of mycelia from A.

315

flavus wh-1 and A. parasiticus in PBS (10−4, 10−3, 10−2, 10−1, 1, 10, 102, 103 µg/mL) or mixed

316

with milled maize or peanut grains (1 mg/mL) were prepared, and the extracts were assayed

317

by the optimized ds-ELISA (See Supporting Information). Mycelia from a non-Aspergillus

318

species, Fusarium verticillioides (Table S1), were similarly mixed with milled maize or

319

peanut grains and served as negative controls. Figure 8 shows that the scFv-AP fusion was

320

able to detect approximately 10−3 µg/mg (10−3 µg/mL) mycelium of two fungi in milled

321

maize or peanut (i.e. 1 µg/g), which was identical to the detection limit in PBS (Fig. 7) but

322

with a lower and slower color development. No reactive signals were detected in negative

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

323

controls. This detection limit is 10 times more sensitive than that of a recent report (10 µg/g

324

in rice).19 The OD405nm values were plotted against the logarithmic concentration of mycelia

325

with a fitting curve by nonlinear regression (R2 > 0.99). R2 values served as measures of

326

goodness of fit. They were similar to other studies,29,30 and therefore, the fungal biomass in

327

contaminated food or feed can be assessed conveniently by ds-ELISA using the AfSA4-AP

328

fusion.

329

To study the feasibility of AfSA4-AP fusion combined with mAb2A8 for rapid

330

immunological detection, ds-ELISA was used to detect 20 naturally Aspergillus contaminated

331

maize and peanut samples collected from different regions and four artificially inoculated

332

samples (maize and peanut were inoculated with A. flavus wh-1 and A. parasiticus,

333

respectively; Table S1). All of the samples (1 mg/mL in PBS) were heat treated (either boiled

334

or roasted) simultaneously before analysis. The results showed that the AfSA4-AP fusion was

335

able to effectively detect the samples prepared by either method without any false-positive

336

signals in blank samples (Tables S2 and S3), confirming that the Aspergillus antigen

337

recognized by the AfSA4 antibody is heat stable. Therefore, the AfSA4-based assay can

338

specifically monitor Aspergillus-contaminated samples in nature.

339

CONCLUSIONS

340

In this study, we isolated and cloned the gene for a high-affinity Aspergillus-specific

341

scFv antibody from an immune-challenged chicken antibody library by phage display, and

342

developed a sensitive direct sandwich immunoassay for aflatoxigenic Aspergillus pathogens.

343

This scFv antibody specifically recognizes a surface antigen within SCWPs that has been

344

proven to be (i) common to A. flavus and A. parasiticus, (ii) constitutively present in their

345

conidiospores and mycelia, and (iii) structurally stable in culture medium, during the fungal

346

colonization of different crops in field and during storage, and under boiled and roasted

347

conditions. Such evidence provides a solid foundation for developing an immunoassay with 14


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348

high reproducibility, sensitivity, and specificity. The scFv antibody fused to AP had an even

349

higher affinity and binding capacity and was able to detect a fungal concentration of 1 µg/g in

350

maize or peanut grains, a level of specificity not possible with conventional methods of

351

immunoassay reported thus far. To our knowledge, this is the first example of the

352

development of an scFv-AP fusion for detection of Aspergillus pathogens. This assay could

353

provide a rapid, specific, and convenient method for routine analysis of food/feed

354

commodities and disease assessment and for patients with potential aflatoxigenesis. Whether

355

the surface antigen recognized by the scFv antibody is conserved across all Aspergillus

356

species including non-pathogenic and non-aflatoxigenic strains remains to be investigated.

357

The isolated scFv antibody could also be used for further investigation of the fungal antigen

358

and its role in pathogenesis and aflatoxigenesis.

