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Bispecific Monoclonal Antibody-based Multi-analyte ELISA for Furaltadone Metabolite, Malachite Green and Leucomalachite Green in Aquatic Products Feng Wang, Hong Wang, Yudong Shen, Yongjun Li, Jie-Xian Dong, Zhenlin Xu, Jinyi Yang, Yuanming Sun, and Zhili Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03233 • Publication Date (Web): 05 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016
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Bispecific Monoclonal Antibody-based Multi-analyte ELISA for Furaltadone
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Metabolite, Malachite Green and Leucomalachite Green in Aquatic Products
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Feng Wang,† Hong Wang,†* Yu-Dong Shen,† Yong-Jun Li,†‡ Jie-Xian Dong,§
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Zhen-Lin Xu,† Jin-Yi Yang,† Yuan-Ming Sun† and Zhi-Li Xiao†*
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† Guangdong Provincial Key Laboratory of Food Quality and Safety, College of
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Food Science, South China Agricultural University, Guangzhou 510642, China
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‡ Zhongshan Quality Supervision & Inspection Institute of Agricultural Products,
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Zhongshan 528403, China
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§ Department of Entomology and Nematology and UCD Comprehensive Cancer
10
Center, University of California, Davis, California 95616, United States
11
Corresponding Author
12
*(H.
13
[email protected].
14
*(Z.
15
[email protected].
Wang)
L.
Tel:
Xiao)
Tel:
+86
+86
2085283448.
2085283448.
Fax:
Fax:
+86
+86
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2085280270.
E-mail:
2085280270.
E-mail:
Journal of Agricultural and Food Chemistry
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ABSTRACT
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A new multi-analyte immunoassay was designed to screen furaltadone metabolite
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5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and
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leucomalachite green (LMG) in aquatic products using a bispecific monoclonal
20
antibody (BsMAb). Gradient drug mutagenesis methods were separately used to
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prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell
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line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient
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and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient.
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BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas
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of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the
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BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3
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ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA,
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3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced
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from MG by potassium borohydride was recognized by BsMAb. Recoveries of
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AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with
31
HPLC analysis. Thus, the multi-analyte ic-ELISA is suitable for rapid quantification
32
of AMOZ, MG and LMG in aquatic products.
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KEYWORDS: furaltadone metabolite, malachite green, leucomalachite green,
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bispecific monoclonal antibody, multi-analyte immunoassay, ELISA
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Introduction
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Immuno-based assays are helpful and convenient for assuring food safety1-3, and
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researches indicate that most current immunoassay methods used to identify
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contaminants are
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immunoassays are based on polyclonal antibody(PAb), monoclonal antibody (MAb)
40
or recombinant antibody (RAb).7-9 However, most immunoassays could only quantify
41
a single analyte, not multiple contaminants in a complex food matrix.10 Thus, a
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multi-analyte immunoassay (MAIA) is needed to address this scientific deficit.11,12
rapid,
sensitive,
specific, and inexpensive4-6. Also,
the
43
Bispecific antibodies (BsAb) could be obtained via chemical cross-linking13,14,
44
molecular biology15,16, or hybrid-hybridoma technology17,18. Bispecific monoclonal
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antibodies (BsMAb) are monoclonals with two different specific antigen-binding sites,
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normally generated via hybrid-hybridomas.19 BsMAb could specifically bind two
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different analytes using its two antigen-binding sites, and thus has a clear advantage in
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analyzing two contaminants. Recently, BsMAb were used for molecular-recognition
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in high-throughput screening of harmful substances in food products. Jin’s group
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developed a highly specific BsMAb that could measure both carbofuran and
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triazophos, and a highly sensitive and rapid enzyme-linked immunosorbent assay
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(ELISA) using the BsMAb could measure the two residues with detection limits
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(LOD) of 1.89 ng/mL for carbofuran and LOD of 0.36 ng/mL for triazophos.20
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Similarly, Guo developed a visual colloidal gold immunochromatographic strip with
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BsMAb to detect the same pesticides, and their LOD was 32 ng/mL and 4 ng/mL for
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carbofuran and triazophos, respectively.21 Li established an ELISA method with
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BsMAb secreted by a trioma LG-D6 cell line, which was prepared using
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hybrid-hybridoma technology, and this was used to analyze imidacloprid and
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parathion-methyl.22
Next,
they
created
a
new
chemiluminescent
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kinetic-resolved MAIA by tagging two haptens of methyl parathion and imidacloprid
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with horseradish peroxidase (HRP) and alkaline phosphatase (ALP), respectively, to
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assay both the two pesticide residues (LOD of 0.33 ng/mL for both).23 The developed
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MAIA assay with a new analytic strategy was more sensitive than Li’s ELISA. Also,
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Hua prepared a BsMAb secreted by a trioma cell line and used BsMAb-based
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ic-ELISA to assess organophosphorus pesticides and two neonicotinoid insecticides.24
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Furaltadone and malachite green (MG) are two veterinary drugs used in
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aquaculture that are absorbed by aquatic animals and rapidly metabolized to
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5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ) and leucomalachite green
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(LMG), respectively (Figure 1).25,26 Researches suggest that these parent compounds
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and their metabolites could reach toxic levels for humans.27,28 Relevant reports
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indicate that MG and LMG are also found in aquatic products.29,30 In order to monitor
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MG, LMG or AMOZ in aquatic products, some instrumental detecting methods such
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as
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chromatography-tandem mass spectrometry (LC–MS/MS) and ultra-high performance
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liquid
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(UHPLC–HRMS) have been developed.
