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Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients
Accepted Manuscript Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients Michalina Oplatowska-Stachowiak, Nermin Sajic, Ya Xu, Simon A. Haughey, Mark Mooney, Yun Yun Gong, Ron Verheijen, Christopher T. Elliott PII:
S0956-7135(15)30312-1
DOI:
10.1016/j.foodcont.2015.11.041
Reference:
JFCO 4770
To appear in:
Food Control
Received Date: 11 May 2015 Revised Date:
21 November 2015
Accepted Date: 28 November 2015
Please cite this article as: Oplatowska-Stachowiak M., Sajic N., Xu Y., Haughey S.A., Mooney M., Gong Y.Y., Verheijen R. & Elliott C.T., Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients, Food Control (2016), doi: 10.1016/j.foodcont.2015.11.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of
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peanuts, maize and feed ingredients
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Michalina Oplatowska-Stachowiaka,b*, Nermin Sajicb, Ya Xua, Simon A. Haugheya, Mark Mooneya, Yun
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Yun Gonga, Ron Verheijenb & Christopher T. Elliotta
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a
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Malone Road, Belfast BT95BN, United Kingdom
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b
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Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, 18-30
EuroProxima B.V., Beijerinckweg 18, 6827 BN Arnhem, The Netherlands
*Corresponding author. Email: [email protected], tel: +44 (0) 289097 6531,
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fax: +44 (0) 289097 6513
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12 Abstract
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Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on
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different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious
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illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical
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methods are required for the identification of contaminated agricultural commodities before they
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are processed into final products and placed on the market. In order to provide new tools for
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aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B1 and the
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other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity
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and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were
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characterized and two antibodies showing IC50 of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B1 were
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applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut
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matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities
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of aflatoxin B1 and total aflatoxins ELISAs were 0.4 µg/kg and 0.3 µg/kg for aflatoxin B1, respectively,
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which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the
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detection of aflatoxins B2, G1 and G2. The application of the developed tests was demonstrated by
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screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further
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applied to analyse naturally contaminated maize porridge and distiller’s dried grain with solubles
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samples and the results were correlated to these obtained by UHPLC-MS/MS method.
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Keywords
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aflatoxins, aflatoxin B1, mycotoxins, ELISA, immunoassay, food, peanuts, maize, DDGS 1
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1. Introduction Aflatoxins (AFB1, AFB2, AFG1 and AFG2) are a group of the most potent carcinogens found in nature
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that are produced mainly by two fungal species of the genus Aspergillus: A. flavus and A. parasiticus.
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The former produces mainly B aflatoxins, while the latter both B and G. Aflatoxins have been
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classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer
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(IARC, 2002; IARC, 2012). The most affected crops are corn, peanuts and cottonseed but also other
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agricultural commodities such as rice, soybean and pistachio can be highly contaminated. AFM1 is a
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metabolite of AFB1 in humans and animals and can be transferred through the food chain due to its
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presence in milk from animals consuming contaminated feed. The recent BIOMIN report (BIOMIN,
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2014) shows that prevalence of aflatoxin contamination in feed grains is the highest in Africa (67% of
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screened samples tested positive), South Asia (59% positive), South-East Asia (59% positive) and
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Southern Europe (55% positive). The contamination of food and feed with aflatoxins is strictly
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controlled in many countries worldwide and maximum limits are established for different types of
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commodities. In the EU the maximum level for AFB1 is between 2 and 8 µg/kg and for the sum of
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AFB1, AFB2, AFG1 and AFG2 between 4 and 15 µg/kg in groundnuts, nuts, dried fruit and cereals. The
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maximum level of AFB1 in baby food is 0.1 µg/kg (Commission Regulation (EC) No 1881/2006). In the
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US the action limit for AFB1 in food is 20 µg/kg (FDA, 2005). The maximum content of AFB1 in feed
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materials has been set to 20 µg/kg in the EU (Commission Regulation 574/2011).
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Due to the common occurrence of aflatoxins fast and easy-to-use screening methods are required.
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Over recent years there have been increased efforts to establish fast and reliable immunochemical
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based screening methods such as ELISAs (Jiang et al., 2013, Kim et al., 2011; Kolosova et al., 2006;
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Lee et al., 2004; Li et al., 2009; Lee & Rachmawati, 2006; Lipigorngoson et al., 2003; Zhang et al.,
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2013, Rossi et al., 2012.) and lateral flow devices (LFDs) (Anfossi et al., 2011; Delmulle et al., 2005;
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Lee et al., 2013; Urusov et al., 2014) for the detection of AFB1 and total aflatoxins. While LFDs are an
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excellent choice for qualitative or semi-quantitative detection on site, reliable fast screening
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quantitative tests are required for other applications when level of contamination needs to
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determine with higher accuracy.
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In order to develop immunochemical methods for the detection of aflatoxins antibodies with good
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sensitivities and cross-reactivates are required. The high sensitivities of the antibodies allow for large
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sample dilution and thus reduction of sample matrix effects. Good cross-reactivities (ideally 100%)
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are needed in tests for total aflatoxins to avoid underestimation of the results when mixtures of
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aflatoxins are present in a sample and the results are calculated based on an AFB1 reference
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standard curve. While antibodies can have good cross-reactivities in buffer system it is also
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important to experimentally determine their performance in sample matrix. To date, Li et al., 2009
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by screening the hybridoma supernatants using AFG2 as a competitor; however the sensitivity of the
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obtained antibody was not high. On the other hand, ultra-sensitive antibody to aflatoxins was
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prepared by Zhang et al., 2009 showing IC50 of 1.2, 1.3, 2.2, 18.0 and 13.2 pg/mL for AFB1, AFB2,
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AFG1, AFG2 and AFM1, respectively in an indirect competitive ELISA.
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ELISA tests for the commercial applications should be easy to use with as few steps as possible and
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simple extraction method. Monoclonal antibodies are desirable as immortal cell lines are the source
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of unlimited supply of the antibodies of exactly the same quality. Our study aimed to develop fast
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and simple monoclonal antibody based ELISAs for the detection of AFB1 and total aflatoxins in food
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and feed. The objective of the present study was to generate a panel of monoclonals to aflatoxins
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from which the best performing antibodies in terms of sensitivity and cross reactivity would be
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selected for incorporation into prototype ELISA kits. These kits would then be subjected to a rigorous
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validation study to determine their fitness for purpose.
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2. Materials and methods
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2.1. Chemicals, consumables & apparatus
Aflatoxins (AF) B2, G1, G2 and M1, methanol, hexane, sulphuric acid, disodium hydrogen phosphate
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dihydrate, sodium dihydrogen phosphate dihydrate, sodium carbonate, sodium bicarbonate, sodium
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chloride, Tween 20, bovine serum albumin and bovine thyroglobulin were purchased from Sigma
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(Dorset, UK). Aflatoxin B1 (AFB1) was obtained from Romer Labs (Tulln, Austria). Marvell dried
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skimmed milk was purchased from Premier International Foods (UK). Rabbit anti-mouse HRP-IgG
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was obtained from Dako (Glostrup, Denmark). Tetramethylbenzidine substrate solution (TMB) was
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purchased from Millipore (Watford, UK). Quill A adjuvant was obtained from Brenntag (Leeds, UK)
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and Pam3Cys-Ser-(Lys)4 (PCSL) adjuvant from EMC Microcollections (Tuebingen, Germany). Two
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peanuts samples used for validation were Jumbo Peanuts and Red Skin Peanuts purchased from
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local stores.
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96-wells Nunc Immuno MaxiSorp plates were obtained from Nunc (Rosklide, Denmark); MAbTrap
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Protein G and Vivaspin concentrators 50 kDa MWCO were purchased from GE Healthcare (Chalfont
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St Giles, UK); slide-A-Lyzer 10K MWCO Dialysis Cassettes 0.5–3 mL capacity were purchased from
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Thermo Scientific (USA); IsoStrip – Mouse Monoclonal Antibody Isotyping Kit was purchased from
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Roche Diagnostics Ltd. (West Sussex, UK).
