Evaluation and Application of Immunochemical Methods for Fumonisin

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 27, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch028

Chapter 28

Evaluation and Application of Immunochemical Methods for Fumonisin B in Corn 1

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Mary W. Trucksess and Mohamed M . Abouzied 1

Division of Natural Products, U.S. Food and Drug Administration, 200 C Street, Southwest, HFS-346, Washington, DC 20204 Neogen Corporation, 620 Lesher Place, Lansing, MI 48912 2

Two immunochemical methods were evaluated and compared with a strong anion exchange (SAX) method for the determination of fumonisin B (FB ) in corn. The two methods were the enzyme-linked immunosorbent assay (ELISA) and the monoclonal antibody-affinity column (IAC) method. The ELISA test is a direct competitive microtiter-well assay in which a strip reader, which measures the color of an enzymatic reaction, is combined with a log/logit data transformation program and linear regression calibration to determine FB concentration. FB in test samples from IAC or SAX isolation is derivatized with o-phthaldialdehyde, and the derivative is separated from other impurities by reversed-phase high-performance liquid chromatography (HPLC) and then quantitated by fluorescence detection. Recoveries of FB from corn spiked over the range of 1-4 µg/g were 73-106, 79-83, and 64-92% for the ELISA, IAC, and SAX methods, respectively. Results of analysis of the same extract from naturally contaminated corn by the three methods were similar. HPLC-electrospray mass spectrometry was used to positively identify the FB isolated with the microtiter wells from an extract of a naturally contaminated corn. 1

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Mycotoxins are natural toxins produced by fungi. The occurrence of mycotoxins in foods and feeds is a worldwide problem. The five most important toxins from an agricultural perspective are the aflatoxins, deoxynivalenol, zearalenone, ochratoxin A , and the fumonisins (7). Analytical methods are well established for the first four toxins. The fumonisins represent the most recendy discovered family of Fusarium toxins (2). They are water-soluble metabolites produced on corn by Fusarium moniliforme, F. proliferatum, and several other fungi (3,4). The most abundant of the fumonisins are fumonisin B (FB ) and fumonisin B x

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0097-6156/96/0621-0358$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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28. TRUCKSESS & ABOUZIED

Immunochemical Methods for Fumonisin Β

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(FBj). The F B / F B ratio in corn is usually about 3:1 (5). ¥B was found to cause equine leucoencephalomalacia (2), porcine pulmonary edema (6), and rodent hepatotoxicity (7) and has been implicated in human esophageal cancer on the basis of epidemiological data (5,9). The International Agency for Research on Cancer, Working Group on the Evaluation of Carcinogenic Risks in Humans, evaluated the available toxicological data and classified the toxins derived from F. moniliforme, which include F B and F B , as possible carcinogens to humans (10). Therefore, great interest has been generated to develop methods of analysis for these toxins. Fumonisins can be separated and identified by high-performance liquid chromatography (HPLC) (77), thin-layer chromatography (72), and gas chromatography-mass spectrometry (GC-MS) (75). Immunochemical methods also have been developed (14-16). With the availability of commercial immunoassay kits, monitoring for fumonisin contamination can be moved from the laboratory to the production facilities. Immunoassays can be used to obtain data quickly, so that immediate action can be taken when necessary. In order to meet the demand for minimum laboratory sample handling and increased numbers of analyses, we evaluated two immunochemical methods: an enzyme-linked immunosorbent assay (ELISA) method (Veratox Quantitative Fumonisin Test, Neogen Corp., Lansing, M I 48912, 1995) and an immunoaffinity column method (IAC) (17). The parameters, such as the capacity, specificity, accuracy, and precision of the two methods, were compared with those obtained for a strong anion exchange column (SAX) method (77). A n advantage of the E L I S A method is that it does not require derivatization of the toxin with o-phthaldialdehyde (ΟΡΑ) and reversed-phase H P L C separation for quantitation with a fluorescence detector. We also investigated the cross-reactivities of the antibodies and the use of an M S technique to confirm the identity of FB isolated from the E L I S A antibodycoated wells. 1

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Experimental Standards. F B and F B were isolated from cultured corn in our laboratory. On the basis of M S ion ratio data, the purities of the compounds were 96 and 90%, respectively. X

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Samples. Corn analyzed by a reference method (77) and shown to contain FB at a level of 150 ng/g was used for the recovery study.