359

Supporting Information Available

360

ACKNOWLEDGMENTS

361

We thank Dr. Carlos F. Barbas III (The Scripps Research Institute, La Jolla, CA, USA) for

362

providing pComb3X. This research was funded by grants from the National Basic Research

363

Program of China (2013CB127801) and the National Natural Science Foundation of China

364

(31272004).

365

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366

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453

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454 455 456 457 458 459 460

Legends of Tables and Figures

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

461

Table 1. The kinetic parameters of AfSA4 scFv antibody and AfSA4-AP fusion determined

462

by SPR measurements.

463

Fig. 1. Alignment of amino acid sequences from four isolated scFv antibodies and chicken

464

germline. Amino acid sequences of light chain (VL) and heavy chain (VH) are compared with

465

that of chicken germline. FR and CDR regions are indicated above the germline sequence.

466

Sequence gaps are indicated by lines. Residues different from germline are shaded.

467

Fig. 2. Three-dimensional structures of four isolated scFv antibodies. (a) Structure model

468

of AfSA4 and close-up views of the L-FR2 and H-CDR3 interactions. Hydrogen bonds are

469

indicated as red lines and a parallel π-stacking interaction is present between Try45 of L-FR2

470

and Trp232 of H-CDR3 (enlarged portion). (b, c and d) Structure models of AfSA5, AfSA8,

471

and AfSD10. Structure models were generated by I-TASSER server. Frameworks and linkers

472

are indicated in green. Different colors for CDRs: L-CDR1 (purple blue), L-CDR2 (red),

473

L-CDR3 (orange), H-CDR1 (purple), H-CDR2 (sky blue), and H-CDR3 (blue). Dashed lines

474

and numbers either above or below the lines indicate the measured distances (Å) between

475

residues. The figures were generated by PyMOL software (version 1.3, Schrödinger, LLC).

476

Fig. 3. Immunofluorescence labeling of mycelia and conidia of A. flavus and A. parasiticus

477

with scFv antibodies. (a–d) Mycelia of A. flavus and (i–l) mycelia of A. parasiticus were

478

exposed to the bacterially expressed AfSA4 (a, b, i, j). (e–h) conidia of A. flavus and (m–p)

479

conidia of A. parasiticus were exposed to the AfSA4 (e, f, m, n). All mycelia and conidia

480

were also exposed to a nonspecific scFv antibody PIPP as negative controls (c, d, g, h, k, l, o,

481

p). These were followed by the addition of mouse anti-HA tag antibody and Cy3-conjugated

482

goat anti-mouse antibody. Bright-field photographs (a, e, i, m, c, g, k, o) are on the left and

483

the third column while fluorescence photographs (b, f, j, n, d, h, l, p) are on the second

484

column and the right. Bar, 20 µm.

20


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485

Fig. 4. Construct structures and SDS-PAGE of AfSA4 antibody and AfSA4-AP fusion. (a)

486

Construct structure of AfSA4 antibody (i), and AfSA4 fusion to alkaline phosphatase (AP)

487

with 218 linker (ii). (b) Image of a Coomassie brilliant blue–stained SDS-PAGE gel of

488

purified AfSA4 antibody and AfSA4-AP fusion. M, protein molecular weight standards. Lane

489

1, AfSA4 antibody. Lane 2, scFv-AP fusion.

490

Fig. 5. Immunoblots of SCWPs from A. flavus and A. parasiticus detected with AfSA4

491

antibody and AfSA4-AP fusion. SCWPs were loaded on a 12% SDS-PAGE gel, separated,

492

and blotted onto nitrocellulose membranes. After blocking, the membranes were incubated

493

with 50 nM AfSA4 (a) followed by addition of mouse anti-HA antibody and AP-labeled goat

494

anti-mouse IgG antibody, or directly detected with the same concentration of AfSA4-AP

495

fusion (b). M, protein molecular weight standards.

