An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

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<ul><li><p>bad</p><p>an, Chaina</p><p>Keywords:</p><p>onome2, Gy (ESA c</p><p>performancewere optimised. Finally, the limits of detectionwere 3 ng/L formilk and 6 ng/L formilk-basedcereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged</p><p>rcinogoxic ms and AAatox</p><p>0.050 lg/L in milk and 0.025 lg/kg in infant formulas (Byrne,2004). However, in China and United States, the regulations keepAFM1 levels below 0.5 lg/L.</p><p>infant milk products.</p><p>2. Materials and methods</p><p>2.1. Chemicals and instruments</p><p>Aatoxin B1, B2, G1, G2 and M1 standard solution, aatoxinM1-BSA conjugate (4 mol aatoxin M1 per mol BSA), goatanti-mouse immunoglobulin horseradish peroxidase (IgG-HRP),</p><p> Corresponding authors. Address: Oil Crops Research Institute, Chinese Academyof Agricultural Sciences, Wuhan 430062, China. Tel.: +86 27 86812943; fax: +86 2786812862 (P. Li). Key Laboratory of Oil Crop Biology of the Ministry of Agriculture,Wuhan 430062, China (Q. Zhang).</p><p>E-mail address: (P. Li).</p><p>Food Chemistry 125 (2011) 13591364</p><p>Contents lists availab</p><p>Food Che</p><p>lse1 Both of Di Guan and Peiwu Li ranked as rst authors.inate awide range of foods and animal feedstuffs. AatoxinB1 (AFB1)is themost toxic. AndWorldHealth Organization classies AFB1 as ahuman carcinogen and proposes no safe dose (Anklam, Stroka, &amp;Boenke, 2002). AFM1 is the hydroxylatedmetabolite of AFB1. Lactat-ing animals that ingest feedstuffs contaminated with AFB1 excreteAFM1 into milk (Polan, Hayes, &amp; Campbell, 1974), and subsequentlyit can be found in a large variety of milk products. Although AFM1 isless carcinogenic and mutagenic than AFB1 (Neal, Eaton, Judah, &amp;Verma, 1998), it is also a health danger and has been classied asGroup 1 carcinogen (IARC, 2002). As milk and milk products arewidely consumed by infants and children who are more susceptibleto adverse effect of mycotoxins, the presence of AFM1 inmilk repre-sents a worldwide concern. The European Union limit for AFM1 is</p><p>These techniques have high sensitivity and accuracy, but requireextensive sample preparation, expensive equipment and well-trained personnel. Recently, ELISA methods have also been de-scribed and were mainly used in routine analysis (Anfossi et al.,2008; Pei, Zhang, Eremin, &amp; Lee, 2009; Thirumala-Devi et al.,2002). These methods have been shown to be simple, the portabil-ity of the equipment, hand-holding validation, and reliable for theanalysis of a large number of samples. Considering the hugerequirement of antibodies, we here prepared a sensitive and spe-cic Mab against AFM1, which has benets of uniform, constantproperties and unlimited production (Li, Zhang, &amp; Zhang, 2009).With these Mabs, we developed, optimised and validated anultra-sensitive competitive immunoassay for AFM1 in milk andAatoxin M1Monoclonal antibodyEnzyme-linked immunosorbent assayMilk</p><p>1. Introduction</p><p>Aatoxins are highly toxic and caare a group of structurally related tAspergillus avus, Aspergillus parasticupy, 2002; Sweeney &amp; Dobson, 1998).0308-8146/$ - see front matter 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.006from91% to 110%. Thirty sampleswere analysed, and concordant resultswere obtainedwhen the datawerecompared with a reference high-performance liquid chromatography method.</p><p> 2010 Elsevier Ltd. All rights reserved.