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Food and Agricultural Immunology

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Development of a noncompetitive idiometricnanobodies phage immumoassay for thedetermination of fumonisin B1

Mei Shu, Yang Xu, Jie-xian Dong, Chan Zhong, Bruce D. Hammock, Wen-junWang & Guo-ping Wu

To cite this article: Mei Shu, Yang Xu, Jie-xian Dong, Chan Zhong, Bruce D. Hammock, Wen-jun Wang & Guo-ping Wu (2019) Development of a noncompetitive idiometric nanobodies phageimmumoassay for the determination of fumonisin B1, Food and Agricultural Immunology, 30:1,510-521, DOI: 10.1080/09540105.2019.1604637

To link to this article: https://doi.org/10.1080/09540105.2019.1604637

© 2019 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup

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Development of a noncompetitive idiometric nanobodiesphage immumoassay for the determination of fumonisin B1Mei Shua, Yang Xub, Jie-xian Dongc, Chan Zhonga, Bruce D. Hammockd,Wen-jun Wanga and Guo-ping Wua

aCollege of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, People’s Republic ofChina; bState Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, People’sRepublic of China; cDepartments of Neurobiology, Physiology and Behavior, and of Physiology andMembrane Biology, University of California, Davis, CA, USA; dDepartment of Entomology and UCDComprehensive Cancer Center, University of California, Davis, CA, USA

ABSTRACTHere we demonstrate a noncompetitive idiometric nanobodiesphage immunoassay for the determination of FB1, utilizing twotypes of anti-idiotypic nanobody, that is specific for differentepitopes within the hypervariable region of the primary mAb. Threealphatype anti-idiotypic nanobodies were obtained from a naivenanobodies phage-display library. A noncompetitive idiometricimmunoassay was established with a combination of betatype anti-idiotypic nanobody and phage-displayed alphatype anti-idiotypicnanobody. The half-maximal saturation of the signal value (SC50)was 0.68 ng/mL, with the limit of detection (LOD) was 0.19 ng/mL.When the noncompetitive assay was compared to the competitiveELISA (LOD = 3.41 ng/mL), an over 17-fold improvement insensitivity was observed. A correlation (R2) was 0.988 between thedata of the noncompetitive assay and LC–MS/MS for thedetermination of FB1 in cereal samples. The noncompetitiveidiometric immunoassay would have a broad utility for monitoringsmall molecules in food and environment.

ARTICLE HISTORYReceived 21 February 2019Accepted 3 April 2019

KEYWORDSFumonisin B1; betatype anti-idiotypic antibody; alphatypeanti-idiotypic antibody;nanobody; thenoncompetitive idiometricimmunoassay

1. Introduction

Immunoassays can be classified as either competitive, in which the measured signal isassociated with a limited amount of antibodies that failed to capture the analyte; or

© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis GroupThis is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in anymedium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

CONTACT Wen-jun Wang wwjun9999@sina.com College of Food Science and Engineering, Jiangxi AgriculturalUniversity, Nanchang 330045, Jiangxi, People’s Republic of China; Guo-ping Wu jdwgp@163.com College of FoodScience and Engineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, People’s Republic of China

Supplemental data for this article can be accessed at https://doi.org/10.1080/09540105.2019.1604637.

FOOD AND AGRICULTURAL IMMUNOLOGY2019, VOL. 30, NO. 1, 510–521https://doi.org/10.1080/09540105.2019.1604637

noncompetitive, in which the measurement of the signal is associated with antibody thatbinds the analyte (Li, Wu, Song, Zheng, & Kuang, 2019; Suryoprabowo et al., 2019).Theoretical study has showed that noncompetitive immunoassays are good sensitivity,precision, a wider working range, and comparability with competitive immunoassays(Gujral, Yoo, Bamdad, Suh, & Sunwoo, 2017). Thus, researchers have sought to developnoncompetitive immunoassays for the determination of small molecules. Despite thesepotential advantages, the noncompetitive immunoassay system is unsuitable for measur-ing small molecules with a single antigenic determinant (epitope) called haptens, such astoxins, vitamins, drugs, eicosanoids, and industrial chemicals (Akter, Vehniäinen, Kan-kaanpää, & Lamminmäki, 2017; Li et al., 2018). Several attempts have been made to over-come this difficulty, and several novel immunoassays that can noncompetitively detecthaptens have been developed. These methodologies are based on chemical modificationto the analytes (Carlomagno et al., 2014; Haasnoot & Verheijen, 2001), idiometricassays with anti-idiotype antibodies (Niwa et al., 2009), the anti-metatype antibody(Omi et al., 2015; Tsutsumi, Nagata, Yoshida, Harada, & Ueno, 2000), the split recombi-nant variable region fragments of the antibody (Chung, Makino, Dong, & Ueda, 2015) orother principles (Saha, Roy, & Dhar, 2013). Although these approaches have been success-fully used to measure several haptens, preparing the reagents and these unconventionalantibodies is complicated and time consuming. These drawbacks have hindered the popu-larization of using these methods to detect other haptens.