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A Laboratory Blender (Christison Particle Technologies, UK) was used for sample blending. DVX-2500
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Multi-Tube vortexer and ELISA plate shaker were obtained from VWR (Lutterworth, UK). The
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centrifuge Sorvall Legend RT was obtained from Thermo Scientific (USA). The ELISA plate readers
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used were Multiskan FC from Thermo Scientific (USA) and Safire2 from Tecan (Switzerland).
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2.2. Cell culture reagents and apparatus Dulbecco’s Modified Eagle’s Medium (DMEM) GlutaMAXTM without sodium pyruvate; penicillin-
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streptomycin (PenStrep); heat inactivated foetal bovine serum (HIFBS); hypoxanthine, aminopterin,
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thymidine supplement (HAT); hypoxanthine, thymidine supplement (HT) were purchased from
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Invitrogen Ltd. (Paisley, UK). Briclone hybridoma cloning medium was obtained from NICB (Dublin,
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Ireland) and polyethylene glycol fusion medium (PEG) was purchased from Immune Systems Ltd.
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(Paignton, UK). Myeloma cells SP2/0-Ag14 were obtained from the European Collection of Animal
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Cell Cultures (ECACC).
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The following media were used for cell culture work: (1) growth medium: DMEM containing 10%
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HIFBS and 1% PenStrep; (2) serum free medium: DMEM containing 1% PenStrep; (3) HAT medium:
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DMEM containing 10% HIFBS, 1% PenStrep, 1% HAT supplement and 5% Briclone; (4) HT medium:
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DMEM containing 10% HIFBS, 1% PenStrep, 1% HT supplement; and (5) cloning medium: DMEM
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containing 10% HIFBS, 1% PenStrep and 5% Briclone.
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Falcon 96 well tissue culture plates and 75 cm2 cell culture flasks were purchased from Becton
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Dickinson Labware (Oxford, UK). 500 cm2 Nunclon triple layer cell culture flasks and 24-well plates
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were obtained from Thermo Scientific (Denmark).
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2.3. Production of the AFB1 conjugates
AFB1 was first converted to AFB1 carboxymethyloxime and then conjugated to BSA, BTG and HRP
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using mixed anhydride method (Biermann and Terplan, 1980).
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2.4. Mice immunization
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Animal experiments were performed under the licence PPL 2682 granted by Department of Health,
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Social Services and Public Safety in the UK. The experiments were carried out in accordance with the
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UK Animals Scientific Procedures Act 1986. Immunisations were performed based on the procedure
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of Stewart et al., 2009. In short, AFB1-BSA and AFB1-BTG were used to immunize 4 female BALB/c
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mice (2 mice per immunogen). 8-week old animals were injected subcutaneously with 15 µg of the
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conjugate mixed with 50 µg of adjuvant in a total volume of 200 µL of sterile saline. The injections
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were repeated every 4 weeks. Quill A adjuvant was used for the first 3 immunizations and then PCSL
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for the 4th one. One of the mice died shortly after first immunization with AFB1-BSA, therefore the
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dose for the second animal was reduced to 7.5 µg for the remainder of the immunizations. Four days 4
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injected intraperitoneally. Blood samples from the tail veins were taken ten days after 2nd, 3rd and 4th
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immunization. The serum was separated by centrifugation (2000×g, 10 min) after overnight
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incubation at 4 °C. The titre (the dilution of the serum giving the absorbance value between 1 and 2
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in the antigen-coated ELISA) and sensitivity (expressed as IC50 for each aflatoxin) were determined
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using antigen-coated ELISA.
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2.5. Monoclonal antibody production
On the day of the fusion the selected mouse was sacrificed by CO2 inhalation and its spleen was
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removed and processed immediately. The splenocytes were fused with SP2 myeloma cells according
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to the procedure first described by Köhler and Milstein (1975) using polyethylene glycol as a
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fusogen. The ratio of the splenocytes to myeloma cells used was 4:1. After the fusion the cells were
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diluted in HAT medium and plated onto twenty 96-well Falcon plates (200 µL per well). The plates
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were incubated for 12 days at 37 °C at 8% CO2. The supernatants from wells containing visible
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colonies were tested for the specific antibodies using an antigen-coated ELISA. Positive colonies
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(producing antibodies binding to the coating antigen) were transferred to the 24-well plates and
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cultured in 1 mL of HAT medium. When the cell lines achieved approximately 70% confluency the
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supernatants were screened again in the competitive antigen-coated ELISA to determine sensitivity
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and cross-reactivity of the antibodies. The selected cell lines were subcloned twice by dilution
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method to assure monoclonality. The final cell lines were grown in triple layer flasks in
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approximately 200 mL of growth medium for 14 days. Then the hybridoma supernatants were
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harvested and centrifuged (200×g, 5 min). The supernatants were concentrated using Vivaspin tubes
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to approximately 5 mL volume. The concentrated antibody was purified using MAbTrap Protein G
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kit. The purified antibody was dialyzed using dialysis cartridges against saline for 2 days. The final
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stock of the antibody was obtained by diluting the concentrated stock to 2 mg/mL with saline. The
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antibodies were stored frozen at -20 °C.
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2.6. Antigen-coated assay – mice bleeds screening, fusion screening and initial characterisation
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One hundred µL of 1 µg/mL solution of coating antigen (AFB1-BSA in case of AFB1-BTG immunized
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mice and AFB1-BSA in case of AFB1-BTG immunized mice) or free protein (BSA or BTG) prepared in
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0.1 M carb/bicarb buffer pH 9.6 was added to the Nunc MaxiSorp 96-well plate. The plate was
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incubated overnight at 4 °C. The solution was then discarded and 200 µL of 1% skimmed milk
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solution prepared in 0.1 M phosphate buffer pH 7.2 was added and incubated on a plate shaker for
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45 min at 37 °C. The blocking buffer was discarded and the plate was washed three times with ELISA 5
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mice bleed (or hybridoma supernatant) diluted in 0.1 M phosphate buffer pH 7.2 and additional 50
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µL of buffer or aflatoxin standard were added to two wells coated with conjugate and two wells
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coated with free protein (to test for unspecific binding). The plates was incubated for further 45 min
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at 37 °C on a plate shaker. After washing three times 100 µL of anti-mouse antibody labelled with
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HRP enzyme was added (diluted 1/2000 in 0.1 M phosphate buffer buffer pH 7.2) and the plate was
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incubated for further 45 min at 37 °C. The plate was washed three times and 100 µL of TMB solution
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was added to each well and then the plate was developed for 10 min at room temperature. The
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absorbance was recorded on a plate reader at 450 nm after adding 25 µL of 2.5 M sulphuric acid.
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The microtitre strips were coated overnight at room temperature with 10 µg/mL polyclonal rabbit
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anti-mouse antibody diluted in phosphate buffered saline (PBS). Seven points standard curve for
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AFB1 in assay buffer (PBS with 1% BSA and 0.01% Tween 20) containing 20% methanol was prepared
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in the range 0–0.2 ng/mL. 50 µL of the standards or extracted samples were added to the wells in
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duplicate. Background control was also prepared by adding 100 µL of assay buffer to two wells. Then
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25 µL the antibody (diluted 1/20000) and 25 µL of the AFB1-HRP (diluted 1/20000) diluted in the
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assay buffer were added to each well except the background control wells. The strips were
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incubated on a shaker at 37 °C in the dark. Then the solution was discarded and the strips were
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washed three times with rinsing buffer (PBS with 0.05% Tween 20). 100 µL of TMB was added to
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each well and then the strips were incubated for 30 min at 20 to 25 °C in the dark. After adding 100
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µL of stop solution (0.5 M sulphuric acid) the strips were read at 450 nm on a microtiter plate
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reader.
