Chapter 27
Immunochemical Methods for Fumonisins in Corn
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Mary W. Trucksess Division of Natural Products, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 200 C Street Southwest, Washington, DC 20204
An enzyme-linked immunosorbent assay (ELISA) and an immunoaffinity column (IAC) cleanup procedure have been successfully applied to the determination of fumonisins in corn. The performance of the ELISA was evaluated by comparison to a reference high-performance liquid chromatographic (HPLC) method. The IAC procedure was coupled with HPLC determination. The recoveries of fumonisin B from corn spiked at the 1,2, and 4μg/g levels were 73-106, 79-83, and 64-92% for the ELISA, IAC, and H P L C methods, respectively. The accuracy and precision of the methods compared favorably. In the comparative studies using naturally contaminated corn samples, the ELISA results were 2-100% higher than those determined by HPLC. The immunoaffinity procedure results were about 71% of the levels observed using HPLC. 1
The fumonisins are a group of structurally related mycotoxins, which are secondary metabolites produced on corn by Fusarium moniliforme (1,2), Fusarium proliferatum (3), and several other flingi (4,5). Of the known naturally occurring fumonisins, fumonisins B (FB ) and Bj (F^ ) are the most abundant (6). In the United States the FB fFB ratio in corn is about 3:1 (7). was found to cause equine leukoencephalomalacia (2), porcine pulmonary edema (8), and rodent hepatotoxicity (9). Cattle and poultry can also be affected, but are not as susceptible to the mycotoxin as horses and swine. The Mycotoxin Committee of the American Association of Veterinary Laboratory Diagnosticians recommends that the total intake of FB! be limited to less than 5 μg/g in the non-roughage diet of horses, 10 μg/g in the total diet of swine and 50 μg/g in the feed for cattle and poultry (10). FBj has also been implicated in human esophageal cancer on the basis of epidemiological data (11,12). The International Agency for Research on Cancer, Working Group on the Evaluation of Carcinogenic Risks in Humans, has classified l
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T h i s c h a p t e r n o t s u b j e c t t o U.S.
copyright
P u b l i s h e d 1996 A m e r i c a n C h e m i c a l S o c i e t y
In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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the toxins derived from F. Moniliforme, which include F B and F B , as possible carcinogens to humans (13). Subsequently many methods have been developed for the determination of these toxins.
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Methods for fumonisin determination include thin-layer, liquid, and gas chromatography, as well as mass spectrometry (14-16). All these methods entail sample preparation, extraction, solid phase purification, chromatographic separation, and derivitization prior to quantitation. Mycotoxin testing by chromatographic methods is thus relatively slow and costly. With the advance of biotechnology, antibodies have been produced against the fumonisins and have been used in both enzyme-linked immunoassays (ELISAs) and immunoaffinity columns. Immunochemical methods can produce results more quickly than the traditional methods (17-22). Immunochemical methods are often the methods of choice for both mycotoxin monitoring and surveillance studies, which require rapid analysis of a large number of samples. Monitoring programs are useful for measuring the effectiveness of milling or food processing in controlling fumonisins in human and animal food. Surveillance studies can identify the incidence and occurrence of the fumonisins as well as the geographical areas where fumonisin contamination is a problem. Data can also be used to estimate human exposure for risk assessments. The validity of the data depends on several factors, including the method of analysis. It is extremely important that the immunochemical methods used in these studies are evaluated for their ability to produce accurate results when compared with a reference method. The objective of the current paper is to compare the performance of immunochemical methods with an Association of Official Analytical Chemists (AOAC) Internationalfirstaction high performance liquid chromatographic (HPLC) method.
Commercial Immunochemical Kits Several immunochemically based commercial kits have been marketed in the United States for detection and cleanup of fumonisins in corn (Table I). Two formats have been used: a competitive ELISA for determination of fumonisins and an immunoaffinity column cleanup procedure . The ELISA is a microtiter-well format. The anti-fumonisin antibodies are bound to the polystyrene microtiter wells. Thefreefumonisins in the extract and the fumonisin B horseradish peroxidase ( F B HRP) conjugate compete for the antibody binding sites. After incubation, washing, and addition of substrate, the color that develops in the wells is inversely related to the amount of toxin in the test sample. In the immunoaffinity column procedure the antibodies are attached to an agarose bead support. The fumonisins can be bound to the specific antibodies conjugated to the column support. The column is then washed, resulting in removal of unbound impurities. The fumonisins can then be desorbed and eluted with a strong organic solvent such as methanol, resulting in purification. The purified fumonisins are then derivatized. Thefluorescentfumonisin derivatives are quantitated by HPLC. r
In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Table L Commercial Immunochemical Methods for Fumonisins in Corn
Kit
Format
ELISA Microwell
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Veratox'
b
Fumonitest
Affinity Column
Ridascreen Fumonisin Fast
ELISA Microwell
Detection Analysis Limit (ng/g) Time/min.
Cost $/Test
500
35
7.00
25
50
10.00
9
65
7.00
c
"Neogen Coφ, Lansing, MI 48912 Viacam, Somerville, MA 02145 Bio-Tek Instruments, Inc., Winooski, VT 05404
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Methods.
Sample Extraction. For all three methods, corn samples (50 g) were extracted with 250 mL methanol water (70/30). Analytical Procedures.