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including lateral flow immunoassay, immunosensor, ELISA, etc. have been raised to
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detect MG, LMG or AMOZ.33-35 These methods reported are all based on
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monospecific antibodies. However, AMOZ and MG (LMG) are two types of illegal
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drugs in aquatic products, and we usually need to monitor both of them in the same
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sample. BsMAb could provide new strategies in multi-residue detection. Based on it,
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we could develop immunoassays analyzing two or more analytes in one test.
surface-enhanced
chromatography
resonance
coupled
raman
with
high 29,31,32
scattering
resolution
(SERRS),
mass
liquid
spectrometry
Recently, immunological methods
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In this study, we developed a BsMAb-based multi-analyte ELISA for joint
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quantitative analysis of AMOZ, MG, and LMG in aquatic products. We first created
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HGRPT- and TK--deficient hybridoma cell lines and fused them. Next, BsMAb was
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raised, screened, and purified, and a BsMAb-based ELISA was developed to analyze
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the residues. The sensitivity, specificity, and recovery of the method were evaluated,
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and the results indicated that,
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detecting contaminants in complex aquatic matrices.
the multi-analyte ic-ELISA could be applied for
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Materials and methods
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Reagents and Materials
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8-Azaguanine,
5-bromo-2’-deoxyuridine,
culture
media
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RPMI-1640,
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hypoxanthine, aminopterin, and thymidine (HAT) medium, hypoxanthine and
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thymidine
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3,3’,5,5’-tetramethylbenzidine
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hydrochloride, and potassium borohydride were purchased from Sigma Chemicals (St.
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Louis, MO). Hyclone culture medium was obtained from Hyclone (Logan, UT).
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Gibco fetal calf serum was purchased from Gibco (Grand Island, NY).
(HT)
medium,
polyethylene (TMB),
glycol
2000
3-nitrobenzaldehyde,
(PEG
2000),
hydroxylamine
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Tween-20, HCl, K2HPO4, NaOH, ethyl acetate, n-hexane, acidic alumina,
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acetonitrile, dichloromethane, p-toluenesulfonic acid, ammonium acetate, and
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ammonium hydroxide were obtained from Shanghai Aladdin Bio-Chem Technology
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Co., Ltd. (Shanghai, China). Horseradish peroxidase-labeled goat anti-mouse IgG
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(IgG-HRP) was obtained from Boster Biotech Co., Ltd. (Wuhan, China). Polystyrene
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ELISA plates were purchased from Jiete Biotech Co., Ltd. (Guangzhou, China).
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Coating antigens AMOZA-OVA and MG-H8-OVA were prepared in our laboratory,
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and all other organic solvents and chemicals were of analytical grade. Female Balb/c
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mice (10-week-old, 25-35 g) were fed in the Guangdong Medical Laboratory Animal
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Centre (Foshan, China).
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Apparatus
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First, 500 mg/3 mL Strata-X-C solid phase extraction (SPE) columns were
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obtained from Phenomenex (Torrance, CA). ELISA plates were washed using a
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Multiskan MK2 microplate washer (Thermo Scientific, USA). Absorbances were
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measured on a Multiskan MK3 microplate reader (Thermo Labsystems, USA). A
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NanoDrop 2000c UV-Vis spectrophotometer was purchased from Thermo Scientific
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(Wilmington, DE). HPLC-MS/MS analysis was performed with a chromatographic
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system (Alliance, USA) including a Waters 2695 separation module equipped with an
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auto-sampler and a Waters 2487 dual lambda absorbance detector (Waters, Milford,
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MA). The HPLC-FLD assay was conducted using a Waters Alliance 2695-2475
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HPLC system and an Agilent SB-C18 column (150 mm × 4.6 mm, 5 µm particle size)
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(Agilent Technologies, CA, USA) with a fluorescent detector (Waters, Milford, MA).