With the development of phage-displayed technology, more researchers have becomeinterested in establishing the noncompetitive phage anti-immune complex assay(PHAIA) (Dong et al., 2014; González-Techera, Vanrell, Last, Hammock, & González-Sapienza, 2007). The peptide has been used in these formats. However, peptide with onlyseveral amino acids must be used to attach to the phage particles or fusion proteins.Recent success in the generation of nanobodies prompted our interest in developing nano-bodies-based immunoassay. Thenanobody is the variable domain of a heavy-chain antibody(VHH) derived from the variable region of the heavy-chain antibody existing in camelids(Hamerscasterman, 1993). Nanobodies exhibit many unique antibody features, such as asmallmolecular weight (15 kDa), high expression in various expression levels, thermostabil-ity, high solubility, and easy genetic manipulation (del Rio et al., 2019; Tu et al., 2012). Theanti-idiotypic nanobody, with these advantages, has been developed for diagnostic, thera-peutic, food safety and environmental immunoassays (Qiu et al., 2018). Themajor differencebetween the peptide and nanobody is related to the affinities that can be achieved, being thenanobody more complex tridimensional structures that can achieve higher affinity.

So we established a noncompetitive idiometric nanobodies phage immunoassay usingtwo types of anti-idiotypic antibody (Ab2) to explore the possibility of expanding thescope of the noncompetitive immunoassay to small molecules. Ab2 is the second antibodythat recognizes different epitopes within the hypervariable region of the primary antibody(Jerne, 1974). Alphatype Ab2 (Ab2α) and betatype Ab2 (Ab2β) are the two main types ofanti-idiotypic antibodie (Lan et al., 2017; Liu et al., 2018). Ab2α recognizes an epitope,which is not part of the binding site (paratope) of the primary antibody, involved inantigen recognition.Meanwhile, Ab2β competes with the analyte for an epitope at the para-tope. Ab2α will bind to the primary antibody in the presence of or absence the analyte andwill not capture onto the Ab2β/primary antibody complex because of steric hindrance,whereas the Ab2β will not bind to the primary antibody in the presence of the analyte.

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In this technique, a noncompetitive idiometric nanobodies phage immunoassay systemsuitable for the measurement of Fuminosin B1 has been generated. The assay was per-formed using these matched antibodies, namely, the primary antibody and two specifictypes of anti-idiotypic nanobodies (the fusion protein of the Ab2β nanobody and thephage of the Ab2α nanobody). The noncompetitive idiometric nanobodies phage immu-noassay is appropriate for determining small molecules in samples.

2. Materials and methods

2.1. Chemicals and reagents

PfuTurbo Cx Hotstart DNA polymerase was purchased from Agilent Technologies Inc.(Santa Clara, CA). Enzymes and M13KO7 helper phage were purchased from NewEngland Biolabs, Inc. (Beverly, MA). Mycotoxin standards, bovine serum albumin(BSA) and TMB liquid-1 component substate were obtained from Sigma–Aldrich(St. Louis, MO). B-PER bacterial protein extraction reagent, HisPur Ni–NTA resin, andSYPRO Ruby protein gel stain were purchased from Thermo Fisher Scientific Inc.(Waltham, MA). HRP conjugated anti–M13 monoclonal antibody was abtained formGE Healthcare (Piscataway, NJ). Primers A–F (5′–CAT GCC ATG ACT GTG GCCCAG CCG GCC CAG KTG CAG CTC GTG GAG TCN GGN GG), A–R1/R2 (A–R1:5′–CAT GCC ATG ACT CGC GGC CCC CGA GGC CTC GTG GGG GTC TTC GCTGTG GTG CG; A–R2: 5′–CAT GCC ATG ACT CGC GGC CCC CGA GGC CTGGCC TTG TTT TGG TGT CTT GGG) were synthesized from Integrated DNA Technol-ogies (Division of Biological Sciences, Automated DNA Sequencing Facility, University ofCalifornia, Davis, CA, USA). The monoclonal antibodies (anti-FB1 mAb 3F11, anti-FB1mAb 1D11, anti-ZEN mAb, anti-DON mAb) and Ab2β nanobody (B26) were stored inDr. Yang Xu’s laboratory. The pComb3x phagemid vector was kindly provided by Dr.Carlos Barbas III (Scripps Research Institute, La Jolla, CA).