496

Fig. 6. Sensitivity and detection limits of AfSA4 antibody and AfSA4-AP fusion by

497

indirect ELISA with different concentrations of A. flavus and A. parasiticus. An aliquot of

498

100 µL A. flavus (●, ○) or A. parasiticus (▲, ∆) was ground and diluted in PBS at indicated

499

concentrations and added into plate wells for detection with 200 nM purified AfSA4 antibody

500

(○, ∆) and AfSA4-AP fusion (●, ▲). Values represent mean ± SD of triplicate assays.

501

Fig. 7. Sensitivity and detection limits of mAb2A8 and pAIgY as capture antibodies and

502

AfSA4-AP fusion as detection antibody in ds-ELISA with different concentrations of A.

503

flavus and A. parasiticus. A 100-µL aliquot of mAb2A8 (solid line) or pAIgY (dashed line)

504

was added into plate wells, followed by addition of 100 µL of A. flavus (●, ○) or A.

505

parasiticus (▲, ∆) that was ground and diluted in PBS at indicated concentrations, and then

506

100 µL of AfSA4-AP fusion (200 nM) were added for detection. Values represent mean ± SD

507

of triplicate assays.

508

Fig. 8. The limits of quantifications of A. flavus and A. parasiticus in maize and peanut

21



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Page 22 of 32

509

grains determined by ds-ELISA with AfSA4-AP fusion. An 100-µL aliquot mAb2A8 was

510

added into plate wells, followed by addition of 100-µL mixtures of maize–A. flavus (solid line,

511

●), maize–A. parasiticus (solid line, ▲), peanut–A. flavus (dashed line, ○), and peanut–A.

512

parasiticus (dashed line, ∆) at indicated concentrations. Then 100 µL of AfSA4-AP fusion

513

(200 nM) were added to each well for detection. Values represent mean ± SD of triplicate

514

assays. Maize–F. verticillioides (solid line, ■) and peanut–F. verticillioides (dashed line, □)

515

were served as negative controls.

516 517 518 519 520 521 522 523 524 525 526 527 528 529

Table 1 Antibody AfSA4

ka (M−1 s−1) 1.96 × 104

kd (s−1) 9.0 × 10−4 22


KA (M−1) 2.18 × 107

Page 23 of 32

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AfSA4–AP

2.04 × 105

1.7 × 10−3

1.20 × 108

530

ka, kinetic association rate; kd, kinetic dissociation rate; KA, equilibrium association constant

531

(KA=ka/kd).

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

560

Figure 1

561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576

24


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


577 578

Figure 2

579 580 581 582 583 584 585 586 587 588

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

589 590 591 592 593 594 595 596 597 598

Figure 3

599 600 601 602 603 604 605 606 607 608 609 26


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


610 611 612 613 614 615 616

Figure 4

617 618 619 620 621 622 623 624 625 626 627 628 629 630 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

631 632 633 634 635 636 637 638

Figure 5

639 640 641 642 643 644 645 646 647 648 649 650 651 28


Page 28 of 32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


652 653 654 655 656 657 658 659 660

Figure 6

661 662 663 664 665 666 667 668 669 670 671 672 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

673 674 675 676 677 678 679 680 681

Figure 7

682 683 684 685 686 687 688 689 690 691 692 693 30


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


694 695 696 697 698 699 700 701 702

Figure 8

703 704 705 706 707 708 709 710 711 712 713 714 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

715 716

For TOC only

717 718 719 720 721 722

TOC graphic

723

A mouse monoclonal antibody, mAb2A8, and a chicken scFv antibody, AfSA4, recognize

724

different surface antigens or epitopes present in A. flavus and A. parasiticus. The mAb2A8 as

725

capture antibody and the scFv AfSA4 fused to alkaline phosphatase (AP) as detection

726

antibody and catalytic enzyme are an ideal combination for detecting aflatoxigenic

727

Aspergillus pathogens in maize and peanut grains.

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Chicken Single-Chain Antibody Fused to Alkaline Phosphatase

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