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Screening of HGRPT and TK Deficient Hybridoma Cell Lines
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Anti-3-NPAMOZ and anti-LMG hybridomas were developed in our laboratory
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[3-NPAMOZ: 5-morpholino-methyl-3-(3-nitrobenzylidenamino)-2-oxazolidone]. First,
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8-azaguanine (8-AG) and 5-bromo-2’-deoxyuridine (5-BrdU), were added at different
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levels to Hyclone culture medium with 20% Gibco fetal calf serum every two weeks,
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to screen HGRPT-deficient 3-NPAMOZ and TK-deficient LMG cells, respectively.
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Cell death from HAT medium was identified to obtain deficient hybridoma cell lines
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in a humidified 5% CO2 incubator at 37 °C. Monoclonal antibody characteristics were
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analyzed using ic-ELISA before and after drug treatment. Limiting dilution assays
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were used to purify the two deficient cell lines for tetradoma production.
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Preparation of Tetradoma Cell Lines and BsMAb
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HGRPT-deficient 3-NPAMOZ hybridoma cells and TK-deficient LMG
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hybridoma cells were fused with PEG 2000 to produce tetradoma cell lines according
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to published methods.36 After 10 days of cell culture, supernatants from all
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microplates were analyzed by both indirect noncompetitive and indirect competitive
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ELISA to confirm that BsMAb recognized and bound to 3-NPAMOZ and LMG. After
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several rounds of cloning with limiting dilutions, tetradoma cell lines stably secreting
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BsMAb both against 3-NPAMOZ and LMG were obtained. Female Balb/c
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mouse-induced antibodies were produced for BsMAb which were purified using
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caprylic acid-ammonium sulfate precipitation. The concentration of BsMAb was
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quantified using a NanoDrop 2000c UV-Vis spectrophotometer and the BsMAb was
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stored at −20 °C.
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BsMAb based Indirect Competitive ELISA
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The ic-ELISA protocol was performed as described previously.37 Briefly, 96-well
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microtiter plates were coated with 100 µL/well of AMOZA-OVA or MG-H8-OVA in
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coating buffer (0.05 mol/L of carbonate buffer, pH 9.6) overnight at 37 °C. After
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washing two times with 300 µL washing buffer (0.05% PBST), 120 µL/well of
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blocking solution (5% skim milk in PBST) was added to microplate wells and
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incubated at 37 °C for 3 h in water bath. Plates were dried for 1 h in a 37 °C oven
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after blocking solution was discarded. Then 50 µL of the standards or sample extracts
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and 50 µL of BsMAb (appropriate dilution) were successively added to blocked plate
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and further incubated at 37 °C for 40 min. After five washes with washing buffer, 100
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µL/well goat anti-mouse IgG-labeled HRP (1:5,000) was immediately added to plates
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with a 30 min incubation at 37 °C. After five washes, freshly prepared TMB substrate
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solution (100 µL/well) was added to all the microplate wells for color development at
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37 °C for 10 min. Enzymatic reaction was halted by adding 50 µL/well of stopping
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solution (2 mol/L H2SO4). Absorption was measured on a plate reader at 450 nm, and
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data analysis was performed with a four-parameter equation to generate a sigmoidal
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curve using Origin 8.5 software.
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Optimization of ic-ELISA method
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A checkerboard titration method was used to determine the optimal
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concentration of coating antigen (AMOZA-OVA or MG-H8-OVA) and BsMAb
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dilution. To develop a sensitive multi-analyte ELISA variable such as the BsMAb
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dilution buffer (PB, PBS, PBST, Tris-HCl), competition time (30-50 min), HRP-IgG
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dilution (1:4000 to 1:8000), HRP-IgG incubation time (30-50 min), working buffer
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for standard analyte (H2O, PB, PBS, PBST), and pH (from 6.0 to 8.4) were rested.
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IC50 and maximum absorbance (Amax) were obtained from ELISA standard curves,
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and Amax, IC50 and Amax/IC50 ratio were used to evaluate the ELISA performance.
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Cross-Reactivity Study
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Cross-reactivity (CR) for several analogues was studied by comparing IC50
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values of the optimized competitive ELISA. CRs were calculated as follows: CR (%)
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= (IC50 of 3-NPAMOZ or LMG / IC50 of analogues) × 100.
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Preparation of Samples
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During sample preparation, AMOZ was derivatized with 3-nitrobenzaldehyde to
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form 3-NPAMOZ for high-affinity binding to BsMAb. However, sample preparation
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for LMG did not require this step, so sample treatments were developed separately.