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Extraction. Test portions of corn spiked at 1, 2, and 4 μg/g and of naturally contaminated corn (50 g) were extracted with 250 m L methanol-water (70/30). The extracts were then diluted as described in the three methods. Five replicates of each test solution (diluted extract) were analyzed by the E L I S A method. Three replicates of each test solution were analyzed by the other two methods.

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

360

IMMUNOASSAYS FOR RESIDUE ANALYSIS

E L I S A Method. The polyclonal antibodies against ¥B were produced in rabbits after immunization with an FB^keyhole limpet hemocyanin conjugate (18). The assay used a microtiter-well format. The corn extracts (100 μΕ) were diluted with 3.9 m L methanol-water (10/90). The diluted extracts and standard solutions were added to the wells, which contained FB horseradish peroxidase (HRP) conjugate in 100 uL buffer. After mixing, the contents of the wells were transferred to the antibody-coated wells. (The wells were coated with sheep antifumonisin polyclonal antibodies in 0.1 M carbonate buffer (pH 9.6) overnight at 40 °C. The wells were washed with deionized water. Nonspecific binding was rniriirnized by blocking the unbound sites of the microtiter wells with 300 μΕ of 1 % polyvinyl alcohol (w/v) in phosphate-buffered saline for 30 min at 37 °C. After washing with deionized water, strips were dried and packed in foil pouches with desiccant, sealed, and stored at 4 °C until used.) The wells were incubated at room temperature for 20 min with mixing for 30 s at 5 min intervals. The wells were then washed five times with water, and 100 pL tetramethylbenzidine-peroxide substrate solution was added. After 10 min the reaction was stopped by adding 100 pL diluted sulfuric acid solution, and the toxins were quantitated from the absorbance, which was measured at 450 nm with an E L I S A reader coupled to a computer using log/logit software. 1


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I A C Method. The column (Vicam, 29 Mystic A v e . , Somerville, M A 02145) employed monoclonal antibody-coated agarose beads. The corn extract (10 mL) was diluted with 40 m L diluting solution (12.5 g sodium chloride, 2.5 g sodium bicarbonate, and 2 drops Tween 20 in 500 m L water). After filtering, 5 m L of the diluted extract (equivalent to 0.2 g test sample) was added to the column. The column was washed with 5 m L diluting solution and 5 m L water, and the toxin was eluted by washing two times with 0.8 m L methanol-water (80/20). The eluate was evaporated, and the residue was redissolved in 200 pL methanol, derivatized, and analyzed by H P L C . S A X Method. The cartridge was packed with 0.5 g quaternary amine (strong anion exchange) in a polypropylene tube with a 10 m L reservoir (Varian). The cartridge was conditioned with 5 m L methanol and 5 m L methanol-water (70/30). The corn extract (10 mL, equivalent to 2 g test sample) was applied to the cartridge. The cartridge was washed with 5 m L methanol-water (70/30) and 3 m L methanol. The toxin was eluted with 10 m L methanol-acetic acid (99/1), and the solvent was evaporated at 60 °C under a stream of nitrogen. The residue was dissolved in 200 pL methanol, derivatized, and analyzed by H P L C . Derivatization and H P L C Analysis. The derivatization reagent consisted of 40 mg ΟΡΑ, 1 m L methanol, 5 m L 0.1 M sodium tetraborate, and 50 uL mercaptoethanol. A Waters 710 Plus autoinjector was used to deliver 100 uL derivatization reagent to 25 pL test solution, and to mix the solution and inject it onto a 25 cm χ 3.9 mm, 5 μιη Waters μBondapak C column. The mobile phase was acetonitrile-water-acetic acid (50/50/1); tne flow rate was 1 mL/min. 1 8


28.