</p><p>enic compounds, whichetabolites produced byspergillus nomius (Crep-ins frequently contam-</p><p>Several methods have been described for the determination ofAFM1, including the uorescence detection after immunoafnityclean-up (Chiavaro, Cacchioli, Berni, &amp; Spotti, 2005), high perfor-mance liquid chromatography with uorescent detector (Beebe &amp;Takahashi, 1980; Dragacci, Grosso, &amp; Gilbert, 2001) or mass spec-trometry (Cavaliere, Foglia, Pastorini, Samperi, &amp; Lagan, 2006).1</p><p>1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that inuence assayAnalytical Methods</p><p>An ultra-sensitive monoclonal antibody-immunoassay for aatoxin M1 in milk an</p><p>Di Guan a,b,c,1, Peiwu Li a,b,c,,1, Qi Zhang b,c,, Wen ZhaOil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062bKey Laboratory of Oil Crop Biology of the Ministry of Agriculture, Wuhan 430062, ChincQuality Inspection and Test Center for Oilseeds Products MOA PRC, Wuhan 430062, Ch</p><p>a r t i c l e i n f o</p><p>Article history:Received 31 December 2009Received in revised form 30 September2010Accepted 3 October 2010</p><p>a b s t r a c t</p><p>A sensitive and specic mselected by semi-solid HATreactivity to aatoxin B1, Blinked immunosorbent assaperformed in the AFM -B</p><p>journal homepage: www.ell rights reserved.sed competitive enzymeinfant milk products</p><p>g a,b, Daohong Zhang a,b, Jun Jiang a,b</p><p>ina</p><p>clonal antibody (Mab) against aatoxin M1 (AFM1), named as 2C9, wasdium. It exhibited high afnity for AFM1 of 1.74 109 L/mol and no cross-1 and G2. Based on the antibody, an ultra-sensitive competitive enzyme-LISA)was developed for AFM1 inmilk and infantmilk products. Assayswereoated (0.0625 lg/mL) ELISA format in which the antibody was dilutedle at ScienceDirect</p><p>mistry</p><p>vier .com/locate / foodchem</p></li><li><p>istrmouse monoclonal antibody ISO2-1 kits, OVA (agrose gel electro-phoresis grade), methyl cellulose, complete and incompleteFreunds adjuvants, 3,30,5,50-tetramethyl benzidine (TMB), hypo-xanthineaminopterinthymidine (HAT), hypoxanthine-thymidine(HT), and polyethylene glycol 1450 (PEG 1450, 50%) were pur-chased from SigmaAldrich (St. Louis, MO). RPMI-1640 mediumwith l-glutamine and HEPES (free acid, 283.3 g/L) were obtainedfrom HyClone. Fetal bovine serum, penicillin (+10,000 U/mL) andstreptomycin (+10,000 lg/mL) were from Gibco. Hybridoma Fu-sion and Cloning Supplement (HFCS) was obtained from Roche(Switzerland). Unless otherwise stated, all other inorganic chemi-cals and organic solvents were of analytical reagent grade or better.Water was obtained from a MilliQ purication system (Millipore).Female Balb/c mice were purchased from Centers for Disease Con-trol and Prevention of Hubei province.</p><p>The absorbance at 450 nm was detected by using a SpectraMaxM2e microplate reader (Molecular Devices, USA). Cell cultureplates (6, 24 and 96 wells) were from Iwaki, Japan. Polystyrene96-well microtiter plates were from Costar (Corning, Massachu-setts, USA). HPLC series (Agilent 1200) were consisted of a uores-cence detector, a ODS-3 column (3 lm particle size, 150 4.6 mmI.D.), for which samples were cleaned up by immunoafnity col-umns supplied by Beijing Chinainvent Instrument Tech. Co. Ltd.(Beijing, PRC).</p><p>2.2. Competitive ELISA</p><p>Plates were coated at 37 C with 100 lL/well of the appropriateAFM1-BSA concentration in 0.05 M carbonatebicarbonate buffer(pH 9.6). After incubation for 2 h, plates were washed three timeswith PBST (PBS with Tween-20: 8 g/L NaCl, 1.15 g/L Na2HPO4,0.2 g/L KH2PO4, 0.2 g/L KCl, and 0.05% Tween-20, v/v) and thenblocking with 1.5% OVA in PBST (200 lL/well) for 1 h at 37 C. Afteranother washing step, 50 lL/well of MAb diluted in PBS and 50 lL/well of analyte solution were added, and incubated for 1 h. Follow-ing a washing step, goat anti-mouse HRP conjugate (1:5000 inPBST, 100 lL/well) was added and incubated for 1 h at 37 C. Theplates were washed again, and 100 lL/well of TMB solution(3.3 lL of 30% H2O2, 400 lL of 0.6% TMB in DMSO per 25 mL of ace-tate buffer, pH 5.5) was added. The color development was stoppedafter 15 min with 2 M H2SO4 (50 lL/well). The absorbance wasmeasured at 450 nm. Sigmoidal curves were tted to a logisticequation (Raab, 1983) from which IC50 values (concentration atwhich binding of the antibody to the coating antigen is inhibitedby 50%) were determined.