2.2. Naive nanobodies phage-displayed library construction

Construction of a naive nanobodies phage-displayed library was described as Faraji (Farajiet al., 2018). In brief, total RNA was extracted using a total RNA isolation system (ThermoFisher Scientific, Waltham, MA, USA) from llama peripheral blood lymphocytes. It wasused to synthesize the first strand of cDNA and was then amplified using VHH IgGspecific primers (Primers A–F and A–R1/R2). The DNA fragments were prepared bythe digestion using SfiI and subsequently ligated into the digested pComb3x constructs.It was transfected into electrocompetent ER2738 cells. The library size was titered byplating on Luria Bertani-ampicillin plates.

2.3. Library panning

For the sceening procedure, two wells of a microtiter were immobilized with purified anti-FB1 mAb at 50, 10, 5, and 1 μg per well for rounds 1, 2, 3, and 4, respectively, and were leftovernight at 4°C. Four additional microwells coated with 100 μL of 3 mg/mL BSA–PBSwere used for pre-absorption and post-absorption. After coating, wells were blocked by

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incubation with 300 μL of 5 mg/mL BSA–PBS at room temperature for 1 h. After washingfive times with PBST (0.01 mol/L PBS pH 7.4 containing 0.05% (v/v) Tween-20), thediluted phage library (2 × 1012 pfu/mL, 100 µL) was dispensed into the pre-absorptionwell. Meanwhile, the anti-FB1 mAb coated well had been incubated with the concentrationof FB1 standard at 0, 10, 25, and 50 ng/mL for rounds 1, 2, 3, and 4, respectively. Afterincubating for 1 h at room temperature, the unbound phage was added to the anti-FB1mAb well and incubated as described before. Wells were washed 10 times with PBST,then the bound phage was eluted with 100 μL of 0.1 mol/L glycine–HCl (pH 2.2) for10 min and neutralized by the addition of 15 μL of 1 mol/L Tris–HCl (pH 8.5). At last,the elution was transferred into the post-absorption plate and incubated at room tempera-ture for 1 h. Individual clones were picked randomly and tested using phage ELISA afterfour rounds of panning-elution selection.

2.4. Preparation of purified of phage Ab2α suspensions

Phage Ab2α clones were selected and individually amplified according to previously report(Barbas, Burton, Scott, & Silverman, 2001). After two times of precipitation with PEG–NaCl (20% (w/v) PEG8000/2.5 mol/L NaCl), the phages were suspended with PBS(0.01M, pH7.4) for storage at 4°C.

2.5. Phage ELISA screening

In particular, the Ab2α that is selected in the assay will not bind to the primary antibody-Ab2β complex due to epitope proximity. In order to choose the Ab2α nanobody, a idiometricphage ELISA that based on the use of three matched antibodies, the primary antibody, Ab2αand Ab2β antibody (B26) was set up according to the principle shown in Figure 1. The anti-

Figure 1. Schematic diagram of the noncompetitive idiometric nanobodies phage ELISA.

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FB1 mAb or BSA (5 µg/mL) in 0.05 M carbonate buffer (pH 9.0, 150 µL) was placed inmicrotiter wells, and left overnight at 4°C. After washing five times with PBST, the wellswere blocked for 1 h at room temperature with 3% (w/v) milk–PBS, then washed threetimes with PBST. PBS containing a solution of B26 (10 μg/mL, 50 µL) which was expressedusing a bacterial system (Shu et al., 2015), with FB1 (10 ng/mL, 50 µL) or without, and theculture of individual amplified phage clones (50 µL), were added to the wells and mixed.These mixtures were incubated for 1 h at room temperature. After washing ten times asdescribed above, Horseradish peroxidase (HRP) conjugated anti–M13 antibody (diluted1:5000 in PBS) (100 μL) was added, and incubated at room temperature for another hour.The bound peroxidase activity was measured colorimetrically using a substrate solution(100 μL/well). After incubating at room temperature for 10 min, the enzymatic reactionwas stopped with 2 mol/L H2SO4 (50 μL/well). The absorbance at 450 nm was measuredwith a microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA).