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2.8. Sample preparation
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Samples (50 to 100 g) of groundnuts, peanut butter, maize porridge and feed ingredient – distiller’s
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dried grain with solubles (DDGS) were blended into homogeneous powder or slurry using a
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laboratory blender. A 3 g of the sample was weighted into a polypropylene centrifuge tube. A 9 mL
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aliquot of 80% (v/v) methanol was added and the mixture was shaken for 10 minutes using multi-
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tube vortex. After centrifugation (10 min at 2000 x g), 250 µL of the supernatant was added to 750
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µL of the assay buffer and mixed. Maize porridge and DDGS samples were ready for the analysis.
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Peanut and peanut butter samples were additionally defatted by adding 1 mL of n-hexane. The
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sample were vortexed for 1 min and centrifuged (10 min, 2000 x g). The hexane layer was discarded
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and the bottom layer was used for the ELISA analysis. The spiked samples were prepared by adding
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aflatoxins standard solutions to the samples before extraction.
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2.9. AFB1 and total AF ELISAs validation The validation of the AFB1 and total AF ELISAs was performed according to the European
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Commission Decision 2002/657/EC using a modified method (Scortichini et al., 2005; Cooper et al.,
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2007). The detection capability (CCβ), recovery and repeatability of the assays were determined. The
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detection capability is ‘‘the smallest content of the substance that may be detected, identified
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and/or quantified in a sample with an error probability β’’. The β error (false compliant rate) should
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be less than 5% as specified in Commission Decision 2002/657/EC. The blank matrix effect was
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determined by analysing ten replicates of two different peanut samples using AFB1 and total AF
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ELISAs, and the mean value for the blank samples was calculated. The same set of samples was
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spiked with AFB1 at various concentrations and analyzed by the ELISA kits to determine the detection
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capability and verify if the β error (false compliant rate) was less than 5%. The spiking levels selected
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were as close to the blank samples results as possible.
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The recovery and repeatability for the AFB1 ELISA were determined by analysing three sets of six
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peanut samples (n=6) spiked with AFB1 at the concentration of 0.5, 1 and 1.5 times the maximum
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permitted level for AFB1. According to the Commission Regulation 1881/2006 the maximum level is 2
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µg/kg for AFB1 and 4 µg/kg for the sum of AFB1, AFB2, AFG1 and AFG2 in peanuts. For the total AF
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ELISA eight sets of two peanut samples (n=2) fortified with two different concentrations of AFB1,
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AFB2, AFG1 (1 and 2 µg/kg) and AFG2 (2 and 4 µg/kg) were analyzed. The mean concentrations, mean
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recoveries, standard deviations (SD) and coefficients of variation (CV) were calculated at each level.
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The procedures for AFB1 and total AF ELISAs recovery and repeatability study were repeated two
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more times to determine the overall means, SD and CV.
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3. Results and Discussion
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3.1. Screening of mice bleeds
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Antibodies specific to the target were detected in mice bleeds just after two immunizations. Two
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fusions were performed using spleens collected from AFB1-BSA immunized mouse after third
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injection and from AFB1-BTG immunized mouse after fourth injection.
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3.2. Fusion screening and characterisation of the final monoclonal antibodies
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A total of 525 hybridomas from the first fusion and 505 from the second fusion were screened by
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the non-competitive antigen-coated ELISA. Four clones from the first fusion and three from the 7
ACCEPTED MANUSCRIPT second fusion showing superior performance in terms of cross-reactivity and sensitivity were
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selected for further work. After two rounds of cloning the final cell lines were established. The cell
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lines produced between 41–90 µg of monoclonal antibody per mL of cell culture medium. The final
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2 mg/mL antibodies stocks were characterized using the competitive antigen-coated ELISA. The
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standard curves for five aflatoxins were prepared in the range 0.001–100 ng/mL. Five antibodies
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showed broad cross-reactivity with the five aflatoxin standards incorporated into the test, while two
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antibodies were more specific to AFB1.
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3.3. AFB1 and total AF ELISAs development
Two antibodies – 1NP-D and 1NP-C were selected for the development of AFB1 and total AF ELISAs,
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respectively. The IC50 for AFB1 for these two antibodies characterised in a final test format were
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0.037 ± 0.002 ng/mL and 0.031 ± 0.001 ng/mL, respectively. Figure 1 shows typical standard curve
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for AFB1. The cross-reactivities (defined as the ratio of IC50 of AFB1 and IC50 of the tested aflatoxin
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multiplied by 100%) with AFB2, AFG1, AFG2 and AFM1 were 30%, 59%, 8% and 4% for 1NP-D and 46%,
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65%, 11% and 7% for 1NP-C.
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3.4. AFB1 and total AF ELISAs validation
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The developed assays were validated in peanut matrix. The summary results for the blank and spiked
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samples are presented in Table 1. Separation of blank and spiked samples was obtained for each
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assay (Fig. 2.). There was no overlap between the blank and fortified samples which means the
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detection capabilities were less than the spiked levels. Both α (false non-compliant rate) and β (false
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compliant rate) errors were found to be zero for both AFB1 and total AF ELISAs.
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Table 1 Determination of the detection capability (CCβ) of the AFB1 and total AF ELISAs.
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blank samples
fortified samples
mean value [µg/g] range [µg/kg] mean value [µg/kg] range [µg/kg]
CCβ [µg/kg]
AFB1 ELISA 0.08 0.00–0.36 0.46 0.41–0.55 <0.4
total AF ELISA 0.03 0.00–0.15 0.34 0.25–0.44 <0.3
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The results of the recovery and repeatability study for AFB1 ELISA are presented in Table 2. The
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overall recoveries (97.1–107.3%) and the CV (7.8–11.0%) were deemed to be highly acceptable for
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all concentration levels tested.
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different concentrations. The results of the recovery and repeatability study for AFB1, AFB2, AFG1 and
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AFG2 are presented in Table 2. The samples were spiked separately with 1 and 2 µg/kg of AFB1, AFB2
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and AFG1, while spiking concentrations for AFG2 were 3 and 4 µg/kg. When calculating from AFB1
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standard curve the mean measured concentrations at two levels were 0.86 and 1.66 µg/kg for AFB1,
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0.30 and 0.49 µg/kg for AFB2, 0.37 and 0.69 µg/kg for AFG1 and 0.22 and 0.25 µg/kg for AFG2. The
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differences in measured and spiked concentrations for AFB2, AFG1 and AFG2 are the results of the
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cross-reactivity profile of the antibody in matrix. The cross-reactivity for AFB2, AFG1 and AFG2
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measured in buffer were 46%, 65% and 11% respectively. The mean recoveries obtained for AFB2
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(24.5–30.0%), AFG1 (34.4–36.8%) and AFG2 (6.3–7.6%) were between 0.5 to 0.7 times the cross-
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reactivity values in buffer and this can be a result of different cross-reactivity profile in matrix and
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also analyte losses during extraction. AFB1 is the most common aflatoxin and it co-occurs with other
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aflatoxins, while AFB2, AFG1 and AFG2 generally do not occur without AFB1. In this assay set-up AFB1
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alone can be detected at as low as 0.3 µg/kg, which is the detection capability of the assay.
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Theoretically, if only AFB2 or AFG1 were present in a sample at the level 2 µg/kg (which is half of the
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maximum limit 4 µg/kg for total aflatoxin content in peanuts), the measured concentration would be
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on average 0.49 and 0.69 µg/kg, respectively which is above the detection capability of the assay. If
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the sample was contaminated with 4 µg/kg of AFG2 alone the measured concentration would be just
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below the detection capability. However, this is unlikely to happen as AFG2 does not occur on its
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own, without the presence of other aflatoxins.
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Table 2 Recovery and coefficient of variation data for the detection of aflatoxins in peanut samples
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by the AFB1 and total AF ELISAs (n=18 for AFB1 ELISA and n=6 for total AF ELISA at each level).
