Chapter 26
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Comparison Study of a Fumonisin Enzyme Immunoassay and High-Performance Liquid Chromatography 1
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Ralf M. Dreher and Ewald Usleber 1
R-Biopharm GmbH, Roesslerstrasse 94, D-64293 Darmstadt, Germany Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, University of Munich, Schellingstrasse 10, 80799 Munich, Germany 2
Corn samples collected from German retail stores were tested for fumonisin using a commercially available enzyme-linked immunosorbent assay (RIDASCREEN Fumonisin) and HPLC. The recoveries for fumonisin B from artificially contaminated corn at levels of 50 and 500 ng/g were 59.7% and 73.2% after the enzyme immunoassay (EIA) analysis of the raw extracts; the corresponding values after S A X extraction and HPLC analysis were 62.3% and 65.6%. The comparison of both methods showed a slight overestimation of the EIA versus HPLC, but the RIDASCREEN Fumonisin can be characterized as a powerful and very acceptable screening method. 1
Fumonisins (Figure 1) are a group of mycotoxins mainly produced by Fusarium moniliforme and have shown to be of high importance as naturally occurring contaminants in corn worldwide (1,2). The known toxic effects like equine leukoencephalomalacia outbreaks, porcine pulmonary edema and heptocarcinogenicity in rats (3,4,5), as well as the naturally occurring levels of fumonisins in corn, present a potential threat to human and animal health. This potential threat suggests the need for screening and analytical methods to routinely monitor for the presence of fumonisins in foods and feeds. Currently, the most frequently used analytical methods for fumonisins are high-performance liquid chromatographic (HPLC) systems (6,7). Costs and time requirements, especially for the sample clean-up steps, make these methods unsuitable to process large numbers of samples. In such a situation, immunochemical approaches have been shown to be good alternatives for the screening of cereals for fumonisins. So far, four enzyme immunoassays have been described which are monoclonal (12-14), and also polyclonal (8), and anti-idiotype/anti-anti-idiotype (15) antibodies have been developed. A membrane-based test for fumonisin Bj (FB^ also has been described (16). The development of such an immunological test by Usleber et al. (8) was used in a 0097-6156/96/0621-0341S15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Fumonisin Enzyme Immunoassay and HPLC
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commercially available test kit, RIDASCREEN Fumonisin. This report describes the specifications of RIDASCREEN Fumonisin. The results of this test were compared to those obtained by a HPLC method which is essentially based on a method originally described by Shephard et al. (6) and improved by Sydenham et al. (9). Materials and Methods F B ! was purchased from Sigma Chemicals, Deisenhofen, Germany. RIDASCREEN Fumonisin kits were obtained from R-Biopharm GmbH, Darmstadt, Germany. A l l chemicals and solvents used were of at least analytical grade. Source of Samples. A total of 19 samples, collected in German retail stores, were analyzed for fumonisins. Some of these samples were imported from another European country. H P L C . A method originally described by Shephard et al. (6) and improved by Sydenham et al (?) was used for HPLC detection of FBj with some modifications. A Merck LichroCart 125-4 column, filled with LiChrospher 100 RP-8 material, was used as the stationary phase. The mobile phase was methanol (669 mL)/0.1 M NaH2P04 (340 mL), adjusted to a pH of 3.4 with orthophosphoric acid and pumped at a flow rate of 1 mL/min. For toxin derivatization, 50 | i L of standard F B j (in methanol) was mixed with 200 uL of o-phthaldialdehyde (ΟΡΑ) solution (9), and then 20 uL was injected into the HPLC system using a Waters Model 712 WISP injector. Derivatized ¥Βγ (retention time 12.8 min) was detected using a Shimadzu RF 535 fluorescence detector (excitation at 335 nm; emission at 440 nm). Toxin concentrations (injection range from 2 to 40 ng) were calculated from peak areas using Nelson E G software (LKB, Bromma, Sweden). R I D A S C R E E N Fumonisin E L I S A . The Fumonisin-ELISA was performed according to the instruction of the producer. The basis of the test is the antigenantibody reaction. The microtiter wells are coated with antibodies directed against FB^. Fumonisin enzyme conjugate, fumonisin standard solutions or sample solutions are added. Free F B j and fumonisin enzyme conjugate compete for the fumonisin binding sites (competitive enzyme immunoassay). Any unbound enzyme conjugate is then removed in a washing step. Enzyme substrate (urea peroxide) and chromogen (tetramethylbenzidine) were added to the wells and incubated. Bound enzyme conjugate converts the colorless chromogen into a blue product. The addition of the stop reagent (1 M H S 0 ) leads to a color change from blue to yellow, which was measured photometrically at 450 nm. The toxin content was quantified using an on line PC and enzyme immunoassay (EIA) software (10), which uses a cubic spline function for calculation of the standard curve. The measuring range of the curve is usually from 20-80% relative binding (B/B x 100). To determine the test specificity, fumonisins B ^ B and B were tested for competition with F B ^ H R P (horseradish peroxidase) under the conditions of the RIDASCREEN instructions. The absorbance values were plotted point-to-point and the relative cross-reactivity of each toxin was 2
