Development of a Screening Fluorescence Polarization Immunoassay

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Development of a screening fluorescence polarization immunoassay for simultaneous detection of fumonisin B1 and B2 in maize Chenglong Li, Tiejun Mi, Gea Oliveri Conti, Qing Yu, Kai Wen, Jianzhong Shen, Margherita Ferrante, and Zhanhui Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01845 • Publication Date (Web): 05 May 2015 Downloaded from http://pubs.acs.org on May 7, 2015

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Journal of Agricultural and Food Chemistry

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Development of a Screening Fluorescence Polarization

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Immunoassay for Simultaneous Detection of Fumonisin B1

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and B2 in Maize

4

Chenglong Li †, Tiejun Mi

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Shen †, #, Margherita Ferrante §, Zhanhui Wang †, #, *

6

†

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Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing

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Laboratory for Food Quality and Safety, 100193 Beijing, People’s Republic of China

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‡

†, ‡

, Gea Oliveri Conti §, Qing Yu †, Kai Wen †, Jianzhong

College of Veterinary Medicine, China Agricultural University, Beijing Key

College of Veterinary Medicine, Northwest A & F University, 712100 Yangling,

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People’s Republic of China

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§

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Environmental and Food Hygiene, University of Catania, 87 avenue S. Sofia, 95123

13

Catania, Italy

14

#

15

People’s Republic of China

16

*

17

Tel: +86-10-6273 4565

18

Fax: +86-10-6273 1032

19

E-mail: [email protected]

Department “GF Ingrassia”, Hygiene and Public Health, Laboratory of

National Reference Laboratory for Veterinary Drug Residues, 100193 Beijing,

Author to whom correspondence should be addressed

1

ACS Paragon Plus Environment


Abstract 20

We report the development of a screening fluorescence polarization immunoassay

21

(FPIA) for simultaneous detection of fumonisin B1 (FB1) and B2 (FB2) in maize.

22

Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC),

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FB1-5-([4, 6-dichlorotriazine-2-yl] amino)-fluorescein (FB1-5-DTAF) and FB1-texas

24

red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select

25

appropriate tracer-antibody pairs using seven previously produced monoclonal

26

antibodies (mAbs). An FPIA employed the pair of FB1-FITC and mAb 4B9 showing

27

98.9% of cross-reactivity (CR) towards FB2 was used to simultaneously detect FB1

28

and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After

29

optimization, the FPIA revealed a limit of detection (LOD) of 157.4 µg/kg for FB1

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and 290.6 µg/kg for FB2, respectively. Recoveries were measured for spiked samples

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of FB1 or FB2 separately, ranging from 84.7-93.6%, with coefficient of variation (CV)

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less than 9.9%. Total time needed for FPIA including sample pretreatment was less

33

than 30 min. The FPIA was used to screen naturally contaminated maize samples.

34

Results detected by FPIA showed good agreement with that of HPLC-MS/MS with a

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fit of R2 0.99 for simultaneous detection of FB1 and FB2. The established method

36

offered a rapid, simple, sensitive and high-throughput screening tool for detection of

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fumonisins in maize.

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Keywords Fumonisin B1; Fumonisin B2; Fluorescence polarization immunoassay;

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Maize; Detection

2


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1. Introduction 40

Fumonisins are a class of mycotoxins produced as secondary metabolites by

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fungi of the genus Fusarium, notably Fusarium verticillioides and Fusarium

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proliferatum.1, 2 Since the first description and characterization of fumonisins in 1988,

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at least 18 different fumonisins analogues classified into A, B, C and P groups have

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been identified so far.3-6 The B group, mainly fumonisin B1 (FB1) and B2 (FB2) with a

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ratio of 10:3, is believed to be the most prevalent and toxic in naturally contaminated

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cereals throughout the world.7, 8

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Fumonisins have received much attention due to their hepatotoxicity and

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carcinogenic effects on animals by interfering with sphingolipid metabolism. For