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AFB1 ELISA
AFB1
AFB1 AFB2
total AF ELISA AFG1 AFG2
fortification mean concentration mean recovery CV [%] level[µg/kg] ±SD [µg/kg] ±SD [%] 1 2 3 1 2 1 2 1 2 3 4
0.97 ± 0.11 2.00 ± 0.16 3.22 ± 0.25 0.86 ± 0.14 1.66 ± 0.15 0.30 ± 0.04 0.49 ± 0.09 0.37 ± 0.07 0.69 ± 0.10 0.22 ± 0.02 0.25 ± 0.04 9
97.1 ± 10.7 100.2 ± 7.8 107.3 ± 8.4 85.7 ± 13.6 83.0 ± 7.5 30.0 ± 4.3 24.5 ± 4.5 36.8 ± 6.9 34.4 ± 4.9 7.6 ± 0.7 6.3 ± 1.1
11.0 7.8 7.8 15.9 9.1 14.1 18.6 18.7 14.4 8.7 15.3
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3.5. Application of the AFB1 and total AF ELISAs for the analysis of peanut samples In order to test the developed kits a survey of both peanut and peanut butter samples purchased
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from different retailers in the UK was performed. A total of 26 peanut samples and 6 peanut butter
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samples were analyzed using both AFB1 and total AF ELISA kits. Two of these samples were found to
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contain aflatoxins at concentrations above the detection capabilities of the assays: 0.38 µg/kg AFB1
293
and 0.43 µg/kg total aflatoxin in the first sample and 0.44 µg/kg AFB1 and 0.44 µg/kg total aflatoxins
294
in the second sample. In both cases the levels were lower than the regulatory limits (2 µg/kg for AFB1
295
and 4 µg/kg for total aflatoxins) what was confirmed by UHPLC-MS/MS analysis (Oplatowska-
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Stachowiak et al., 2015). UHPLC-MS/MS method detected the presence of 0.28 µg/kg and 0.67 µg/kg
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of AFB1 in first and second sample, respectively. AFB2, AFG1 and AFG2 were found to be below the
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limit of detection.
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3.6. Application of the total AF ELISA for the analysis of naturally contaminated DDGS and maize porridge samples
Twenty five DDGS and 20 maize porridge samples were analyzed by the total AF ELISA. The results
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were correlated to these obtained by UHPLC-MS/MS method (Oplatowska-Stachowiak et al., 2015).
304
There was a good agreement between the total AF ELISA and UHPLC-MS/MS methods as the square
305
of the correlation coefficients were 0.97 for DDGS and 0.95 for maize porridge samples (Fig. 3.). The
306
slopes of the regression lines were 0.74 for DDGS and 0.62 for maize porridge samples, indicating
307
underestimation of the total aflatoxin content by ELISA. While UHPLC-MS/MS can quantitate each
308
aflatoxin separately, ELISA measures total aflatoxins content as AFB1 equivalent. Therefore, if a
309
mixture of aflatoxins is present in a sample the result by ELISA will be lower due to the different
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cross-reactivity of the antibody with each aflatoxin. According to the UHPLC-MS/MS results AFB1
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constituted 69–100%, AFB2 0–31%, AFG1 0–20% and AFG1 0–3% of the total aflatoxins content in
312
DDGS. As for maize porridge samples the percentages of each AFB1, AFB2, AFG1 and AFG2 in respect
313
to the total aflatoxins content were 50–100%, 0–7%, 0–50% and 0–4%, respectively.
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3.7. Proficiency testing of the total AF ELISA
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Total AF ELISA was tested in three proficiency testing schemes and z-scores were satisfactory in each
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case (│z│<2) (Table 3). As the mixtures of aflatoxins were present in each sample the results
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obtained by ELISA were lower than the total AF assigned values due to the different cross-reactivity
319
of the antibody with each aflatoxin. The best result was obtained for rye flour. This sample
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contained the highest percentage of AFB1 with which the antibody has the highest cross-reactivity. 10
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The method was demonstrated to be suitable for the screening of various matrices for total AF
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content.
323 Table 3 Results of the proficiency tests for the total AF ELISA in different matrices.
AFB1 AFB2 AFG1 AFG2 total AF
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22.34 1.714* 10.48 0.5* 33.84
32.05
FAPAS® Proficiency Test 2014 (04243) (peanut) zscore
-0.24
measured value [µg/kg]
3.32 2.29 3.19 1.48 10.32
6.90
*median of reference dataset (no assigned value)
zscore
assigned value [µg/kg]
measured value [µg/kg]
zscore
3.80 1.89 1.07 1.00 7.67
-1.5
5.60
-1.2
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assigned value [µg/kg]
FAPAS® Proficiency Test 2015 (04261) (maize)
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CODA-CERVA Multimycotoxin Proficiency Testing 2014 (rye flour) assigned measured value value [µg/kg] [µg/kg]
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4. Conclusions
Seven monoclonal antibodies against aflatoxins were produced. Two of the best performing
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antibodies in terms of sensitivity and cross-reactivity were used to develop AFB1 and total AF ELISA
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prototype test kits. These kits were validated for the detection of aflatoxin contamination in
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peanuts, demonstrating the applicability of the method for the detection of AFB1 and sum of
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aflatoxins below the maximum levels set by the Commission Regulation (EC) No 1881/2006 (2 and
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4 µg/kg, respectively). The test kits were used to screen 32 samples purchased from stores in the UK.
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Two samples contained low levels of AFB1 which were subsequently confirmed by UHPLC-MS/MS
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analysis. The total AF ELISA was also applied to test naturally contaminated maize porridge and
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DDGS samples and a good correlation between the ELISA results and UHPLC-MS/MS analysis was
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found. The method trueness was confirmed in three proficiency tests in rye, peanut and maize
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matrices. These new methods will be transformed into commercial aflatoxin fast screening tools for
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the protection of consumers in the EU and worldwide.
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Acknowledgements
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The authors would like to thank Dr Caroline Frizzell and Dr Rachel Clarke for their help with the
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monoclonal antibody production.
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Köhler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495–497. Kolosova, A. Y., Shim, W.-B., Yang, Z.-Y., Eremin, S. A., & Chung, D.-H. (2006). Direct competitive
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Lee, N. A., Wang, S., Allan, R. D., & Kennedy, I. R. (2004). A rapid aflatoxin B1 ELISA: development
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Li, P., Zhang, Q., Zhang, W., Zhang, J., Chen, X., Jiang, J., Xie, L., & Zhang, D. (2009). Development of a
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class-specific monoclonal antibody-based ELISA for aflatoxins in peanut. Food Chemistry, 115(1),
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Lipigorngoson, S., Limtrakul, P., Suttajit, M., & Yoshizawa, T. (2003). In-house direct cELISA for
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determining aflatoxin B1 in Thai corn and peanuts. Food Additives and Contaminants, 20(9),
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Rossi, C. N., Takabayashi, C. R., Ono, M. A., Saito, G. H., Itano, E. N., Kawamura, O., Hirooka, E. Y., &
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Stewart, L. D., Elliott, C. T., Walker, A. D., Curran, R. M., & Connolly, L. (2009). Development of a
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monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their
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toxicity equivalence factors. Toxicon, 54(4), 491–498. 13
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Urusov, A. E., Zherdev, A. V., & Dzantiev, B. B. (2014). Use of gold nanoparticle-labeled secondary
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antibodies to improve the sensitivity of an immunochromatographic assay for aflatoxin B1.
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Microchimica Acta, 181(15), 1939–1946. Zhang, D., Li, P., Zhang, Q., Zhang, W., Huang, Y., Ding, X., & Jiang, J. (2009). Production of
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screening procedure. Analytica Chmica Acta, 636(1), 63–69.
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Figure captions
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Fig. 1. Typical standard curves for AFB1 (n=5).