</p><p>2.3. Non competitive ELISA</p><p>A non competitive ELISA was used to determine the antibody ti-ters of mouse sera or cell culture supernatants. The procedureswere identical with that of competitive ELISA except for the detec-tion of the antigen, AFM1 and its related competitors.</p><p>2.4. Preparation and characterisation of monoclonal antibodies</p><p>2.4.1. ImmunisationThree six-week-old female Balb/c mice were subcutaneously</p><p>immunised with the immunogen at approximately three-weekintervals. The immunogen used was AFM1-BSA conjugate (50 lg)dissolved in 0.2 mL of sterilised 0.85% NaCl solution and emulsiedwith an equal volume of Freunds adjuvant. The initial injectionwas given using Freunds complete adjuvant and the rest twoinjections were given with Freunds incomplete adjuvant. The</p><p>1360 D. Guan et al. / Food Chemmouse with the highest titer of antiserum was given booster injec-tion three days before cell fusion. No adjuvant was used for thebooster injection.2.4.2. Monitoring antiserum titers by two-step procedureAntiserum was collected from the tail vein of each mouse. BSA</p><p>was added to eliminate the effect of anti-BSA in serum. The opti-mum concentration of BSA was chosen by non competitive ELISAwith 1 lg/mL BSA coated. Then, the anti-AFM1 titers were checkedby non competitive ELISA as described in Section 2.3.</p><p>2.4.3. Cell fusion and selectionThis protocol was modied from Davis, Pennington, Kubler, and</p><p>Conscience (1982). Briey, the mouse with the highest antibody ti-ter was sacriced, spleen was removed aseptically and splenocyteswere fused with SP2/0 myeloma cells using 50% (v/v) PEG1450.Then the fused cells were mixed with selective semi-solid media(RPMI 1640medium supplemented with 20% (v/v) foetal bovineserum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 1% (v/v) HEPES, 1% (w/v) methyl cellulose, 2% (v/v) HFCS, HAT), and pla-ted to 6-well plate (1.5 mL/well). After two weeks, lots of whitedots visible to the naked eyes were removed into 96-well plates,respectively.</p><p>Supernatants from 96-well plates were checked by two-stepscreening procedure. First of all, supernatants were determinedby non competitive ELISA. Then, the positive well was checkedby competitive ELISA using AFM1 as competitor.</p><p>2.4.4. Antibody production and puricationThe antibodies were prepared on a large scale as ascetic uid, by</p><p>inoculating the hybridoma cells into pristine-treated BALB/c mice.The IgG fractions were prepared by ammonium sulfate precipita-tion followed by the protein A column (Kobayashi, Oiwa, Kubota,Sakoda, &amp; Goto, 2000).</p><p>2.5. Characterisation of antibodies</p><p>The isotypes of the Mabs were performed with the isotyping kitaccording to the protocol provided by the manufacturer. To evalu-ate the cross-reactivity of the antibodies, tests were made usingaatoxin M1, B1, B2, G1 and G2. The protocol used was competitiveELISA as described above. These data were converted to a plot ofantibody inhibition, expressed as B/B0%, where B was the absor-bance at each concentration of analyte and B0 was the absorbancein the absence of analyte. CR for different aatoxins was deter-mined by comparing the IC50 values of analytes and calculatedas: CR (%) = [IC50 (AFM1)/IC50 (analyte)] 100 (Kolosova, Shim,Yang, Eremin, &amp; Chung, 2006). The afnities of the antibodies weredetermined by indirect non-competitive ELISA (Beatty, Beatty, &amp;Vlahos, 1987).</p><p>2.6. Optimisation of a Competitive ELISA</p><p>With the checkerboard procedure, the appropriate concentra-tions of coating antigen were prepared by serial dilutions from0.5 to 0.0625 lg/mL of AFM1-BSA with a dilution factor of 2 inthe carbonate-bicarbonate buffer and primary antibody (seriallydiluted puried monoclonal antibodies with 2-fold dilution from1:2500 to 1:30,000 in the PBS). The optimum reagent concentra-tions were dened as those, which give maximum absorbancearound 1.0 in the absence of analyte with minimum reagent ex-penses (Zeng et al., 2007).