49

examples, fumonisins have been implicated as contributors to leukoencephalomalacia

50

in equines and rabbits, pulmonary edema syndrome and hydrothorax in swine and

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apoptosis in the liver of rats.4,

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consumption of FB1 highly contaminated home-grown maize and an incidence of

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esophageal and liver cancer in those regions of China.10 Based on these available data,

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the International Agency for Research on Cancer has assessed the cancer risk of FB1

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and rated it as group 2B human carcinogen.11

9

In humans, there is a correlation between the

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In order to protect public health from unacceptable contamination, guidance or

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regulations for fumonisins have been enforced in many countries. The Food and Drug

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Administration issued recommended maximum levels of 2-4 mg/kg for total FB1, FB2

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and fumonisin B3 (FB3) in human foods.12 The scientific committee for food of the

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European Commission has set action levels for the sum of FB1 and FB2 which ranged

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from 200 µg/kg in processed maize-based foods and baby foods to 4000 µg/kg in

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unprocessed maize.13, 14

3



63

Several analytical methods for fumonisins detection have currently been

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developed, including high-performance liquid chromatography (HPLC) with

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fluorescence detection, liquid chromatography coupled to mass spectrometry (LC-MS)

66

or tandem mass spectrometry (LC-MS/MS).15-17 These instrumental methods,

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generally suffering from sophisticated instrumentation, tedious sample preparation

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and highly trained/skilled technicians, especially are expensive and time-consuming

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for routine screening of suspected contaminants in many samples. Immunoassays,

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mainly enzyme-linked immunosorbent assay (ELISA), have frequently been reported

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for the determination of fumonisins because they are highly sensitive, cost effective

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and easy to use.18-24 However, ELISA as a heterogeneous method in solid phase,

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which is limited by requiring multiple incubation and washing steps, and generally

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needed from 15-60 minutes for the analysis of fumonisins. Thus, with the increasing

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demand for quicker, easier and high-throughput screening of contaminants, much

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effort has been focused on exploring alternatives.

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Fluorescence polarization immunoassay (FPIA) is a homogeneous assay based

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on the competition between unlabeled analyte and fluorescein-labeled tracer for

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specific antibody in solution phase, which uses changes in fluorescence polarization

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(FP) to detect the presence of target.25 Compared with ELISA, FPIA only takes as

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little as a few seconds or minutes before measuring and no separation or washing

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steps are required, which is rapid, simple with high-throughput suitable for analysis of

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large numbers of samples. Recently, FPIA has been applied to detect mycotoxins,

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including aflatoxins (AFs), fumonisins, deoxynivalenol (DON), T-2 toxin, ochratoxin

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A (OTA) and zearalenone (ZEA).18, 19, 25-32 Maragos et al.18 initially developed an

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FPIA method in tube format for the measurement of FB1 by utilizing tracer FB1-6-([4,

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6-dichlorotriazine-2-yl] amino)-fluorescein (FB1-6-DTAF) and monoclonal antibody 4


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(mAb) P2A5-3-F3 with a limit of detection (LOD) of 500 µg/kg in maize. After this

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pioneering work, Nasir et al.19 improved the sensitivity of the assay which employed

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the same immunoreagents with an LOD of 100 µg/kg by refining the tracer and each

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step of the FPIA. However, the FPIAs reported were both performed in glass tubes,

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which are low-throughput as samples need to be inserted into the instrument one after

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another, and only FB1 was detected in maize.