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Grass carp and tilapia were purchased from a local supermarket. After their skins
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and bones were removed, muscles were weighed and homogenized. For AMOZ
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analysis in aquatic products, samples were prepared as described in the literature with
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slight modifications.31 First, 1 g homogenized fish was transferred into 50 mL
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centrifuge tubes, spiked with several concentrations of AMOZ, and 4 mL deionized
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water and 0.5 mL of 1 mol/L HCl were mixed with samples. After the addition of 100
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µL of 10 mmol/L freshly prepared 3-nitrobenzaldehyde in methanol, samples were
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incubated at 55 °C for 2 h for derivatizing AMOZ and then 5 mL of 0.1 mol/L
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K2HPO4, 0.4 mL of 1 mol/L NaOH, and 6 mL ethyl acetate were added. Tube contents
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were mixed vigorously for 5 min and centrifuged (4000 rpm, 10 min). The upper
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organic layer was removed to 15 mL centrifuge tubes and evaporated to dryness under
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nitrogen at 45 °C. Finally, the dry residues were reconstituted with 1 mL PBS and
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mixed with addition of 1 mL n-hexane. Samples were shaken vigorously for 1 min,
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and then the lower supernatant was separated from the mixture by centrifugation
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(4000 rpm, 10 min) and determined by ic-ELISA and HPLC-MS/MS.
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According to procedures of Dong’s group38, the pretreatment method for LMG
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analysis was carried out as follows: 3 g homogenized fish was weighted and
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transferred into 50 mL centrifuge tubes, spiked with several concentrations of LMG,
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MG, or LMG-MG mixtures. After the addition of 1 g disodium EDTA, 3 g acidic
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alumina, and 8 mL acetonitrile, tubes were vigorously vortexed for 30 s. Then 0.5 mL
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of 20% hydroxylamine hydrochloride, 1 mL of 0.05 mol/L p-toluene sulfonic acid,
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and 1.5 mL ammonium acetate buffer (0.125 mol/L, pH 4.5) were added, shaken for 5
199
min, and the supernatant was collected by centrifugation (4000 rpm, 10 min). Another
200
4 mL acetonitrile were added to the sediment, and the samples were vigorously
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vortexed for 2 min and centrifuged again for another 2 min. Finally, supernatants were
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merged.
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Then, 1 mL of 0.2 mol/L potassium borohydride was added into supernatant and
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incubated in the dark for 10 min. Mixture sample was shaken vigorously for 2 min
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after the addition of 10 mL dichloromethane and centrifuged for 10 min. The lower
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organic layer was then transferred to another new tube and evaporated to dryness
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under nitrogen gas at 40 °C. Finally, 2 mL acetonitrile was used to dissolve the
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residue for clean-up. Strata-X-C SPE columns were pre-activated with 5 mL
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acetonitrile. Samples were loaded onto columns, 2 mL acetonitrile-ammonium
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hydroxide (9:1, v/v) was eluted, and eluents were collected, dried under nitrogen. A
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final dry residue was reconstituted in 1 mL acetonitrile for ic-ELISA and HPLC
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analysis.
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Analysis of Samples by ic-ELISA and HPLC
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For ic-ELISA analysis of AMOZ and LMG, sample extracts were analyzed at
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appropriate dilution. And for HPLC analysis, the extracts was filtered through a
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0.22-µm microporous membrane prior to use. AMOZ was derivatized to 3-NPAMOZ
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and HPLC-MS/MS conditions were modified from published reports32 as follows: An
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Aglient XDB-C18 (4.6 mm × 50 mm, 1.8 µm particle size) column was purchased
219
from Agilent (Santa Clara, CA) with a mobile phase B of acetonitrile and a mobile
220
phase A of 0.1% formic acid (0.25 mL/min) as follows: 0 min, 22% B; 0–6 min,
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22–99% B; 6–9 min, 99% B; 9–9.1 min, 99–22% B; and 9.1–15 min, 22% B. The
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injection volume of 5 µL analytes was determined by ESI-MS/MS in a positive mode.
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For LMG analysis, a conventional HPLC method with a fluorescent detector
224
(HPLC-FLD) was used based on published methods38. Preparative HPLC was
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performed on an Agilent SB-C18 column (4.6 mm × 150 mm, 5 µm particle size). The
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isocratic mobile phase was a mixture of acetonitrile and 0.125 mol/L ammonium
227
acetate (7:3, v/v, pH 4.5) with a flow-rate of 1.0 mL/min and an total injection volume
228
of 20 µL sample extract. The FLD excitation wavelength was 265 nm and emission
229
wavelength was 360 nm.
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Results and discussion
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Drug treatment of hybridomas and preparation of BsMAb
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To obtain deficient hybridoma cell lines, Figure 2 depicted a gradient drug
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mutagenesis application as depicted in Methods. The anti-3-NPAMOZ hybridoma cell
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line was treated as depicted in Methods (5, 10, 20, 40 µg/mL of 8-AG medium for
235
least 8 weeks), and an anti-LMG hybridoma cell line was cultivated as depicted in
236
Methods (25, 50, 100 µg/mL of 5-BrdU medium for least 6 weeks). Deficient cells
237
were confirmed with cell culture in HAT medium. Figure 3 depicted titers of both
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monoclonal antibodies before and after drug treatment, and no differences were noted
239
between antibodies in either hybridoma cell line before and after drug treatment. An
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HGRPT-deficient 3-NPAMOZ hybridoma cell line and a TK-deficient LMG
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hybridoma cell were fused as depicted in Methods and screened with both
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non-competitive and competitive indirect ELISA.