2.6. A noncompetitive idiometric nanobodies phage ELISA

To select a suitable combination of Ab2α and B26, the concentrations of coating anti-FB1mAb, B26 and serial dilutions of each phage Ab2α particles in the absence or presence ofFB1 were determined in checkerboard experiments. A noncompetitive idiometric nanobo-dies phage ELISA was set up, which use three matched antibodies, namely, anti-FB1 mAb,phage Ab2α, and B26. The microwells were coated with the anti-FB1 mAb (300 ng) andblocked as described above. After washing five times with PBST (10 mM, pH7.4 PBS +0.05% Tween 20), the following three types of solution were added and mixed: (1) aseries of FB1 standard solutions (or extracted samples) (50 μL), (2) a solution containingB26 (50 μL), and (3) a solution containing the Ab2α phage (50 μL). The wells were incu-bated for 1 h at room temperature, and washed ten times with PBST. Then a 1:5000dilution of HRP conjugated anti-M13 antibody was added and incubated at room temp-erature for another hour. The bound peroxidase activity was measured as described before.

2.7. Cross-reactivity evaluation for the noncompetitive idiometric nanobodiesphage ELISA

The cross reactivities of the primary mAb (3F11) for a group of mycotoxins (FB2, FB3,AFB1, OTA, and ZEN) in the range of 0.1–1000 ng/mL and different primary monoclonalantibodies were determined using the noncompetitive idiometric nanobodies phageELISA. The cross-reactivity (%CR) was calculated: SC50 (FB1)/SC50 (cross-reacting com-pound) × 100%.

2.8. Sample analysis and validation

The specificity of the noncompetitive immunoassay performance was evaluated by spiking10–1000 μg/kg concentrations of FB1 to corn blank sample extraction. Incurred sampleswere prepared as our previous report (Shu et al., 2016). The supernatant was loadedonto the strong anion exchange–solid phase extraction (SPE) columns (Agilent Technol-ogies, Palo Alto, CA, USA) preconditioned with 5 mL of 75% methanol in water (v/v). Upto 2 mL of the sample solution (acetic acid/methanol, 10:90, v/v) was added to the column

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and evaporated to dryness under a stream of nitrogen. Thereafter, the dry residue was dis-solved in 2 mL of 5% methanol, 10 mM PBS (pH 7.4) for the noncompetitive idiometricnanobodies phage ELISA and LC–MS/MS analysis.

3. Result and discussion

3.1. Panning and sequence alignment of Ab2α nanobody

A naive nanobodies phage-displayed library with an estimated size of 4.2 × 108 Colony-Forming Units (CFU) was constructed on the pComb3X vector. In order to obtain strongAb2α phage clones, four cycles of panning were conducted with increasing concentrationsof the FB1 standard for binding in each round. In addition, vigorous washing steps and pre/post-absorption were used to minimize no or infirm binding. During four rounds of biopan-ning, the eluted phages were increased from 1.5 × 105–4.6 × 108 /mL phage particles (datanot shown), indicating that phages were enriched during each panning. Using a idiometricphage ELISA, up to 90 clones were selected from the third and fourth round and screenedfor binding to the coating anti-FB1 mAb in the presence or absence of FB1. Fifteen out of 90clones showed negligible binding to the anti-FB1 mAb in the absence of FB1, although theybinded to the coating antibody in the existence of 50 ng/mL FB1 (Figure 2(a)).

The plasmids of the positive clones were isolated and sequenced. The amino acidsequences were aligned using the software of BioEdit (Carlsbad Biosciences, CA, USA).Analysis of the nanobody sequences revealed three unique sequences were obtained.Among them, T8 contains the possession of amino acid residue F(Phe), E(Glu), R(Arg),and F in the FR2 and 21 amino acids in CDR3 regions, which are the primary featuresof heavy-chain antibodies. T3 and T9 possess G and P instead of E and F in FR2 and arelatively short CDR3 region (13 amino acids) (Figure S1).