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In the current work, an FPIA in a microplate reader format was developed for

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simultaneously detection of FB1 and FB2 in maize samples. For this purpose, we

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synthesized three tracers and paired them with seven already produced mAbs. After

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optimization, the developed FPIA was applied to detect FB1 and FB2 in spiked maize

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and in naturally contaminated maize samples. 2. Materials and Methods 2.1. Reagents and materials

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FB1 was purchased from Pribolab Pte. Ltd. (Singapore City, Singapore) and FB2

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was obtained from LKT Laboratories, Inc. (St. Paul, MN). FB3, fluorescein

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isothiocyanate isomer I (FITC), and 5-([4, 6-dichlorotriazine-2-yl] amino)-fluorescein

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hydrochloride (5-DTAF) were supplied by Sigma-Aldrich (St. Louis, MO). Texas

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red-X succinimidyl ester (TRX) was acquired from Life Technologies (Carlsbad, CA).

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Aflatoxin B1 (AFB1), ZEA, OTA, DON, and T-2 toxin were purchased from

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Fermentek Biotechnology (Jerusalem, Israel). Water was obtained from a Milli-Q

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system (Bedford, MA). All other reagents were analytical grade or better unless

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specified otherwise, which were acquired from Sinopharm Chemical Reagent Co., Ltd

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(Shanghai, China). Pre-coated TLC silica gel 60 F254 aluminium sheets were acquired

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from Merck (Darmstadt, Germany). Black opaque 96-well microtiter plates with a 5



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non-binding surface were purchased from Corning (Oneonta, NY). The 0.45 µm

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syringe filters were obtained from Tianjin Jinteng Experiment Equipment Co., Ltd

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(Tianjin, China).

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Seven mAbs to FB1, named 2B9, 4B9, 7C9, 2D7, 4F5, 5F8 and 2H8 were

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previously prepared.23 Two batches of flour samples of naturally contaminated maize

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were kindly provided by Prof. Sarah De Saeger (Ghent University) and were stored at

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-20 °C.33

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Borate buffer (50 mM, pH 8.0) with 0.1% sodium azide was used as a working

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buffer solution in this study. Stock solutions (5 mg/mL) of FB1 and FB2 were

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prepared by dissolving 1 mg of standard in 200 µL of methanol respectively and were

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stored at -20 °C until use. 2.2. Apparatus

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FPIA was performed using a spectramax M5 microplate reader obtained from

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Molecular Devices (Sunnyvale, CA) to measure FP and fluorescence intensity (FI)

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signal. 2.3. Preparation of fluorescein-labeled FB1 tracers

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As FB1 has a single primary amine to enable coupling reactions, it was directly

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conjugated to fluorescein according to the protocol with minor modifications.34

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Briefly, stock solution of FB1 (50 µL) was added to FITC (2 mg) dissolved in 50 µL

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of methanol followed by the addition of triethylamine (10 µL). After overnight

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reaction at room temperature (RT) in the dark, a small portion (20 µL) of crude

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product

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trichloromethane/methanol/acetic acid (40:10:1, v/v/v) mobile phase. The major

was

purified

by

thin

layer

chromatography

6


(TLC)

using

a

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yellow band at Rf 0.1 was scraped from the plate and eluted with 100 µL of methanol.

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Other tracers FB1-5-DTAF, FB1-TRX were prepared in the same way and were stored

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in the dark at -20 °C. All the tracer solutions were further diluted with borate buffer to

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get the working concentration with FI about 20 times that of borate buffer

135

background. 2.4. Sample preparation

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An amount of 1 g of maize flour samples was added into a 50 mL plastic

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centrifuge tube. Then the samples were extracted with 5 mL of methanol/water (2:3,

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v/v) at RT. After vortexing for 1 min and ultrasonication for 5 min, the mixtures were

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centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant (2 mL) was filtered

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through a 0.45 µm syringe filter and 1 mL of filtrate was mixed with 3 mL of borate

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buffer. Finally, 70 µL of the diluted extract was analyzed by FPIA without further

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treatment.