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Different concentration of chemical mutagens produces various mutagenic
244
effects on hybridoma cells. If mutagens level is too low, mutagenesis time will last a
245
long time and the biological stability of cells may be decreased, which is
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disadvantageous to the drug treatment. Conversely, under a high mutagens level,
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mutagenesis time will be shortened, but it could induce more gene mutations and
248
result in cellular properties loss.
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Optimization of BsMAb based ic-ELISA
250
Formats of multi-analyte ELISAs normally exist in two ways: antigen-coated
251
ic-ELISA and antibody-coated direct competitive ELISA (dc-ELISA),11 Considering
252
the factors such as stability reduction of coating antibody, inconvenience in
253
preparation of multi tracers, interaction effect of several response signals and others
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under the dc-ELISA model, we selected ic-ELISA analysis strategy as the ELISA
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format for further study.
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Firstly, BsMAb was produced and purified as described in Methods, and optimal
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ic-ELISA conditions for BsMAb and the coating antigen (AMOZA-OVA or
258
MG-H8-OVA) were tested using chessboard titration. Data showed that the
259
AMOZA-OVA at 250 ng/mL and a 1:16000 BsMAb dilution were the optimal
260
combination. For MG-H8-OVA, 500 ng/mL was best, with a 1:8000 dilution of
261
BsMAb was used in future experiments.
262
To optimize the ELISA Amax values, the lowest IC50 value and the highest ratio of
263
Amax/IC50 were studied, and Table 1 summarized the optimization results for ic-ELISA.
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For AMOZ analysis, it showed that BsMAb dilution in Tris-HCl buffer (pH 7.4),
265
HRP-IgG antibody (1:9000), a competition reaction time of 30 min, an HRP-IgG
266
incubation time of 30 min, and a standard working solution with PB buffer (pH 6.4)
267
was best. For LMG, data depicted that BsMAb dilution in PB buffer (pH 7.4),
268
HRP-IgG antibody (1:5000), a standard dilution PBST buffer (pH 7.0), and the same
269
HRP-IgG incubation time and competition reaction as before were optimal.
270
Sensitivity and specificity of ic-ELISA for AMOZ and LMG
271
Figure 4 showed standard curves of the ic-ELISA for AMOZ and LMG, fit using
272
Origin Pro 8.5 software. As shown in Table 1, the IC50 value of ic-ELISA for
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3-NPAMOZ was 1.7 ng/mL (Equivalent to AMOZ concentration of 1.30 ng/mL) with
274
a detection limit (LOD, IC10) of 0.2 ng/mL (Equivalent to AMOZ concentration of
275
0.15 ng/mL). And the IC50 value and LOD of ic-ELISA for LMG were 45.3 ng/mL
276
and 4.8 ng/mL. It indicated the established ic-ELISA had high sensitivity and a wide
277
linear range. Ic-ELISA specificity was measured using CR and data showed that
278
(Table 2), there was no obvious CR with the analogues, but there was CR with
279
furaltadone (32.6% of CR) and AMOZ (2.1% of CR). However, furaltadone was
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rapidly metabolized to AMOZ, and the high CR of furaltadone would not affect the
281
performance of ic- ELISA. For BsMAb specificity for LMG analysis, MG, and LCV
282
had low CR (3.3% and 4.8%, respectively). The BsMAb-based ic-ELISA for
283
measuring AMOZ and LMG was highly specific for the tested compounds.
284
Confirmation of drug-spiked aquatic products with ic-ELISA and HPLC
285
Sample matrix effects were studied using standard curves of blank aquatic
286
products. Ic-ELISA standard curves based on blank fish sample extracts at several
287
dilutions were compared with curves generated from working buffers. Ic-ELISA
288
standard curves appeared in Figure 5. The results indicated that a 1:6 dilution with PB
289
(pH 6.4) for AMOZ analysis could eliminate the matrix effect. And sample extraction
290
clean-up by SPE could be directly used to assay LMG with ic-ELISA. Also, the
291
spiked sample extraction of AMOZ, LMG, MG or LMG-MG were measured with
292
HPLC. For instrument validation, AMOZ was measured using HPLC-MS/MS and
293
LMG by HPLC-FLD (See Table 3 and 4). The recovery of AMOZ in grass carp and
294
tilapia ranged from 72.4% to 101.0% with recovery of MG, LMG and MG-LMG
295
ranged from 75.0% to 108.0%, and the coefficient of variation was less than 15%.
296
Figure 6 showed correlation of ic-ELISA and HPLC-MS/MS or HPLC-FLD analysis.