</p><p>Assay optimisation was performed using AFM1 as the competi-tor analyte. A set of experimental parameters (ionic strength, pH,and blocking reagent) were studied sequentially to improve thesensitivity of the immunoassay. The main criterion used to evalu-ate immunoassay performance was IC50. To determine the effect of</p><p>y 125 (2011) 13591364salt concentration on the assay performance, PBS at 0, 10, 20, 40,80, and 160 mM with constant pH of 7.0 was tested. The effect ofpH was evaluated using different PBS solutions, ranging from pH</p></li><li><p>5.0 to 9.0. Finally, the inuence of blocking reagent (1.5% gelatin,1.5% BSA, and 1.5% OVA) was investigated.</p><p>2.7. Analysis of samples</p><p>Samples were obtained from local markets and supermarketsand analysed before their expiration dates. Infant formula andmilk-based cereal weaning food (10 g) were suspended in 100 mLof warm deionised water. Subsequently, these samples as well asliquid milk samples were centrifuged at 3500g for 10 min at 4 C.The upper fat layer was completely removed, and the aqueouslayer was directly used for the analysis.</p><p>The quantitative analysis of AFM1 in samples was performed byindirect competitive ELISA. Standard curves were obtained usingAFM1 standard solutions prepared in deionised water or in AFM1-free milk samples extract.</p><p>The matrix effect, the recovery percentage and the limit ofdetection were assayed using blank samples.</p><p>To evaluate the accuracy and to validate the method, a compar-ative study using both the developed competitive ELISA and a HPLCreference procedure (Wang, Zhang, Zhang, &amp; Shao, 2003) was per-formed. A HPLC system equipped with a 150 4.6 mm, particlesize 3 lm, ODS-3 column was used. The mobile phase consistedof acetonitrile and water at a volume ratio of 33:67, delivered tothe column at a rate of 1 mL/min. Detection was made by a spec-</p><p>trouorometer, the excitation and emission wavelengths beingset at 365 and 435 nm, respectively.</p><p>3. Results and discussion</p><p>3.1. Monitoring antiserum titers</p><p>Since AFM1 is low molecular weight and devoid of antigenicity,it must be coupled to protein carrier in order to elicit an immuneresponse. Antibodies produced against a hapten-carrier antigenresult in antibodies against for the hapten, the carrier and alsofor various mixtures of these molecules (Heussner, Moeller, Day,Dietrich &amp; OBrien, 2007). So the carrier protein used for monitor-ing and screening is different from the immunisation. But commer-cial AFM1-carrier is only AFM1-BSA, we have to prepare AFM1conjugating with other carrier proteins or develop other methods.Preparation of AFM1-protein conjugates is high cost (AFM1, RMB0.1 million per mg) and harmful to the operators. Therefore wedeveloped some other methods to solve this problem.</p><p>Two-step procedure was applied to monitor antiserum titers.Mice were immunised with AFM1-BSA, so BSA was added in serumat rst to eliminate the effect of anti-BSA. With 20 mg/mL BSA, theabsorbance was decreased to a level similar to blank control value.Therefore the antiserum containing 20 mg/mL BSA was chosen.Then, titers of anti-AFM1 were checked by non competitive ELISA.</p><p>D. Guan et al. / Food Chemistry 125 (2011) 13591364 1361Fig. 1. Inuence of different of factors, coating antigen and antibodies ratio (a), pH (b), ionmeans of three independent experiments.ic strength (c) and blocking reagents (d) on the performance of assay. Results are the</p></li><li><p>3.4. Optimisation of a competitive ELISA</p><p>The optimal coating antigen and antibodies ratio were checkedby...</p></li></ul>


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