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In order to acquire the accuracy and repeatability of the FPIA method, 1 g of

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blank maize flour was spiked with known amounts of standard solutions of FB1 or

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FB2 to obtain samples with different concentrations ranging from 300-2000 µg/kg

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before extraction. 2.5. Fluorescence polarization immunoassay 2.5.1. Antibody dilution curve

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The antibody dilution curve was performed by mixing 70 µL per well of tracer

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solution at working concentration with 70 µL per well of two-fold serially diluted

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mAb. Borate buffer was added to reach an overall volume of 210 µL per well. 2.5.2. Maize matrix-based calibration curve 7



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The concentration of fumonisins in naturally contaminated maize samples was

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determined relative to the maize matrix-based calibration curve of FB1 or FB2

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prepared in blank matrix extracts.

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The assay was conducted by adding 70 µL per well of tracer working solution

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and 70 µL per well of prepared standards (or samples) to the microplate, followed by

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the addition of 70 µL per well of optimal diluted mAb corresponding to a 70% tracer

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binding based on the antibody dilution curve. After the mixture was shaken for 10 s in

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the microplate reader, FP values were measured at λex 485 nm, λem 530 nm with

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emission cutoff of 515 nm for FB1-FITC and FB1-5-DTAF, or at λex 585 nm, λem 620

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nm with emission cutoff of 610 nm for FB1-TRX, respectively.

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The sigmoidal curve was obtained by plotting the measured FP values against the

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concentration of fumonisins and fitting them to a four-parameter logistic equation by

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OriginPro 8.0 (Northampton, MA). The LOD was experimentally defined as IC10, the

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concentration of standard inhibiting 10% of tracer binding with antibody, i.e. the

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concentration that corresponds to 10% inhibition of the maximal FP signal.35 The

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detectable range corresponds to the concentration of standard varying from IC20-IC80. 2.5.3. Cross-reactivity determination

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To determine the specificity of this method, cross-reactivity (CR) with other

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mycotoxins including FB2, FB3, AFB1, ZEA, OTA, DON and T-2 toxin were

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calculated by the following equation, where IC50 value was the concentration of

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standard inhibited tracer binding by 50%.

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CR %= (IC50 of FB1 )⁄(IC50 of other mycotoxins) ×100% 2.6. Accuracy and precision

8


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The accuracy (expressed as recovery), the precision containing repeatability

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(denoted by coefficient of variation (CV)) and reproducibility were measured for the

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developed FPIAs. Correlation studies were conducted to determine the reproducibility.

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Samples of naturally contaminated maize already detected by HPLC-MS/MS, were

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analyzed by the developed FPIA. 3 Results and Discussion 3.1. Synthesis and characterization of tracers In previous reports, several tracers of FB1 were prepared and used in FPIA for

176 177

the

determination

of

fumonisins,

including

FB1-DTAF,

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FB1-iodoacetamido-fluorescein (FB1-IAF) and FB1-carboxy-fluorescein (FB1-FAM).18,

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19

180

mAb P2A5-3-F3. Among these tracers, FB1-DTAF had the most rigid structure as

181

well as the least “propeller effect”, and was found to be the best one.

Different tracers have shown different performance in FPIA when using the same

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In this study, three tracers (hapten labeled with three fluorescein including FITC,

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5-DTAF and TRX) were synthesized in order to achieve the most sensitive FPIA

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(Figure 1). After purification by TLC, tracers were primarily characterized for binding

185

with seven mAbs by the addition of saturating amount of mAbs (dilution of 1/100) to

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tracer working solutions. The main bands of TLC at Rf 0.1 for both FB1-FITC and

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FB1-5-DTAF, Rf 0.5 for FB1-TRX were used as tracers in the following studies on

188

account that only they have an affinity with antibodies since FP signals significantly

189

increased before and after mAbs were added (Figure 2). The couple of FB1-TRX and

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mAb 2H8 gave the largest increase in FP signal with δ FP (FPbind minus FPfree) about

191

392.4 mP (millipolarization units) showing the highest affinity. Meanwhile, FP values

192

of other combinations were also increased and ranged from 49-218 mP. 9



3.2. Selection of tracer-antibody pairs 193

It is reported that the combination of tracer and antibody may have a significant