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Fig. 2. Determination of the detection capability of AFB1 and total AF ELISAs.
423 Fig. 3. Correlation between total aflatoxins concentration (sum of AFB1, AFB2, AFG1 and AFG2) in
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naturally contaminated a) DDGS (n=25) and b) maize porridge samples (n=20), as determined by
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UHPLC-MS/MS method (x-axis) and total AF ELISA (y-axis). Results are in µg/kg. Solid line represents
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regression through the origin.
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ACCEPTED MANUSCRIPT Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients
Michalina Oplatowska-Stachowiaka, Nermin Sajicb, Ya Xua, Simon A. Haugheya, Mark Mooneya, Yun
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Yun Gonga, Ron Verheijenb & Christopher T. Elliotta
Highlights
Seven highly sensitive monoclonal antibodies against aflatoxins were produced
•
Fast aflatoxin B1 and total aflatoxin ELISAs were developed and validated in peanuts
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The detection capabilities for aflatoxin B1 were very low: 0.4 µg/kg and 0.3 µg/kg
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32 peanut samples were screened and two low contaminated samples were identified
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High accuracy of the developed assay was confirmed in proficiency tests
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S0956-7135(15)30312-1
DOI:
10.1016/j.foodcont.2015.11.041
Reference:
JFCO 4770
To appear in:
Food Control
Received Date: 11 May 2015 Revised Date:
21 November 2015
Accepted Date: 28 November 2015
Please cite this article as: Oplatowska-Stachowiak M., Sajic N., Xu Y., Haughey S.A., Mooney M., Gong Y.Y., Verheijen R. & Elliott C.T., Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients, Food Control (2016), doi: 10.1016/j.foodcont.2015.11.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of
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peanuts, maize and feed ingredients
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Michalina Oplatowska-Stachowiaka,b*, Nermin Sajicb, Ya Xua, Simon A. Haugheya, Mark Mooneya, Yun
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Yun Gonga, Ron Verheijenb & Christopher T. Elliotta
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a
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Malone Road, Belfast BT95BN, United Kingdom
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b
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Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, 18-30
EuroProxima B.V., Beijerinckweg 18, 6827 BN Arnhem, The Netherlands
*Corresponding author. Email: [email protected], tel: +44 (0) 289097 6531,
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fax: +44 (0) 289097 6513
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12 Abstract
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Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on
15
different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious
16
illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical
17
methods are required for the identification of contaminated agricultural commodities before they
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are processed into final products and placed on the market. In order to provide new tools for
19
aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B1 and the
20
other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity
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and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were
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characterized and two antibodies showing IC50 of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B1 were
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applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut
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matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities
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of aflatoxin B1 and total aflatoxins ELISAs were 0.4 µg/kg and 0.3 µg/kg for aflatoxin B1, respectively,
26
which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the
27
detection of aflatoxins B2, G1 and G2. The application of the developed tests was demonstrated by
28
screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further
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applied to analyse naturally contaminated maize porridge and distiller’s dried grain with solubles
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samples and the results were correlated to these obtained by UHPLC-MS/MS method.
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Keywords
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aflatoxins, aflatoxin B1, mycotoxins, ELISA, immunoassay, food, peanuts, maize, DDGS 1
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1. Introduction Aflatoxins (AFB1, AFB2, AFG1 and AFG2) are a group of the most potent carcinogens found in nature
36
that are produced mainly by two fungal species of the genus Aspergillus: A. flavus and A. parasiticus.
37
The former produces mainly B aflatoxins, while the latter both B and G. Aflatoxins have been
38
classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer
39
(IARC, 2002; IARC, 2012). The most affected crops are corn, peanuts and cottonseed but also other
40
agricultural commodities such as rice, soybean and pistachio can be highly contaminated. AFM1 is a
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metabolite of AFB1 in humans and animals and can be transferred through the food chain due to its
42
presence in milk from animals consuming contaminated feed. The recent BIOMIN report (BIOMIN,
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2014) shows that prevalence of aflatoxin contamination in feed grains is the highest in Africa (67% of
44
screened samples tested positive), South Asia (59% positive), South-East Asia (59% positive) and
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Southern Europe (55% positive). The contamination of food and feed with aflatoxins is strictly
46
controlled in many countries worldwide and maximum limits are established for different types of
47
commodities. In the EU the maximum level for AFB1 is between 2 and 8 µg/kg and for the sum of
48
AFB1, AFB2, AFG1 and AFG2 between 4 and 15 µg/kg in groundnuts, nuts, dried fruit and cereals. The
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maximum level of AFB1 in baby food is 0.1 µg/kg (Commission Regulation (EC) No 1881/2006). In the
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US the action limit for AFB1 in food is 20 µg/kg (FDA, 2005). The maximum content of AFB1 in feed
51
materials has been set to 20 µg/kg in the EU (Commission Regulation 574/2011).
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Due to the common occurrence of aflatoxins fast and easy-to-use screening methods are required.
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Over recent years there have been increased efforts to establish fast and reliable immunochemical
54
based screening methods such as ELISAs (Jiang et al., 2013, Kim et al., 2011; Kolosova et al., 2006;
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Lee et al., 2004; Li et al., 2009; Lee & Rachmawati, 2006; Lipigorngoson et al., 2003; Zhang et al.,
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2013, Rossi et al., 2012.) and lateral flow devices (LFDs) (Anfossi et al., 2011; Delmulle et al., 2005;
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Lee et al., 2013; Urusov et al., 2014) for the detection of AFB1 and total aflatoxins. While LFDs are an
58
excellent choice for qualitative or semi-quantitative detection on site, reliable fast screening
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quantitative tests are required for other applications when level of contamination needs to
60
determine with higher accuracy.
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In order to develop immunochemical methods for the detection of aflatoxins antibodies with good
62
sensitivities and cross-reactivates are required. The high sensitivities of the antibodies allow for large
63
sample dilution and thus reduction of sample matrix effects. Good cross-reactivities (ideally 100%)
64
are needed in tests for total aflatoxins to avoid underestimation of the results when mixtures of
65
aflatoxins are present in a sample and the results are calculated based on an AFB1 reference
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standard curve. While antibodies can have good cross-reactivities in buffer system it is also
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important to experimentally determine their performance in sample matrix. To date, Li et al., 2009
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by screening the hybridoma supernatants using AFG2 as a competitor; however the sensitivity of the
70
obtained antibody was not high. On the other hand, ultra-sensitive antibody to aflatoxins was
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prepared by Zhang et al., 2009 showing IC50 of 1.2, 1.3, 2.2, 18.0 and 13.2 pg/mL for AFB1, AFB2,
72
AFG1, AFG2 and AFM1, respectively in an indirect competitive ELISA.
73
ELISA tests for the commercial applications should be easy to use with as few steps as possible and
74
simple extraction method. Monoclonal antibodies are desirable as immortal cell lines are the source
75
of unlimited supply of the antibodies of exactly the same quality. Our study aimed to develop fast
76
and simple monoclonal antibody based ELISAs for the detection of AFB1 and total aflatoxins in food
77
and feed. The objective of the present study was to generate a panel of monoclonals to aflatoxins
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from which the best performing antibodies in terms of sensitivity and cross reactivity would be
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selected for incorporation into prototype ELISA kits. These kits would then be subjected to a rigorous
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validation study to determine their fitness for purpose.