3.2. Ab2α and Ab2β nanobodies based noncompetitive assay

To select a suitable combination of Ab2α and Ab2β, three dilutions of phage particles (3 ×1011, 3 × 1010, 3 × 109 pfu/mL) were added to the wells of the plate coated with four differentconcentrations of coating anti-FB1 mAb (10, 5, 2.5, and 1.25 µg/mL), Ab2β nanobody (20,10, 5, 0 µg/mL) in the absence or presence of FB1 (0, 0.1, 0.5, 1, 5,10, 20 ng/mL). Figure S2shows the result with clone T8. Similar results were evaluated for the other two clones (datanot shown). The maximal intensity of spots difference was obtained at 5 µg/mL coating anti-FB1 mAb with 3 × 1010 pfu/mL phage particles and 10 µg/mL B26. With a higer quantity ofphage Ab2α nanobody, the intensity of spots was observed increasing background absor-bance value. The intensity of spots difference gradually decreased with decreasing the con-centrations of coating anti-FB1 mAb and increased with decreasing the concentrations ofB26. It is insensitive for the phage Ab2α nanobody without adding B26.

Based on the results described above, the assay sensitivities for each of the three cloneswere estimated. As shown in Figure 2(b), the 50% of the maximal signal (SC50) were 1.37± 0.61, 0.68 ± 0.03 and 1.47 ± 0.67 ng/mL for Ab2α T3/B26, Ab2α T8/B26 and Ab2α T9/B26, respectively. Among them, Ab2α T8/Ab2β that provided low background signals,which correspond to the absorbance value at the zero analyte concentration of FB1,were selected to set up the noncompetitive idiometric nanobodies phage immunoassay.

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The noncompetitive assay exhibited the lowest the limit of detection (LOD) of 0.19 ng/mLand the linear range was 0.31–1.74 ng/mL. It presented a 17-fold improvement in the non-competitve assay sensitivity as compared to the LOD of 3.41 ng/mL on the competiviteELISA that set up with the same primary mAb.

3.3. Cross-reactivity

The selectivity of the noncompetitive idiometric phage ELISA was determined by per-forming cross-reactivity analyses with other mycotoxins and different primary

Figure 2. (a) Phage ELISA results with several single clones towards 3F11 with 50 ng/mL FB1 (redcolumn), without FB1 (black column) or BSA (slash column). (b) Noncompetitive idiometric nanobodiesphage ELISA for FB1 set up with different phage Ab2α. Data are reported as an average ± standarderror.

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antibodies. As shown in Figure 3(a), 6.89% and 2.93% cross-reacivity was observed forFB2 and FB3, respectively, but no cross-reactivity (<0.01%) with other compounds(AFB1, ZEN and OTA). And for other primiary monoclonal antibodies, the cross-reac-tivity was negligible (Figure 3(b)). Its specificity should be mostly dependent on therecognition properties of the primary antibody and B26. The results suggesting thatwhen the same primary antibody is used the noncompetitve assay allows us toacquire not only a significant improvement in assay sensitivity but also acceptablespecificity.

Figure 3. (a) Cross-reactivity of the noncompetitive idiometric nanobodies phage ELISA with othermycotoxins. (b) Cross-reactivity of the noncompetitive idiometric nanobodies phage ELISA withprimary antibodies and mycotoxins.

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3.4. Matrix effect

Matrix effects can substantially interfere with enzyme activity, binding between antibodyand analyte on ELISAs. For the purpose of minimizing matrix effects, it seems that it isessential to use solid-phase extraction (SPE) as sample pre-treatment method. The dilutionof sample extracts with PBS by 5, 10, and 20 times was examined subsequently to evaluatethe dilution effect. All dilution times showed a similar behaviour, with SC50 values 0.96 ng/mL (1:1), 0.82 ng/mL (1:5), 0.71 ng/mL (1:10), and 0.81 ng/mL (1:20), compared with0.66 ng/mL in PBS (Figure 4). These results indicate that the matrix effects of these cornsamples were completely eliminated. using SPE as a cleanup step before assay. Thus farin order to minimize the matrix effects, the extracts were diluted 10-fold with PBS.

The accuracy and reproducibility of the noncompetitive idiometric nanobodies phageELISA format were conducted by evaluating the spiked corn samples with variousamounts (10–1000 μg/kg) of FB1. Satisfactory recovery rate (mean 74.10%–110.92%)with coefficient variation (mean CV 2.79%–10.95%) for within-assay evaluation wereobtained. Meanwhile, the recovery rate estimated for between-assay evaluation was71.60%–115.34%, with the CV level ranging from 3.17%–11.35% (Table 1). Theseresults suggested the accuracy and reproducibility of corn samples were achieved usingthe noncompetitive idiometric nanobodies phage ELISA.