194

impact on the sensitivity and specificity when developing an FPIA.35, 36 In this assay,

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IC50 values of FPIA calibration curves for FB1 in borate buffer which were obtained

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by using working concentration of tracers and diluted mAbs that corresponds to a 50%

197

tracer binding was mainly applied to select the optimal tracer-antibody pairs. The

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highest sensitivity (lowest IC50 18.8 ng/mL) was achieved when using FB1-FITC and

199

the 4F5 mAb produced by immunogen FB1-KLH via conjugating the carboxylic

200

group of FB1 to the carrier protein (Table 1). Also this combination gave the widest

201

assay window (δ FP, FPmax minus FPmin) and lowest background compared with other

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mAbs and FB1-FITC pairs. Among the tracers we prepared, FB1-FITC gave the best

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performance followed by FB1-5-DTAF and then FB1-TRX when seven mAbs were

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used (Table 1). It should be noted that FB1-TRX could specifically bind to all of these

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mAbs and gave an adequate increase in FP signals. However, obvious inhibitions of

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FB1 were not found for most of these antibodies. This maybe because FB1-TRX has

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the most “propeller effect” based on its flexible chemical structure in accordance with

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previous studies.19

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Remarkable differences in sensitivity and specificity may be achieved by using

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the same tracer and different antibodies.37 The specificity of FB1-FITC and mAb

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combinations was obtained by evaluating CR with other occurring mycotoxins (FB2,

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FB3, AFB1, ZEA, OTA, DON and T-2 toxin) by FPIA. Unlike the 4F5 mAb, 4B9 was

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obtained using immunogen FB1-GA-BSA through conjugation of the amino group of

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FB1 to the carrier protein as reported.23 The sensitivity of FB1-FITC and 4B9 pair was

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lower than FB1-FITC and 4F5 combination. However, high CR to FB2 (98.9% in

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maize matrix) was acquired when using the FB1-FITC and 4B9 combination (Table 2). 10


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Thus, method 1 employing FB1-FITC and 4B9 was used for simultaneous detection of

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FB1 and FB2 in maize. In addition, method 2 employing the pair of FB1-FITC and 4F5

219

could be used for specific determination of FB1 due to its low CR towards FB2 (2.3%

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in maize matrix). The results also showed negligible CR of both mAbs 4F5 and 4B9

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coupled with FB1-FITC to AFB1, ZEA, OTA, DON and T-2 toxin. 3.3. Investigation of matrix effect

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In general, fumonisins can be extracted from maize with a mixture of organic

223

solvent and water (methanol-water or acetonitrile-water).38 In this study,

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methanol/water (2:3, v/v) was used to extract fumonisins from maize samples

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according to the previous report.24

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As a homogeneous assay, FPIA was more susceptible to interference by matrix

227

effects than other heterogeneous assays. Therefore, the investigation of matrix effect

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is an important part of FPIA. For the purpose of acquiring information on matrix

229

effects of maize samples, calibration curves were performed in both borate buffer and

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diluted sample extracts. Results show that the matrix effect could not be reduced

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simply through a dilution of the extract. The main reason may be that varieties of

232

colored or fluorescent contents are also extracted along with fumonisins when using

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organic solvent.37 In FPIA, PBS substantially free of organic solvent was also used for

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extraction of FB1 in maize samples, but the matrix effect still exists with the protocol

235

that 150 µL of extract was added to 150 µL of antibody in PBS and then 200 µL was

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taken out and mixed with 1.4 mL of tracer.18 Therefore, a maize matrix-based

237

calibration curve was used to determine the concentration of fumonisins in naturally

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contaminated maize samples to reduce the background interference of maize extracts. 3.4. Development of FPIA in maize matrix 11



239

Since tracer concentration influences the sensitivity of an assay markedly, the

240

lowest possible concentration should be used to get the most sensitive assay, but this