HPLC. The reference HPLC method was carried out according to an Association of Official Analytical Chemistsfirstaction method (25). The HPLC method uses a strong anion exchange column (SAX) for purification. ELISA Procedure. The ELISA (Veratox, Neogen, Corp.) was performed as follows: Corn extract (100μί) were diluted with 3.9 mL methanol-water (10/90). The diluted extracts and standard solutions were added to microwell plates and mixed with FBhorseradish peroxidase conjugate. After mixing, the contents of the wells were transferred to anti-fumonisin antibody coated plates. The plates were incubated at room temperature for 20 minutes with mixing for 30 seconds at 5 minute intervals.The wells were washed 5 times with water, and 100 yiL tetramethylbenzidine substrate solution was added to each well. After 10 minutes, the reaction was stopped by addition of 100 //L dilute sulfuric acid. Absorbance of the wells was measured at 650 nanometers. Standard curves were generated using a log/logit fit. r
Immunoaffinity Column Cleanup and Derivatization.
Ten mL of corn extract
In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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was diluted with 40 mL of diluting solution (12.5 g sodium chloride, 2.5 g sodium bicarbonate, 2 drops Tween 20 in 500 mL water). After filtration, 5 mL of the diluted extract was placed on the column. The column was washed with 5 mL diluting solution and 5 mL water. The fumonisin was eluted (2 χ 0.8 mL) with methanol-water (80/20). The eluate was evaporated and then redissolved in 200 μL methanol. A Waters model 710 Plus autoinjector was used to deliver 100 μL of derivatization reagent (40 mg o-phthaldialdehyde, 1 mL methanol, 5 mL 0.1 M sodium tetraborate, and 50 / / L mercaptoethanol) to 25 μL of extract prior to injection onto the HPLC). Results Initial studies (23) were aimed at determining cross-reactivity with structurally related fumonisins as well as other mycotoxins, spike recovery, and immunoaffinity column capacity. In addition, immunochemical results were compared with results generated by the reference HPLC method. Cross Reactivity. For the ELISA method, the relative cross-reactivities of FBj, FBfc and F B were found to be 100, 24, and 30%, respectively. The relative crossreactivity of each fumonisin was calculated by comparing the fumonisin concentration necessary to inhibit the ELISA response by 50%. The crossreactivities of the fumonisins on the immunoaffinity columns were determined indirectly by comparing the recoveries of added FBj and FB^ from columns. The average recoveries of FT*! and F B simultaneously added to the columns at various combinations totaling 1 μg/g were 89 and 79%, respectively. Similar recoveries were obtained in a previous study (26). The results suggested that the monoclonal antibodies in the immunoaffinity column have similar binding affinities with both FB! and FB . The antibodies of both procedures do not react with other Fusarium toxins, such as deoxynivalenol and zearalenone. 3
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Analytical Range. The applicable range for the ELISA method was 0.1-2.5 ng/mL. This is equivalent to 0.5-10 μg/g of corn. The immunoaffinity column has a maximum binding capacity of 1 μg/g F B according to the manufacturer. The manufacturer recommends applying the equivalent of 0.2 g corn extract to the column. The upper limit of determination was 5 μg/g. However, the maximum sample loading capacity of the column was determined to be about 2 g corn extract (27). The lower limit of determination was 25 ng/g. The limit of determination of the H P L C method depends on the S A X purification cartridge. On the basis of loading 5 g equivalent of test portion extract onto the S A X cartridge the applicable range of the method was 25-15,000 ng/g. t
Recoveries of Fumonisin B, from Spiked Corn. Each corn extract was analyzed by the three methods to eliminate sampling and extraction variability. Recoveries of ΈΒ from com spiked over the range of 1- 4 μg/g were 73-106, 79-83, and 64-92% for the ELISA, IAC, and HPLC methods, respectively; the respective relative standard deviations were 4.9-7.2, 0.9-3.5 and 4.7-8.9%. ι
In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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Analysis of Naturally Contaminated Corn. Ten naturally contaminated corn samples were extracted and each extract was analyzed by ELISA, the immunoaffinity based method and by HPLC. The results obtained by the ELISA were about 38% higher than those obtained by the HPLC method. This difference probably was caused by the cross-reactivity of the antibodies with compounds structurally related to or the loss of FI*! in the solid-phase purification prior to HPLC analysis. The slope of the ELISA concentration vs. the HPLC concentration was 1.3765 (Y = 1.3765X + 0.0456); the correlation coefficient was 0.9964. Results obtained by the IAC method were about 71% of those obtained by the HPLC method. The slope of the IAC concentration vs. the H P L C concentration was 0.7135 (Y = 0.7135X + 0.1169); the correlation coefficient was 0.9674. Since the H P L C method (including the IAC extract) quantitatively determined FI*! alone, and the ELISA quantitatively determined "total" fumonisins, the overall agreement of the results obtained by the three methods was considered acceptable. The polyclonal antibody ELISA method was further evaluated (24) with an additional 18 naturally contaminated corn samples that contained total fumonisin (B +B ,+B ) levels ranging from 0.1 to > 5 μg/g. The samples were extracted as described and analyzed by both ELISA and HPLC. The correlation coefficient between the results generated by HPLC and ELISA was 0.967. In 3 of 18 samples the fumonisin levels determined by ELISA were 85-100% higher than those determined in the same extracts by HPLC; in 13 of 18 samples ELISA results were 2-53% higher than those by HPLC; and in 2 of 18 samples ELISA results were 10% and 20% lower than those by HPLC. No recovery data were given in this study. l
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Conclusion Results indicated that the polyclonal antibody ELISA method is suitable for use as a screening method for fumonisins in corn and that the immunoaffinity column method can be used for the determination of fumonisin I*! in corn. The immunoaffinity column method has the added advantage over the HPLC method because it can be used to determine ¥B in canned corn and frozen corn. The HPLC method gave poor recoveries for added fumonisin 1^ (
