Validation of the Total Aflatoxin ELISA test in cereals, peanut and feed. Nermin Sajic, Lucia Streppel, Lisa Overgoor and Liberty Sibanda EuroProxima, Beijerinckweg 18, 6827 BN Arnhem, The Netherlands 1. Abstract The Total Aflatoxin ELISA test from EuroProxima (product code 5121AFT1p) was validated to measure total aflatoxins. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. Aflatoxins were extracted from ground samples with 80% methanol and centrifuged. The sample was diluted and was added to the antibody coated micro-wells together with the aflatoxin-conjugate and mouse monoclonal anti-aflatoxin antibodies. The ELISA was found to have an average recovery of 94% from spiked samples. In addition, 24 samples were analyzed and confirmed to be negative. The obtained percentage recovery efficiency ranged from 78% to 120% across the different spiked concentrations. The assay was found to have an LOD of 0.4 ppb with a 100% accuracy rate. In addition, four FAPAS (Food Analysis Performance Assessment Scheme) proficiency test samples were used to validate the performance of the ELISA test. The recovery and quantification of the Total Aflatoxin contents of these samples equals the values obtained by confirmative methods. 2. Introduction Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungi Aspergillus flavus, A. parasiticus, A. niger and A. nomius. Food products contaminated with aflatoxins include cereal (maize, sorghum, pearl millet, rice, wheat), oilseeds (peanut, soybean, sunflower), spices (chillies, black pepper, coriander, turmeric, zinger), tree nuts (almonds, pistachio, walnuts, coconut) and milk. Among 18 different types of aflatoxins identified, major members are aflatoxin B 1, B 2, G 1 and G 2. Aflatoxins B1 (AFB1) is normally predominant in amount in cultures as well as in food products. Aflatoxin M1 and M2 are major metabolites of aflatoxin B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins. Because of its high toxicity, regulatory limits for aflatoxins in food and feed have been laid down in many countries aiming at reducing human and animal exposure to aflatoxins. Aflatoxins often occur in crops in the field prior to harvest. Post harvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mold growth. The monitoring of aflatoxins which are present in food and feed usually in ug/kg depends on precise, reliable and efficient methods for their accurate determination. The major problem associated with most analytical methods for aflatoxins determination is the presence of coextracted sample interferences, which requires multiple extraction and clean-up steps before quantification. Rapid test kits are available for screening of aflatoxins, such as enzyme- linkedimmunosorbent assay (ELISA), Flow-Through Rapid Test (FTRT) and Lateral Flow Dipsticks. This paper reports on the validation of a recently developed ELISA test for the screening of Total Aflatoxins. 1
3. Materials and Methods If not indicated otherwise, sample treatment and assay protocol were performed as described in the instruction for use 5121AFT1p[1]09.08. The Total Aflatoxin ELISA utilizes antibodies raised in mouse against protein conjugated Aflatoxin and the cross-reactivity of the monoclonal antibody for the respective Aflatoxins are illustrated below. Cross contamination was determined against an Aflatoxin standard obtained from the Sigma. AFB 1 100% AFB 2 30% AFG 1 65% AFG 2 10% AFM1 50% 3.1 Materials Total Aflatoxin EIA 5121AFT1p (EuroProxima, product code 5121AFT1p) Blank wheat samples (local store) Standard material AFB1 for spiking the samples (Sigma, A6636) FAPAS proficiency test samples numbers 04122, 04118, 04102, 04107. Individual aflatoxin derivates used to determine the cross reactivity of AFB2, AFG1, AFG2 and AFM1 (Sigma A9887, A0138, A0263, A9401 respectively) 3.2.1. Sample treatment Feed and food - 9 ml of 80% (v/v) methanol was added to 3 g of ground sample - The mixture was shaken thoroughly at room temperature during 30 min - The mixture was centrifuged (room temperature, 10 min, 2000 x g). - After centrifugation an aliquot of 50 μl of the supernatant was taken - The 50 μl aliquot was diluted with 150 μl of dilution buffer - 50 μl of the diluted supernatant was pipette in the respective well of the EIA plate 3.2.2. Preparation of spike samples A blank wheat sample is divided into two test portions of the respective masses. One portion is the blank whereas the other is spiked with a known aflatoxin concentration, e.g. 0.5 ppb. Two extracts of the test portions of the respective volumes, blank and spike are obtained after extraction and purification steps of the method described in 3.2.1. Both extracts are assayed with the Total Aflatoxin EIA and to yield an analytical response (extressed in ppb). 3.2.3. Assay protocol Pipette 100 µl of standard dilution buffer in duplicate (wells A1, A2, blank). Pipette 50 µl of standard dilution buffer in duplicate (wells B1, B2; Bmax). Pipette 50 µl of each of the Aflatoxin standard solutions in duplicate (wells C1,2 to H1,2 i.e. 0.0313, 0.0625, 0.125, 0.25, 0.5 and 1.0 ng/ml). Pipette 50 µl of each sample solution in duplicate into the remaining wells of the microtitre plate (40 samples; 80 wells). Add 25 µl of conjugate (Aflatoxine-HRP) to all wells, except wells A1 and A2. Add 25 µl of antibody solution to all wells, except wells A1 and A2. 2