241

also resulted in low precision of FP signal.35, 36, 40 Generally, optimal concentration of

242

tracer was chosen when FI was at least 10 times higher than that of borate buffer

243

background.41 According to the manual of the microplate reader used in this assay, it

244

was recommended the precision expressed as standard deviation (SD) of FP be less

245

than 5 mP. Working concentration of tracer was optimized in borate buffer when FI of

246

tracer was about 5, 10, 20 and 40 times higher than background following the

247

procedure previously published.42 The results show that the precision of the FP signal

248

depended on tracer concentration. When FI was about 20 times higher than that of

249

background, tracer at this concentration was selected as working concentration

250

because SD of FP values of both free and bound tracer was less than 5 mP.

251

Antibody dilution curves in the maize matrix were obtained for the FB1-FITC

252

and 4B9 combination, as well as the pair of FB1-FITC and 4F5. Theoretically, the

253

optimal concentration of antibody corresponding to a 50% tracer binding would

254

receive the best sensitivity. However, in this research antibody dilution that

255

corresponds to 70% tracer binding was chosen as optimum concentration so as to get

256

a wider analytical range according to previous studies.43 The best antibody dilution

257

was observed for FB1-FITC with 4B9 (1/46) and 4F5 (1/2900) in maize matrix.

258

Under optimal conditions, calibration curves were plotted in blank maize matrix

259

(Figure 3). FP signals were read every 5 min for 90 min. No incubation time was

260

needed as the equilibrium of competition was quickly completed just after samples,

261

tracer and antibody were mixed (data not shown). Method 1 employing the

262

combination of FB1-FITC and 4B9 could be used to detect FB1 and FB2 in maize

263

samples simultaneously with LOD of 157.4 µg/kg for FB1 or 290.6 µg/kg for FB2 12


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264

respectively (Table 3). Meanwhile method 2 showed an LOD of 53.6 µg/kg for FB1

265

specifically in maize samples by using FB1-FITC and 4F5 pair. This LOD is about

266

2-fold lower than that of the published FPIA method (0.1 mg/kg in maize samples) for

267

the determination of FB1.19 Also, the detectable range in method 1 was 426.8-13166

268

µg/kg for FB1 and 632.8-9072 µg/kg for FB2 respectively, while in method 2 it was

269

108.0-1201 µg/kg for FB1 in maize. Moreover, the whole analysis could be completed

270

within 30 min, including extraction time. Its unrivalled speed and high-throughput

271

makes it ideally suited for screening for targets in large numbers of samples. 3.5. Recovery and precision study

272

The recoveries of method 1 spiked with FB1 or FB2 separately at 500, 1000 and

273

2000 µg/kg were 84.7-93.6%, with CV values less than 9.9% (Table 4). In spiked

274

maize samples of FB1 at 300, 500 and 1000 µg/kg, the recoveries of method 2 ranged

275

from 68.5-87.0%, with CV values of no more than 3.6%. Acceptable recoveries and

276

CV values were acquired for this assay system.

277

Two batches of naturally contaminated maize samples, one contaminated with

278

multi-mycotoxins including FB1 and FB2, the other with FB1 and DON, were

279

analyzed by FPIA and HPLC-MS/MS (Table 5). The total concentrations of FB1 and

280

FB2 in the first batch of maize were determined relative to the maize matrix-based

281

calibration curve of FB1 standard using the FB1-FITC and 4B9 combination.