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2. Materials and methods
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2.1. Chemicals, consumables & apparatus
Aflatoxins (AF) B2, G1, G2 and M1, methanol, hexane, sulphuric acid, disodium hydrogen phosphate
85
dihydrate, sodium dihydrogen phosphate dihydrate, sodium carbonate, sodium bicarbonate, sodium
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chloride, Tween 20, bovine serum albumin and bovine thyroglobulin were purchased from Sigma
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(Dorset, UK). Aflatoxin B1 (AFB1) was obtained from Romer Labs (Tulln, Austria). Marvell dried
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skimmed milk was purchased from Premier International Foods (UK). Rabbit anti-mouse HRP-IgG
89
was obtained from Dako (Glostrup, Denmark). Tetramethylbenzidine substrate solution (TMB) was
90
purchased from Millipore (Watford, UK). Quill A adjuvant was obtained from Brenntag (Leeds, UK)
91
and Pam3Cys-Ser-(Lys)4 (PCSL) adjuvant from EMC Microcollections (Tuebingen, Germany). Two
92
peanuts samples used for validation were Jumbo Peanuts and Red Skin Peanuts purchased from
93
local stores.
94
96-wells Nunc Immuno MaxiSorp plates were obtained from Nunc (Rosklide, Denmark); MAbTrap
95
Protein G and Vivaspin concentrators 50 kDa MWCO were purchased from GE Healthcare (Chalfont
96
St Giles, UK); slide-A-Lyzer 10K MWCO Dialysis Cassettes 0.5–3 mL capacity were purchased from
97
Thermo Scientific (USA); IsoStrip – Mouse Monoclonal Antibody Isotyping Kit was purchased from
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Roche Diagnostics Ltd. (West Sussex, UK).
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A Laboratory Blender (Christison Particle Technologies, UK) was used for sample blending. DVX-2500
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Multi-Tube vortexer and ELISA plate shaker were obtained from VWR (Lutterworth, UK). The
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centrifuge Sorvall Legend RT was obtained from Thermo Scientific (USA). The ELISA plate readers
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used were Multiskan FC from Thermo Scientific (USA) and Safire2 from Tecan (Switzerland).
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2.2. Cell culture reagents and apparatus Dulbecco’s Modified Eagle’s Medium (DMEM) GlutaMAXTM without sodium pyruvate; penicillin-
106
streptomycin (PenStrep); heat inactivated foetal bovine serum (HIFBS); hypoxanthine, aminopterin,
107
thymidine supplement (HAT); hypoxanthine, thymidine supplement (HT) were purchased from
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Invitrogen Ltd. (Paisley, UK). Briclone hybridoma cloning medium was obtained from NICB (Dublin,
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Ireland) and polyethylene glycol fusion medium (PEG) was purchased from Immune Systems Ltd.
110
(Paignton, UK). Myeloma cells SP2/0-Ag14 were obtained from the European Collection of Animal
111
Cell Cultures (ECACC).
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The following media were used for cell culture work: (1) growth medium: DMEM containing 10%
113
HIFBS and 1% PenStrep; (2) serum free medium: DMEM containing 1% PenStrep; (3) HAT medium:
114
DMEM containing 10% HIFBS, 1% PenStrep, 1% HAT supplement and 5% Briclone; (4) HT medium:
115
DMEM containing 10% HIFBS, 1% PenStrep, 1% HT supplement; and (5) cloning medium: DMEM
116
containing 10% HIFBS, 1% PenStrep and 5% Briclone.
117
Falcon 96 well tissue culture plates and 75 cm2 cell culture flasks were purchased from Becton
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Dickinson Labware (Oxford, UK). 500 cm2 Nunclon triple layer cell culture flasks and 24-well plates
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were obtained from Thermo Scientific (Denmark).
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2.3. Production of the AFB1 conjugates
AFB1 was first converted to AFB1 carboxymethyloxime and then conjugated to BSA, BTG and HRP
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using mixed anhydride method (Biermann and Terplan, 1980).
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2.4. Mice immunization
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Animal experiments were performed under the licence PPL 2682 granted by Department of Health,
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Social Services and Public Safety in the UK. The experiments were carried out in accordance with the
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UK Animals Scientific Procedures Act 1986. Immunisations were performed based on the procedure
129
of Stewart et al., 2009. In short, AFB1-BSA and AFB1-BTG were used to immunize 4 female BALB/c
130
mice (2 mice per immunogen). 8-week old animals were injected subcutaneously with 15 µg of the
131
conjugate mixed with 50 µg of adjuvant in a total volume of 200 µL of sterile saline. The injections
132
were repeated every 4 weeks. Quill A adjuvant was used for the first 3 immunizations and then PCSL
133
for the 4th one. One of the mice died shortly after first immunization with AFB1-BSA, therefore the
134
dose for the second animal was reduced to 7.5 µg for the remainder of the immunizations. Four days 4
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injected intraperitoneally. Blood samples from the tail veins were taken ten days after 2nd, 3rd and 4th
137
immunization. The serum was separated by centrifugation (2000×g, 10 min) after overnight
138
incubation at 4 °C. The titre (the dilution of the serum giving the absorbance value between 1 and 2
139
in the antigen-coated ELISA) and sensitivity (expressed as IC50 for each aflatoxin) were determined
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using antigen-coated ELISA.
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2.5. Monoclonal antibody production
On the day of the fusion the selected mouse was sacrificed by CO2 inhalation and its spleen was
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removed and processed immediately. The splenocytes were fused with SP2 myeloma cells according
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to the procedure first described by Köhler and Milstein (1975) using polyethylene glycol as a
146
fusogen. The ratio of the splenocytes to myeloma cells used was 4:1. After the fusion the cells were
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diluted in HAT medium and plated onto twenty 96-well Falcon plates (200 µL per well). The plates
148
were incubated for 12 days at 37 °C at 8% CO2. The supernatants from wells containing visible
149
colonies were tested for the specific antibodies using an antigen-coated ELISA. Positive colonies
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(producing antibodies binding to the coating antigen) were transferred to the 24-well plates and
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cultured in 1 mL of HAT medium. When the cell lines achieved approximately 70% confluency the
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supernatants were screened again in the competitive antigen-coated ELISA to determine sensitivity
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and cross-reactivity of the antibodies. The selected cell lines were subcloned twice by dilution
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method to assure monoclonality. The final cell lines were grown in triple layer flasks in
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approximately 200 mL of growth medium for 14 days. Then the hybridoma supernatants were
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harvested and centrifuged (200×g, 5 min). The supernatants were concentrated using Vivaspin tubes
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to approximately 5 mL volume. The concentrated antibody was purified using MAbTrap Protein G
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kit. The purified antibody was dialyzed using dialysis cartridges against saline for 2 days. The final
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stock of the antibody was obtained by diluting the concentrated stock to 2 mg/mL with saline. The
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antibodies were stored frozen at -20 °C.
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One hundred µL of 1 µg/mL solution of coating antigen (AFB1-BSA in case of AFB1-BTG immunized
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mice and AFB1-BSA in case of AFB1-BTG immunized mice) or free protein (BSA or BTG) prepared in
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0.1 M carb/bicarb buffer pH 9.6 was added to the Nunc MaxiSorp 96-well plate. The plate was
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incubated overnight at 4 °C. The solution was then discarded and 200 µL of 1% skimmed milk
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solution prepared in 0.1 M phosphate buffer pH 7.2 was added and incubated on a plate shaker for
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45 min at 37 °C. The blocking buffer was discarded and the plate was washed three times with ELISA 5
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mice bleed (or hybridoma supernatant) diluted in 0.1 M phosphate buffer pH 7.2 and additional 50
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µL of buffer or aflatoxin standard were added to two wells coated with conjugate and two wells
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coated with free protein (to test for unspecific binding). The plates was incubated for further 45 min
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at 37 °C on a plate shaker. After washing three times 100 µL of anti-mouse antibody labelled with
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HRP enzyme was added (diluted 1/2000 in 0.1 M phosphate buffer buffer pH 7.2) and the plate was
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incubated for further 45 min at 37 °C. The plate was washed three times and 100 µL of TMB solution
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was added to each well and then the plate was developed for 10 min at room temperature. The
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absorbance was recorded on a plate reader at 450 nm after adding 25 µL of 2.5 M sulphuric acid.