297
Correlation coefficients between the detecttion results of ic-ELISA and HPLC
298
analysis were all over 0.95, which were satisfactory for further research. The analysis
299
results of multi-analyte ic-ELISA were in good agreement with that from HPLC.
300
Compared with other detection methods, including FPIA developed by Yan39,
301
CLEIA by Xu40 and HPLC by Zhu41, the developed multi-analyte ic-ELISA in this
302
study showed lower detection limit, excellent specificity and high recovery. The
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multi-analyte ic-ELISA was successfully used to analyze AMOZ, MG and LMG in
304
aquatic products with appropriate specificity, recovery, and accuracy.
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As one of the immunological methods, the established method is time-saving,
306
sensitive, specific, and low-cost compared with most instrumental methods,
307
applicable for screening of AMOZ, MG and LMG residues in aquatic products. In
308
addition, using BsMAb as a bispecific bio-recognition element, combined with the
309
pretreatment method of derivatization, we could detect AMOZ, MG and LMG in one
310
microtiter plate, with the same reagents except sample solutions and standards.
311
Therefore, this method is more convenient and rapid over single-analyte
312
immunoassays.
313
The developed ic-ELISA was suited as a tool for rapid multi-residue detection of
314
AMOZ, MG, and LMG, and this established assay could provide references for
315
analyzing small molecular contaminants in foods. Moreover, BsMAb could be applied
316
in new detection strategies for analysis of related contaminant and be studied for a
317
comprehensive analysis of the antibody structure-function relationship.
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318
Acknowledgments
319
This work was supported by the National Natural Science Foundation of China
320
(31271866),
321
2012CB720803), Science and Technology Planning Project of Guangdong Province
322
(2014A050503059),
323
Guangzhou
324
(S2012010010323, 2014A030311043). We also greatly thank Professor Wen-Tong
325
Gao for his kind help in manuscript revision.
326
Notes
327
The authors declare no competing financial interest.
National
Basic
Research
Program
of
China
(973
Program,
Science and Technology Planning Project of Program of
(2014J4100185),
Guangdong
Natural
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Science
Foundation
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References
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Chromatogr., 2014, 32(4): 419-425.
469
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Figure Captions
471
Figure 1. Structures of furaltadone, AMOZ, MG and LMG.
472
Figure 2. Protocols for selection of an HGPRT-deficient 3-NPAMOZ hybridoma cell
473
line using 8-AG and a TK-deficient LMG hybridoma cell line with 5-BrdU.
474
Figure 3. Identification of a 3-NPAMOZ hybridoma cell line (A) and an LMG
475
hybridoma cell line (B) before and after drug treatment with 8-AG and 5-BrdU,
476
respectively.
477 478
Figure 4. BsMAb based ic-ELISA standard curves for AMOZ (A) and LMG (B) analysis
(n = 3).
479
Figure 5. Standard curves of ic-ELISA based on buffers and blank fish sample extract
480
with several-fold dilution buffer solutions (n = 3). (A) matrix effect with grass
481
carp extract at 1:2, 1:4, and 1:6 dilutions for AMOZ analysis; (B) matrix effect
482
with tilapia extract at 1:2, 1:4, and 1:6 dilutions for AMOZ analysis; (C) matrix
483
effect with grass carp extract with and without SPE extraction at 1:1 and 1:2
484
dilutions for LMG analysis; (D) matrix effect with tilapia extract with and
485
without SPE extraction at 1:1 and 1:2 dilutions for LMG analysis.
486
Figure 6. Correlations of analysis of spiked samples with ic-ELISA and HPLC (A)
487
samples spiked with AMOZ; (B) samples spiked with LMG; (C) samples spiked
488
with MG; (D) samples spiked with LMG-MG.
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Table 1. Optimization results of experimental parameters for ic-ELISA ic-ELISA for analyte AMOZa
Parameters
a
LMG
Coating antigen
AMOZA-OVA
MG-H8-OVA
Coating concentration (ng/mL)
250
500
Dilution of BsMAb
1:16000
1:8000
BsMAb dilution buffer
Tris-HCl (pH 7.4)
PB (pH 7.4)
Competition time (min)
30
30
HRP-IgG dilution
1:9000
1:5000
HRP-IgG incubation time (min)
30
30
Working buffer for standard analyte
PB (pH 6.4)
PBST (pH 7.0)
IC50 (ng/mL)
1.7
45.3
LOD (IC10, ng/mL)
0.2
4.8
Linear response range (IC20 - IC80, ng/mL)
0.4-8.3
7.0-291.7
For AMOZ analysis, the target analyte was a stable AMOZ derivative 3-NPAMOZ.