Furthermore, we attempted to examine FB1 levels in 25 naturally contaminated cerealsamples using the noncompetitive idiometric nanobodies phage ELISA and LC–MS/MS.Results are shown that among the 25 samples, corn and wheat samples were FB1 positive,which three corn samples surpassed the maximum limit for FB1 in cereal (1000 μg/kg),and five rice samples were all FB1 negative (Table 2). The correlation (R2) was 0.988between the two methods (Figure S3). It should be noted that the noncompetitive

Figure 4. Calibration curves of FB1 in PBS and in sample extracts of different dilutions with or withoutsolid phase extraction.

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idiometric nanobodies phage ELISA based on two types of anti-idiotypic nanobody is anacceptable method to detect FB1 in cereal samples.

4. Conclusions

The current work described a sensitive and specific noncompetitive idiometric nanobodiesphage immunoassay based on two types of anti-idiotypic nanobodies for the quantitativedetermination of FB1. The SC50 of the noncompetitive nanobodies phage ELISA was

Table 1. Recoveries of FB1 from the spiked corn samples by the noncompetitive idiometric nanobodiesphage ELISA.FB1 added (µg/kg) ELISA (µg/kg) Average recovery (%) CV (%)

10 Within assay (n = 3)a 7.41 ± 0.73 74.10 9.8550 43.63 ± 3.79 87.26 8.69100 92.68 ± 10.15 92.68 10.95500 530.43 ± 26.73 106.09 5.041000 1084.78 ± 30.29 110.92 2.7910 Between assay (n = 5)b 7.16 ± 0.91 71.60 12.7150 41.58 ± 4.40 83.16 10.58100 90.59 ± 10.28 90.59 11.35500 484.50 ± 29.65 96.90 6.121000 1153.42 ± 36.54 115.34 3.17aEach assay was conducted in triplicates on the same day.bEach assay was performed on five different days.

Table 2. Analysis results of the cereal samples by the noncompetitiveidiometric nanobodies phage ELISA and LC–MS/MS.

Samples NumberELISAa (n = 3)b

(µg/kg)LC–MS/MS(µg/kg)

C1 175.5 ± 3.5 163.9 ± 0.32C2 452.1 ± 6.9 441.1 ± 0.58C3 1500.6 ± 10.6 1224.8 ± 1.43C4 6.6 ± 0.3 NDc

Corn C5 56.2 ± 4.5 65.7 ± 0.21C6 2.31 ± 0.8 NDC7 440.4 ± 3.9 538.2 ± 0.52C8 2380.5 ± 19.3 2235.7 ± 2.68C9 8.4 ± 0.3 12.8 ± 0.08C10 973.3 ± 13.2 1109.0 ± 0.99W1 35.9 ± 2.3 28.6 ± 0.36W2 26.4 ± 1.9 25.9 ± 0.29W3 22.9 ± 5.8 30.4 ± 0.71W4 1.3 ± 0.2 ND

Wheat W5 6.7 ± 0.5 3.9 ± 0.33W6 1347.0 ± 30.6 1200.0 ± 1.8W7 89.4 ± 4.3 55.6 ± 0.47W8 15.2 ± 1.0 9.2 ± 0.26W9 1.4 ± 0.3 NDW10 58.8 ± 2.6 45.9 ± 0.61R1 ND NDR2 ND ND

Rice R3 ND NDR4 ND NDR5 ND ND

aThe noncompetitive idiometric nanobodies phage ELISA.bEach assay was conducted in triplicates on the same day.cNot detectable.

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0.68 ng/mL, with the limit of detection (LOD) of 0.19 ng/mL and the line arc scale rangedfrom 0.31 ng/mL to 1.74 ng/mL. This assay provided an over 17-fold higher sensitivity,comparing to the competitive ELISA (LOD = 3.41 ng/mL). Using LC–MS/MS for FB1detecion, the proposed method was successfully validated in actual cereal samples.Overall, it was indicated that the noncompetitive idiometric nanobodies phage ELISA isa prospect format for actual FB1 analysis in cereals. The assay serves as a rapid approachto convert competitive formats to noncompetitive assays for monitoring small molecules,providing significantly improved sensitivity and adaptability to other immunoassaydetections.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This study was financially supported by the National Natural Science Foundation of China [grantnumbers: 31760488, 31560480], the Education Department of Jiangxi Province [grant number:GJJ170309].

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