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The microtitre strips were coated overnight at room temperature with 10 µg/mL polyclonal rabbit
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anti-mouse antibody diluted in phosphate buffered saline (PBS). Seven points standard curve for
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AFB1 in assay buffer (PBS with 1% BSA and 0.01% Tween 20) containing 20% methanol was prepared
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in the range 0–0.2 ng/mL. 50 µL of the standards or extracted samples were added to the wells in
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duplicate. Background control was also prepared by adding 100 µL of assay buffer to two wells. Then
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25 µL the antibody (diluted 1/20000) and 25 µL of the AFB1-HRP (diluted 1/20000) diluted in the
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assay buffer were added to each well except the background control wells. The strips were
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incubated on a shaker at 37 °C in the dark. Then the solution was discarded and the strips were
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washed three times with rinsing buffer (PBS with 0.05% Tween 20). 100 µL of TMB was added to
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each well and then the strips were incubated for 30 min at 20 to 25 °C in the dark. After adding 100
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µL of stop solution (0.5 M sulphuric acid) the strips were read at 450 nm on a microtiter plate
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reader.
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Samples (50 to 100 g) of groundnuts, peanut butter, maize porridge and feed ingredient – distiller’s
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dried grain with solubles (DDGS) were blended into homogeneous powder or slurry using a
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laboratory blender. A 3 g of the sample was weighted into a polypropylene centrifuge tube. A 9 mL
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aliquot of 80% (v/v) methanol was added and the mixture was shaken for 10 minutes using multi-
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tube vortex. After centrifugation (10 min at 2000 x g), 250 µL of the supernatant was added to 750
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µL of the assay buffer and mixed. Maize porridge and DDGS samples were ready for the analysis.
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Peanut and peanut butter samples were additionally defatted by adding 1 mL of n-hexane. The
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sample were vortexed for 1 min and centrifuged (10 min, 2000 x g). The hexane layer was discarded
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and the bottom layer was used for the ELISA analysis. The spiked samples were prepared by adding
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aflatoxins standard solutions to the samples before extraction.
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2.9. AFB1 and total AF ELISAs validation The validation of the AFB1 and total AF ELISAs was performed according to the European
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Commission Decision 2002/657/EC using a modified method (Scortichini et al., 2005; Cooper et al.,
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2007). The detection capability (CCβ), recovery and repeatability of the assays were determined. The
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detection capability is ‘‘the smallest content of the substance that may be detected, identified
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and/or quantified in a sample with an error probability β’’. The β error (false compliant rate) should
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be less than 5% as specified in Commission Decision 2002/657/EC. The blank matrix effect was
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determined by analysing ten replicates of two different peanut samples using AFB1 and total AF
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ELISAs, and the mean value for the blank samples was calculated. The same set of samples was
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spiked with AFB1 at various concentrations and analyzed by the ELISA kits to determine the detection
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capability and verify if the β error (false compliant rate) was less than 5%. The spiking levels selected
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were as close to the blank samples results as possible.
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The recovery and repeatability for the AFB1 ELISA were determined by analysing three sets of six
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peanut samples (n=6) spiked with AFB1 at the concentration of 0.5, 1 and 1.5 times the maximum
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permitted level for AFB1. According to the Commission Regulation 1881/2006 the maximum level is 2
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µg/kg for AFB1 and 4 µg/kg for the sum of AFB1, AFB2, AFG1 and AFG2 in peanuts. For the total AF
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ELISA eight sets of two peanut samples (n=2) fortified with two different concentrations of AFB1,
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AFB2, AFG1 (1 and 2 µg/kg) and AFG2 (2 and 4 µg/kg) were analyzed. The mean concentrations, mean
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recoveries, standard deviations (SD) and coefficients of variation (CV) were calculated at each level.
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The procedures for AFB1 and total AF ELISAs recovery and repeatability study were repeated two
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more times to determine the overall means, SD and CV.
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3. Results and Discussion
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3.1. Screening of mice bleeds
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Antibodies specific to the target were detected in mice bleeds just after two immunizations. Two
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fusions were performed using spleens collected from AFB1-BSA immunized mouse after third
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injection and from AFB1-BTG immunized mouse after fourth injection.
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3.2. Fusion screening and characterisation of the final monoclonal antibodies
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A total of 525 hybridomas from the first fusion and 505 from the second fusion were screened by
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the non-competitive antigen-coated ELISA. Four clones from the first fusion and three from the 7
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selected for further work. After two rounds of cloning the final cell lines were established. The cell
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lines produced between 41–90 µg of monoclonal antibody per mL of cell culture medium. The final
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2 mg/mL antibodies stocks were characterized using the competitive antigen-coated ELISA. The
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standard curves for five aflatoxins were prepared in the range 0.001–100 ng/mL. Five antibodies
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showed broad cross-reactivity with the five aflatoxin standards incorporated into the test, while two
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antibodies were more specific to AFB1.
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3.3. AFB1 and total AF ELISAs development
Two antibodies – 1NP-D and 1NP-C were selected for the development of AFB1 and total AF ELISAs,
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respectively. The IC50 for AFB1 for these two antibodies characterised in a final test format were
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0.037 ± 0.002 ng/mL and 0.031 ± 0.001 ng/mL, respectively. Figure 1 shows typical standard curve
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for AFB1. The cross-reactivities (defined as the ratio of IC50 of AFB1 and IC50 of the tested aflatoxin
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multiplied by 100%) with AFB2, AFG1, AFG2 and AFM1 were 30%, 59%, 8% and 4% for 1NP-D and 46%,
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65%, 11% and 7% for 1NP-C.
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3.4. AFB1 and total AF ELISAs validation
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The developed assays were validated in peanut matrix. The summary results for the blank and spiked
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samples are presented in Table 1. Separation of blank and spiked samples was obtained for each
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assay (Fig. 2.). There was no overlap between the blank and fortified samples which means the
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detection capabilities were less than the spiked levels. Both α (false non-compliant rate) and β (false
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compliant rate) errors were found to be zero for both AFB1 and total AF ELISAs.
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Table 1 Determination of the detection capability (CCβ) of the AFB1 and total AF ELISAs.
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blank samples
fortified samples
mean value [µg/g] range [µg/kg] mean value [µg/kg] range [µg/kg]
CCβ [µg/kg]
AFB1 ELISA 0.08 0.00–0.36 0.46 0.41–0.55 <0.4
total AF ELISA 0.03 0.00–0.15 0.34 0.25–0.44 <0.3
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The results of the recovery and repeatability study for AFB1 ELISA are presented in Table 2. The
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overall recoveries (97.1–107.3%) and the CV (7.8–11.0%) were deemed to be highly acceptable for
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all concentration levels tested.
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different concentrations. The results of the recovery and repeatability study for AFB1, AFB2, AFG1 and
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AFG2 are presented in Table 2. The samples were spiked separately with 1 and 2 µg/kg of AFB1, AFB2
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and AFG1, while spiking concentrations for AFG2 were 3 and 4 µg/kg. When calculating from AFB1
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standard curve the mean measured concentrations at two levels were 0.86 and 1.66 µg/kg for AFB1,
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0.30 and 0.49 µg/kg for AFB2, 0.37 and 0.69 µg/kg for AFG1 and 0.22 and 0.25 µg/kg for AFG2. The
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differences in measured and spiked concentrations for AFB2, AFG1 and AFG2 are the results of the
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cross-reactivity profile of the antibody in matrix. The cross-reactivity for AFB2, AFG1 and AFG2
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measured in buffer were 46%, 65% and 11% respectively. The mean recoveries obtained for AFB2
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(24.5–30.0%), AFG1 (34.4–36.8%) and AFG2 (6.3–7.6%) were between 0.5 to 0.7 times the cross-
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reactivity values in buffer and this can be a result of different cross-reactivity profile in matrix and
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also analyte losses during extraction. AFB1 is the most common aflatoxin and it co-occurs with other
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aflatoxins, while AFB2, AFG1 and AFG2 generally do not occur without AFB1. In this assay set-up AFB1
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alone can be detected at as low as 0.3 µg/kg, which is the detection capability of the assay.