Seal the microtitre plate and shake the plate for a few seconds. Incubate for 1 hour in the dark at 37 C. Discard the solution from the microtitre plate and wash 3 times with rinsing buffer. Pipette 100 µl of substrate solution into each well. Incubate 30 min. at room temperature (20 C 25 C). Add 100 µl of stop solution to each well. After adding the stop solution, immediately read the absorbance values at 450 nm. 4. Results The limit of detection (LOD), limit of quantification (LOQ) and % recovery of the Total Aflatoxin EIA 5121AFT1p were determined by means of calibration curves with a concentration range as illustrated in Figure 1. Figure 1. Calibration curve of the Total Aflatoxin EIA 3
The LOD was calculated as the mean value of twenty four blank wheat samples, plus 3σ (standard deviation). Table 1. The analytical response for 24 blank wheat samples tested with the Total Aflatoxin EIA SAMPLE ABSORBANCE CONCENTRATION (ppb) Wheat 1 1,952 0,296 Wheat 2 2,032 0,240 Wheat 3 2,080 0,200 Wheat 4 1,902 0,344 Wheat 5 2,042 0,232 Wheat 6 1,957 0,296 Wheat 7 1,756 0,464 Wheat 8 1,816 0,408 Wheat 9 1,900 0,336 Wheat 10 1,953 0,304 Wheat 11 1,942 0,304 Wheat 12 1,925 0,328 Wheat 13 1,961 0,296 Wheat 14 1,976 0,280 Wheat 15 1,939 0,312 Wheat 16 2,005 0,264 Wheat 17 2,307 0,240 Wheat 18 2,007 0,256 Wheat 19 1,966 0,288 Wheat 20 2,057 0,224 Wheat 21 1,983 0,272 Wheat 22 1,990 0,264 Wheat 23 2,044 0,232 Wheat 24 1,978 0,280 The obtained LOD values are depicted in table 2. The LOQ was calculated as the mean value of twenty four blank wheat samples plus 6σ (standard deviation). The obtained LOQ values are depicted in table 2. Table 2. LOD and LOQ for blank wheat samples analyzed by the Total Aflatoxin EIA TOTAL AFLATOXIN LOD 0,462 ppb LOQ 0.633 ppb 4
The calculation of the % recovery is as follows: (Mean) response of the spiked sample mines the (mean) response of the blank sample, divided through the know spiked concentration, multiplied by 100. In formula: % Recovery = (mean) response spiked sample (ppb) (mean) response blank sample (ppb) x 100% (mean) response spiked sample (ppb) In this particular case, the % recovery was calculated as the mean of the recoveries of six samples that have been spiked with either 0.5, 1, 1.5, 2, 3, and 6 ng/ml B1. The obtained recoveries are shown in the table 3. Table 3. Recovery of Aflatoxin B 1 spiked in wheat matrix. Aflatoxin B 1 Mean Absorbency Aflatoxin Conc. Recovery % Spiking level ng/ml OD ppb 0,5 1,595 0,514 102 1 1,425 0,998 100 1,5 1,371 1,254 84 2 1,063 1,595 80 3 0,870 2,350 79 6 0,468 4,689 78 Table 4. Performance characteristics of the Total Aflatoxin ELISA test. FAPAS (04122, 04118, 04102, 04107) samples with known Total Aflatoxin concentrations were tested in the Total Aflatoxin EIA. FAPAS round Cross reactivity 04122 04118 04102 04107 SAMPLE of the Aflatoxin Total EIA Maize AFB1 100 3.63 3.63 AFB2 30 0.94 0.28 AFG1 65 1.93 1.25 AFG2 10 1.12 0.112 Total ppb FAPAS 7.52 5.27 Peanut 6.52 6.52 1.81 0.54 2.66 1.73 1.84 0.18 12.8 8.97 Animal Feed 18.1 18.1 1.51 0.45 5.86 3.81 Animal Feed 7.29 7.29 1.69 0.51 6.43 4.18-1.06 24.8 22.36 0.11 17.1 12.09 5
Total ppb in 5.38 7.08 22.74 14.65 Aflatoxin Total EIA Recovery% 102 79 102 121 *Bolt: Assigned values are a result of the average values of AFB1, AFB2, AFG1, AFG2 given in the FAPAS reports 04122, 04118, 04102, 04107. ** Cursive: Calculated values according to cross reactivity. N.B. Only the total Aflatoxin contents of the sample have been measured directly. Individual values have been calculated using the % cross-reactivities as indicated in paragraph 3. 5. Discussion and conclusion The results of the Total Aflatoxin ELISA kit show that the test has a very high sensitivity as well as a very high specificity. This conclusion is supported by the 0% false positive results from the natural blank samples and the results of the known positive FAPAS samples. The recovery and quantification of the individual Aflatoxins and their summation is identical to the assigned FAPAS values obtained by HPLC and LC-MS. Therefore, the Total Aflatoxin ELISA kit has proven to perform with a high accuracy, recovery and low LODs and LOQs. 6. Literature Anon. 1989. Mycotoxins, Economic and Health Risks. Council for Agricultural science and Technology, Report No.116 pp91. Eaton, D.L. and Groopman, J.D. 1994. The Toxicology of Aflatoxins. Academic Press, New York. 383-426. Finley, J.W.,Robinson, S.F. and Armstrong, D.J. 1992. Food Safety Assessment. American Chemical Society, Washington, D.C. 261-275. Schatzki TF (2001) Dependence of aflatoxin in almonds on the type and amount of insect damage. Journal of Agricultural and Food chemistry 49, 4513 4519. Tubajika KM, Damann KE (2001) Sources of resistance to aflatoxin production in maize. Journal of Agricultural and Food chemistry 49, 2652 2656. 6