282

Meanwhile the concentrations of FB1 in the second batch of maize were determined

283

by the maize matrix-based calibration curve of FB1 standard using the combination of

284

FB1-FITC and 4F5. The results between FPIA and HPLC-MS/MS were compared

285

using a correlation test which showed good agreement with a fit of R2 0.99 for

286

simultaneous detection of FB1 and FB2 in maize and 0.80 for specific determination of 13



287

FB1 in maize samples, respectively. Therefore, the developed FPIA could be

288

potentially applied to screen fumonisins in maize. Acknowledgment

289

We are very thankful to Prof. Sarah De Saeger (Laboratory of Food Analysis,

290

Faculty of Pharmaceutical Sciences, Ghent University) for providing naturally

291

contaminated maize samples. Funding Sources

292

This work was supported by grants from Natural Science Foundation of China

293

(U1301214), Special Fund for Agro-scientific Research in the Public Interest

294

(201203040) and the International Science & Technology Cooperation Program of

295

China (2012DFG31840). Safety information

296

Mycotoxins and related samples used in studies should be handled with extreme

297

caution to avoid exposure to all of these contaminants.

298

Supporting Information Available: Table of data for three tracers binding with

299

100-fold diluted mAbs and figures depicting selection of tracer/antibody combinations,

300

matrix effect, optimization of tracer/antibody concentration and correlation analysis

301

between HPLC-MS/MS and developed FPIA. This material is available free of charge

302

via the Internet at http://pubs.acs.org.

14


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Marasas, W. F. O.; Miller, J. D.; Riley, R. T.; Visconti, A., Fumonisins -

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occurrence, toxicology, metabolism and risk assessment, in: Summerell, B. A.; Leslie,

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J. F.; Backhouse, D.; Bryden, W. L.; Burgess, L. W. (Eds.), Fusarium: Paul E. Nelson

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by Fusarium species. Appl. Environ. Microbiol. 2002, 68, 2101-2105.

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Marasas, W. F. O.; Spiteller, G.; Vleggaar, R., Structure elucidation of the fumonisins,

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mycotoxins from Fusarium moniliforme. J. Chem. Soc. Chem. Commun. 1988,

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cancer-promoting activity produced by Fusarium moniliforme. Appl. Environ.

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Microbiol. 1988, 54, 1806-1811.

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fumonisins and other metabolites produced by Fusarium moniliforme and related

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species on corn, in: Jackson, L. S.; DeVries, J. W.; Bullerman, L. B. (Eds), Advances

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in Experimental Medicine and Biology: Fumonisins in Food, Plenum Press Div

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Plenum Publishing Corp, New York, 1996, pp. 57-64.

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Page 21 of 31


FIGURE CAPTIONS 434

Figure 1 Chemical structures of fluorescein (FITC, 5-DTAF and TRX) and FB1 used

435

in this study

436

Figure 2 Results of three tracers binding with 100-fold diluted mAbs

437

Figure 3 Calibration curves based on optimal conditions in maize matrix of: A.

438

FB1-FITC and 4B9 pair; B. FB1-FITC and 4F5 pair

439

Each point represents the average of triplicates for a given concentration (n = 3)

21



Page 22 of 31

Table 1 IC50 Parameter of Competitive FPIA Calibration Curves of FB1 in Borate Buffer Using Three Tracers and Seven mAbs mAbs Tracers 2B9 4B9 7C9 2D7 4F5 5F8 2H8 (ng/mL)

FB1-FITC 23.3 90.1 491.3 23.9 18.8 50.7 FB1-5-DTAF 34.4 93.0 552.4 24.2 21.4 246.1 FB1-TRX -a -a -a 79.0 60.9 -a a No obvious concentration-dependent FP changes were observed

22


158.4 202.0 223.0

Page 23 of 31


Table 2 Cross-reactivity of FB1-FITC and mAb Combinations to FB1 and Other Mycotoxins by FPIA in Maize Matrix mAbs 4B9 4F5 Mycotoxins Structures

a

IC50 (ng/mL)

CR (%)

IC50 (ng/mL)

CR (%)

FB1

118.5

100.0

18.0

100.0

FB2

119.8

98.9

770.7

2.3

FB3

203.7

58.2

28.1

64.1

AFB1

-a

< 1%

-a

Development of a Screening Fluorescence Polarization Immunoassay

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