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Theoretically, if only AFB2 or AFG1 were present in a sample at the level 2 µg/kg (which is half of the
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maximum limit 4 µg/kg for total aflatoxin content in peanuts), the measured concentration would be
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on average 0.49 and 0.69 µg/kg, respectively which is above the detection capability of the assay. If
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the sample was contaminated with 4 µg/kg of AFG2 alone the measured concentration would be just
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below the detection capability. However, this is unlikely to happen as AFG2 does not occur on its
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own, without the presence of other aflatoxins.
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Table 2 Recovery and coefficient of variation data for the detection of aflatoxins in peanut samples
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by the AFB1 and total AF ELISAs (n=18 for AFB1 ELISA and n=6 for total AF ELISA at each level).
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total AF ELISA AFG1 AFG2
fortification mean concentration mean recovery CV [%] level[µg/kg] ±SD [µg/kg] ±SD [%] 1 2 3 1 2 1 2 1 2 3 4
0.97 ± 0.11 2.00 ± 0.16 3.22 ± 0.25 0.86 ± 0.14 1.66 ± 0.15 0.30 ± 0.04 0.49 ± 0.09 0.37 ± 0.07 0.69 ± 0.10 0.22 ± 0.02 0.25 ± 0.04 9
97.1 ± 10.7 100.2 ± 7.8 107.3 ± 8.4 85.7 ± 13.6 83.0 ± 7.5 30.0 ± 4.3 24.5 ± 4.5 36.8 ± 6.9 34.4 ± 4.9 7.6 ± 0.7 6.3 ± 1.1
11.0 7.8 7.8 15.9 9.1 14.1 18.6 18.7 14.4 8.7 15.3
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3.5. Application of the AFB1 and total AF ELISAs for the analysis of peanut samples In order to test the developed kits a survey of both peanut and peanut butter samples purchased
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from different retailers in the UK was performed. A total of 26 peanut samples and 6 peanut butter
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samples were analyzed using both AFB1 and total AF ELISA kits. Two of these samples were found to
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contain aflatoxins at concentrations above the detection capabilities of the assays: 0.38 µg/kg AFB1
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and 0.43 µg/kg total aflatoxin in the first sample and 0.44 µg/kg AFB1 and 0.44 µg/kg total aflatoxins
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in the second sample. In both cases the levels were lower than the regulatory limits (2 µg/kg for AFB1
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and 4 µg/kg for total aflatoxins) what was confirmed by UHPLC-MS/MS analysis (Oplatowska-
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Stachowiak et al., 2015). UHPLC-MS/MS method detected the presence of 0.28 µg/kg and 0.67 µg/kg
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of AFB1 in first and second sample, respectively. AFB2, AFG1 and AFG2 were found to be below the
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limit of detection.
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Twenty five DDGS and 20 maize porridge samples were analyzed by the total AF ELISA. The results
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were correlated to these obtained by UHPLC-MS/MS method (Oplatowska-Stachowiak et al., 2015).
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There was a good agreement between the total AF ELISA and UHPLC-MS/MS methods as the square
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of the correlation coefficients were 0.97 for DDGS and 0.95 for maize porridge samples (Fig. 3.). The
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slopes of the regression lines were 0.74 for DDGS and 0.62 for maize porridge samples, indicating
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underestimation of the total aflatoxin content by ELISA. While UHPLC-MS/MS can quantitate each
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aflatoxin separately, ELISA measures total aflatoxins content as AFB1 equivalent. Therefore, if a
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mixture of aflatoxins is present in a sample the result by ELISA will be lower due to the different
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cross-reactivity of the antibody with each aflatoxin. According to the UHPLC-MS/MS results AFB1
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constituted 69–100%, AFB2 0–31%, AFG1 0–20% and AFG1 0–3% of the total aflatoxins content in
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DDGS. As for maize porridge samples the percentages of each AFB1, AFB2, AFG1 and AFG2 in respect
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to the total aflatoxins content were 50–100%, 0–7%, 0–50% and 0–4%, respectively.
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3.7. Proficiency testing of the total AF ELISA
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Total AF ELISA was tested in three proficiency testing schemes and z-scores were satisfactory in each
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case (│z│<2) (Table 3). As the mixtures of aflatoxins were present in each sample the results
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obtained by ELISA were lower than the total AF assigned values due to the different cross-reactivity
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of the antibody with each aflatoxin. The best result was obtained for rye flour. This sample
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contained the highest percentage of AFB1 with which the antibody has the highest cross-reactivity. 10
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The method was demonstrated to be suitable for the screening of various matrices for total AF
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content.
323 Table 3 Results of the proficiency tests for the total AF ELISA in different matrices.
AFB1 AFB2 AFG1 AFG2 total AF
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22.34 1.714* 10.48 0.5* 33.84
32.05
FAPAS® Proficiency Test 2014 (04243) (peanut) zscore
-0.24
measured value [µg/kg]
3.32 2.29 3.19 1.48 10.32
6.90
*median of reference dataset (no assigned value)
zscore
assigned value [µg/kg]
measured value [µg/kg]
zscore
3.80 1.89 1.07 1.00 7.67
-1.5
5.60
-1.2
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FAPAS® Proficiency Test 2015 (04261) (maize)
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CODA-CERVA Multimycotoxin Proficiency Testing 2014 (rye flour) assigned measured value value [µg/kg] [µg/kg]
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4. Conclusions
Seven monoclonal antibodies against aflatoxins were produced. Two of the best performing
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antibodies in terms of sensitivity and cross-reactivity were used to develop AFB1 and total AF ELISA
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prototype test kits. These kits were validated for the detection of aflatoxin contamination in
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peanuts, demonstrating the applicability of the method for the detection of AFB1 and sum of
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aflatoxins below the maximum levels set by the Commission Regulation (EC) No 1881/2006 (2 and
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4 µg/kg, respectively). The test kits were used to screen 32 samples purchased from stores in the UK.
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Two samples contained low levels of AFB1 which were subsequently confirmed by UHPLC-MS/MS
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analysis. The total AF ELISA was also applied to test naturally contaminated maize porridge and
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DDGS samples and a good correlation between the ELISA results and UHPLC-MS/MS analysis was
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found. The method trueness was confirmed in three proficiency tests in rye, peanut and maize
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matrices. These new methods will be transformed into commercial aflatoxin fast screening tools for
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the protection of consumers in the EU and worldwide.
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Acknowledgements
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The authors would like to thank Dr Caroline Frizzell and Dr Rachel Clarke for their help with the
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monoclonal antibody production.
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Figure captions
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Fig. 1. Typical standard curves for AFB1 (n=5).
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Fig. 2. Determination of the detection capability of AFB1 and total AF ELISAs.
423 Fig. 3. Correlation between total aflatoxins concentration (sum of AFB1, AFB2, AFG1 and AFG2) in
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naturally contaminated a) DDGS (n=25) and b) maize porridge samples (n=20), as determined by
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UHPLC-MS/MS method (x-axis) and total AF ELISA (y-axis). Results are in µg/kg. Solid line represents
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regression through the origin.
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ACCEPTED MANUSCRIPT Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients
Michalina Oplatowska-Stachowiaka, Nermin Sajicb, Ya Xua, Simon A. Haugheya, Mark Mooneya, Yun
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Highlights
Seven highly sensitive monoclonal antibodies against aflatoxins were produced
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Fast aflatoxin B1 and total aflatoxin ELISAs were developed and validated in peanuts
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The detection capabilities for aflatoxin B1 were very low: 0.4 µg/kg and 0.3 µg/kg
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32 peanut samples were screened and two low contaminated samples were identified
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High accuracy of the developed assay was confirmed in proficiency tests
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•