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calculated on the basis of the concentration necessary to inhibit 50% F B H R P binding. Typical results for F B i , F B and F B are 100%, 40% and 100%. r
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Corn Sample Extraction. Ground corn samples (25 g) were extracted with 50 mL of methanol/water (75/25) for 20 min on a magnetic stirrer. For the spiking experiments, the corresponding F B i concentrations (50 or 500 ng/g) were added to the weighed corn samples and immediately thereafter extracted. The extract was centrifuged and the supernatant filtered through a paper filter. A n aliquot of this raw extract was adjusted to a methanol content of 10% with PBS, further diluted at least 1:3 in 10% methanol/PBS, and directly assayed by RIDASCREEN Fumonisin EIA. An aliquot of the raw extract (10 mL) was further purified using strong anion exchange (SAX) cartridges [Adsorbex (Merck); 400 mg per cartridge] according to the detailed instructions by Sydenham et al (Ρ). The final volume of the purified and concentrated extract was 0.2 mL of methanol, corresponding to 5 g of sample. For the determination of F B i by HPLC, purified extract (50 uJL) was added to 200 uJL of ΟΡΑ solution and further treated as described for toxin standards. Recovery (EIA, HPLC) of F B i from artificially contaminated corn samples was studied at concentrations of 50 and 500 ng/g, respectively. Food samples (popcorn; corn grit; corn semolina, in Germany kown as polenta) were purchased from local retail stores from October 1992 to March 1993 and analyzed by EIA and HPLC. Five of these samples were imported products from another European country. Results and Discussion The intraassay and interassay coefficients of variations of the RIDASCREEN Fumonisin EIA were usually below 7% and 12%, respectively. The mean detection limit and 50% dose were found at 0.2 and 2 ng/mL, respectively. A typical standard curve is shown in Figure 2. When specificity was checked under the conditions of the EIA, the relative cross-reactivity with F B ^ F B and F B were found to be 100%, 40% and 100%, respectively. Due to the high sensitivity of the assay, a simplified sample preparation procedure was sufficient for E I A analysis, resulting in a detection limit for ΡΒχ in corn of about 10 ng/g (10 ppb). To check the data obtained by E I A using this rapid extraction procedure, the raw extracts were additionally analyzed by H P L C after S A X purification. The recoveries for at the 50 and 500 ng/g level, as determined by the two analyses, are listed in Table I. In general, the data shows overall agreement between both sample preparation procedures and methods. In our experiments, magnetic stirring of the sample-solvent mixture for 20 min was not very efficient (recovery 59-73%) but gave consistent results within the concentration range of interest. It also was very convenient for the simultaneous extraction of more than 10 samples; an important aspect for a large-scale screening method. The EIA values of naturally contaminated samples (Table Π) were somewhat higher than those obtained for S A X extracts and HPLC. At least for the lower F B i values (< 100 ng/g), it cannot be excluded that some samples had a higher matrix effect which led to an overestimation of the toxin content as previously mentioned. For higher toxin concentrations, however, the sample extracts were diluted 1:1000 and 2
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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Fumonisin Enzyme Immunoassay and HPLC
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100 η 90 £
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Fumonisin B,
[pg/kg]
(ppb)
Figure 2. Typical standard curve for RIDASCREEN Fumonisin. The x-axis (logscale) indicates the toxin concentration. The y-axis indicates the relative binding of FBHRP (B/B). r
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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Table L Comparison of the Recovery of Fumonisin Β χ from Artificially Contaminated Corn Samples Using SAX-Purified Extracts (HPLC) and Raw Methanolic Extracts (EIA). F B i Found (ng/g)
F B i Added (ng/g)
HPLC (SAX)
0 50 50 50 50 50