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Table 2. CR of ic-ELISA for AMOZ and LMG Tested Compounds
IC50 (ng/mL)
CR (%)
Tested Compounds
IC50 (ng/mL)
CR (%)
3-NPAMOZ
1.7
100
Leucomalachite Green (LMG)
45.3
100
Nitrofurazone
>1000
< 0.1
Malachite green (MG)
1372.7
3.3
Nitrofurantoin
>1000
< 0.1
Brilliant Green
>10000
< 0.1
Furaltadone
5.2
32.6
Leucorilliant Green
>10000
< 0.1
Furazolidone
>1000
< 0.1
Crystal Violet (CV)
>10000
< 0.1
Semicarbazide (SEM)
>1000
< 0.1
Leucocrystal Violet (LCV)
943.8
4.8
1-Aminohydantoin (AHD)
>1000
< 0.1
Parafuchsin
>10000
< 0.1
3-Amino-2-oxazolidone (AOZ)
>1000
< 0.1
Methylene Blue
>10000
< 0.1
AMOZ
81.1
2.1
b
>1000
< 0.1
3-NPAHDb
>1000
< 0.1
3-NPAOZb
>1000
< 0.1
>1000
< 0.1
3-NPSEM
3-Nitrobenzaldehyde b
3-NPSEM, 3-NPAHD, 3-NPAOZ were the derivatives of SEM, AHD, AOZ by
3-nitrobenzaldehyde.
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Table 3. Recovery of AMOZ from spiked samples by ic-ELISA and HPLC-MS/MS ic-ELISA
HPLC-MS/MS
Aquatic
Spiked level
Found±SD c
Recovery±CV d
Found±SD
Recovery±CV
products
(ng/g)
(ng/g)
(%)
(ng/g)
(%)
Grass carp
1
0.83±0.065
83.0±7.8
0.95±0.038
95.0±4.0
2
2.02±0.087
101.0±4.3
1.79±0.057
89.5±3.2
8
6.88±0.19
86.0±2.8
6.52±0.35
81.5±5.4
32
28.72±1.32
89.8±4.6
30.00±1.11
93.8±3.7
1
0.92±0.042
92.0±4.6
0.91±0.033
91.0±3.6
2
1.62±0.10
81.0±6.2
1.97±0.10
98.5±5.1
8
6.85±0.58
85.6±8.5
6.41±0.13
80.1±2.0
32
23.18±1.83
72.4±7.9
30.40±1.92
95.0±6.3
Tilapia
c
SD: sanderd deviation.
d
CV: coefficient of variation.
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Table 4. Recovery of LMG, MG or MG-LMG from spiked samples by ic-ELISA and HPLC-FLD ic-ELISA Aquatic
Analyte
Spiked
Found±SD
Recovery±CV
Found±SD
Recovery±CV
level (ng/g)
(ng/g)
(%)
(ng/g)
(%)
2
1.70±0.082
85.0±4.8
1.48±0.65
74.0±4.4
5
3.92±0.13
78.4±3.3
4.75±0.34
95.0±7.2
15
13.01±0.87
86.7±6.7
11.42±0.63
76.1±5.5
100
93.14±5.96
93.1±6.4
94.44±4.44
94.4±4.7
2
1.50±0.096
75.0±6.4
1.46±0.031
73.0±2.1
5
4.82±0.21
96.4±4.4
4.55±0.059
91.0±1.3
15
14.21±0.81
94.7±5.7
11.16±0.64
74.4±5.7
100
83.71±7.20
83.7±8.6
108.24±4.22
108.2±3.9
LMG-MG
2
1.60±0.098
80.0±6.1
1.56±0.11
78.0±7.1
(1:1)
5
4.18±0.34
83.6±8.1
4.55±0.11
91.0±2.4
15
16.01±0.54
106.7±3.4
10.89±0.73
72.6±6.7
100
90.59±3.71
90.6±4.1
108.66±9.56
108.7±8.8
2
1.65±0.11
82.5±6.7
1.82±0.086
91.0±4.7
5
4.97±0.22
99.4±4.4
3.64±0.35
72.8±9.6
15
12.01±1.00
80.1±8.3
10.11±0.99
67.4±9.8
100
79.16±3.96
79.2±5.0
84.96±4.59
85.0±5.4
2
1.60±0.085
80.0±5.3
1.59±0.038
79.5±2.4
5
4.25±0.12
85.0±2.8
3.70±0.30
74.0±8.1
15
12.36±1.04
82.4±8.4
10.57±0.94
70.5±8.9
100
94.31±5.85
94.3±6.2
87.51±4.90
87.5±5.6
LMG-MG
2
2.16±0.11
108.0±5.1
2.05±0.13
102.5±6.4
(1:1)
5
3.96±0.32
79.2±8.1
5.08±0.56
101.6±11.0
15
12.16±0.47
81.1±3.9
10.77±0.88
71.8±8.2
100
92.95±1.95
93.0±2.1
82.53±4.87
82.5±5.9
products Grass carp
LMG
MG
Tilapia
HPLC-FLD
LMG
MG
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Figure 1. Structures of furaltadone, AMOZ, MG and LMG.
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8-AG 5-BrdU
100 Durg Concentration (µg/mL)
Page 30 of 35
80 60 40 20 0 0
2
4
6
8
Induction Time (week) Figure 2. Protocols for selection of an HGPRT-deficient 3-NPAMOZ hybridoma cell line using 8-AG and a TK-deficient LMG hybridoma cell line with 5-BrdU.
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3.5 (A)
3.5 (B)
culture supernatants of 3-NPAMOZ cell line 50 ng 3-NPAMOZ+culture supernatants of 3-NPAMOZ cell line culture supernatants of 3-NPAMOZ-HGPRT¯ cell line 50 ng 3-NPAMOZ+culture supernatants of 3-NPAMOZ-HGPRT¯ cell line
3.0
3.0
2.5
2.5
2.0 A450 nm
A450 nm
culture supernatants of LMG cell line 50 ng LMG+culture supernatants of LMG cell line culture supernatants of TK¯-LMG cell line 50 ng LMG+culture supernatants of TK¯-LMG cell line
1.5 1.0
2.0 1.5 1.0
0.5 0.5
0.0 0.0
0
20
40 60 80 100 120 Dilution of culture supernatants
140
0
16
32 48 64 80 96 112 128 Dilution of culture supernatants
Figure 3. Identification of a 3-NPAMOZ hybridoma cell line (A) and an LMG hybridoma cell line (B) before and after drug treatment with 8-AG and 5-BrdU, respectively.
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(A)
1.0
(B)
1.0 0.8
0.6
0.6
B/B0
B/B0
0.8
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0.4
0.4
0.2
0.2
0.0 0.0
0.1
1
10
100
1000
1
Concentration of 3-NPAMOZ (ng/mL)
10
100
1000
Concentration of LMG (ng/mL)
Figure 4. BsMAb based ic-ELISA standard curves for AMOZ (A) and LMG (B) analysis
(n = 3).
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1.5
PB (pH 6.4)
(A)
Grass carp extract (1:2)
1.5 (B)
PB (pH 6.4)
1.2
Tilapia extract (1:4) Tilapia extract (1:6)
Tilapia extract (1:2)
Grass carp extract (1:4) Grass carp extract (1:6)
A450 nm
A 450 nm
1.2 0.9
0.9
0.6
0.6
0.3
0.3
0.0
0.0
0.1
1
10
100
1000
0.1
Concentration of 3-NPAMOZ (ng/mL)
1.0 (C)
PBST (pH 7.0) Grass carp extract without SPE extraction Grass carp extract with SPE extraction Grass carp extract with SPE extraction (1:2)
0.6 0.4
0.0
1000
Tilapia extract with SPE extraction (1:2)
1
Concentration of LMG (ng/mL)
PBST (pH 7.0)
0.4
0.0 100
1000
0.6
0.2
10
100
Tilapia extract without SPE extraction Tilapia extract with SPE extraction
0.8
0.2
1
10
1.0 (D)
A 450 nm
A 450 nm
0.8
1
Concentration of 3-NPAMOZ (ng/mL)
10
100
1000
Concentration of LMG (ng/mL)
Figure 5. Standard curves of ic-ELISA based on buffers and blank fish sample extract with several-fold dilution buffer solutions (n = 3). (A) matrix effect with grass carp extract at 1:2, 1:4, and 1:6 dilutions for AMOZ analysis; (B) matrix effect with tilapia extract at 1:2, 1:4, and 1:6 dilutions for AMOZ analysis; (C) matrix effect with grass carp extract with and without SPE extraction at 1:1 and 1:2 dilutions for LMG analysis; (D) matrix effect with tilapia extract with and without SPE extraction at 1:1 and 1:2 dilutions for LMG analysis.
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( A) AMOZ
300
y= -0.3721+1.154x
32
2
( B) LMG
y= -2.488+1.046x 2
R = 0.9976
250
HPLC-FLD (ng/g)
HPLC-MS/MS (ng/g)
R = 0.9726 24
16
200 150 100
8
50 0
0 0
8
16
24
0
32
50
350
( C) MG
350
y= -3.421+1.098x R2= 0.9516
150 100
150 100
0
0 200
2
200
50
150
300
250
50
100
250
R = 0.9589
200
50
200
y=-2.910+1.046x
250
0
150
( D) LMG-MG
300
HPLC-FLD (ng/g)
300
100
ic-ELISA (ng/g)
ic-ELISA (ng/g)
HPLC-FLD (ng/g)
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250
300
0
50
100
150
200
250
300
ic-ELISA (ng/g)
ic-ELISA (ng/g)
Figure 6. Correlations of analysis of spiked samples with ic-ELISA and HPLC (A) samples spiked with AMOZ; (B) samples spiked with LMG; (C) samples spiked with MG; (D) samples spiked with LMG-MG.
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Table of Contents Graphic
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