TRUCKSESS & ABOUZIED

Immunochemical Methods for Fumonisin B

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A Waters 470 fluorescence detector was used at an excitation wavelength of 355 nm and an emission wavelength of 440 nm. Confirmation of Identity of F B ^ Test solution (100 J U L ) and 100 /xL buffer were added to each of the five antibody-coated wells. After incubating at room temperature for 20 min with mixing for 1 min at 3 min intervals, the wells were washed five times with water. The toxin was eluted by adding 100 methanol to each well and mixing for 1 min. The combined methanol was transferred to a 1.5 m L Eppendorf tube and evaporated on a steam bath under a stream of nitrogen. The wells were rinsed twice with methanol. The methanol was added to the same tube and evaporated. The residue was redissolved and analyzed by HPLC-electrospray M S by using a Finnigan TSQ 7000 mass spectrometer (Musser, S. M . , Food and Drug Administration, personal communication, 1995). Results and Discussion Various ratios of methanol/water or acetonitrile/water have been used to extract fumonisins from corn and corn-based products. Although the E L I S A , I A C , and S A X methods use methanol/water in ratios of 7/3, 8/2, and 7.5/2.5 for extractions, respectively, the test portions in this evaluation were extracted with methanol/water (7/3). The same extract was used for the three methods to eliminate variability in extraction efficiency and sampling. We examined the immunochemical methods for their cross-reactivity with structurally related analytes, cross-reactivity with dissimilar analytes, and capacity. We compared the accuracy and precision of the results from analyses of spiked corn by these methods and by the reference method. The correlation coefficients of the immunochemical methods and the reference method were determined from comparisons of results obtained from analyses of naturally contaminated corn. For the E L I S A method, the relative cross-reactivities of F B ^ F B , and F B were found to be 100, 24, and 30%, respectively. In Figure 1 the x-axis (log scale) indicates the toxin concentration. The y-axis indicates the relative binding of toxin-HRP (B/B ) (absorbance of standard toxin concentration/absorbance of toxin negative buffer solution) χ 100. The relative cross-reactivity of each fumonisin was calculated on the basis of concentration necessary to inhibit 50% of the F B H R P binding. The relative cross-reactivities of the fumonisins on the immunoaffinity columns were not provided by the manufacturer. The recoveries of F B and F B added to the columns are shown in Table I. The average recovery of FB added to methanol/water was 89% in the range of 0.3-1.0 μg, whereas the average recovery of F B for the same range was 79%. The average recovery of F B added to corn extract (same levels as in methanol/water) was 90%, whereas the average recovery of F B was 77%. The recoveries of both toxins added to methanol/water and to corn extract were similar from the immunoaffinity column, indicating that the matrix did not affect the antibodies. This finding also 2

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362

IMMUNOASSAYS FOR RESIDUE ANALYSIS

Figure 1. Cross-reactivities of F B ( · - · ) , F B ( • - • ) , and F B ( Α - A ) in the E L I S A . X

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Immunochemical Methods for Fumonisin B

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Table I. Recoveries of F B and F B Added to Methanol/Water and to C o r n Extract from Immunoaffinity Column X

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Amount Added (us) FB FB 1

MeOH/H 0 Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 27, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch028

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1.0 0.7 0.5 0.3 0

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Recovery (%) FB FBj

83 90 90 93

0 0.3 0.5 0.7 1.0

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A v . 89 Corn extract

1.0 0.7 0.5 0.3 0

97 87 88 87

0 0.3 0.5 0.7 1.0

A v . 90

83 76 83 75 79

74 84 74 76 77

suggested that the antibodies might have similar cross-reactivity with F B and FB . It is important to demonstrate that the analyte of concern can be identified in the presence of dissimilar compounds. More than one mycotoxin can be found in the same grain. Many other Fusarium toxins, such as deoxynivalenol and zearalenone, are often found in corn; cross-reactivity with these compounds is undesirable. The ELISA method provided similar results from test solutions containing F B ^ F B and deoxynivalenol, ¥B and zearalenone, and all three furmonisins. The I A C also showed no binding with deoxynivalenol and zearalenone. The applicable range of the ELISA method was 0.1-2.5 ng/mL, which is equivalent to 0.5-10 /xg/g corn. The I A C has a maximum binding capacity of 1 μg FB according to the manufacturer. When an extract (equivalent to 1 g) of corn contaminated with F B at 1.90 μg/g was added to the immunoaffmity column, the corn was found to contain ΈΒ at 0.92 μg/g. However, when an extract (equivalent to 1 g corn) containing 5 μg F B ^ g , the analysis should be repeated using the equivalent of 23 °C yes

25-5000 79-83 0.9-3.5 corn B!:B =100:90 50 LC laboratory ambient yes

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Recovery. N A = not applicable.

analyte for M S analysis. Thus, the immunochemical methods not only can provide a rapid on-site field test, but they also can effectively complement the standard H P L C procedure currently used in routine monitoring for fumonisins in foods. Literature Cited 1. 2.

3. 4.

5. 6.

Miller, J. D. In The Toxicology Forum; Caset Assoc. Ltd.: Fairfax, V A , 1994; pp 3-9. Gelderblom, W. C. Α.; Jaskiewicz, K.; Marasas, W. F. O.; Thiel, P. G.; Horak, R. M.; Vleggaar, R.; Kriek, N . P. J. Appl. Environ. Microbiol. 1988, 54, 1806-1811. Nelson, P. E.; Plattner, R. D.; Shackelford, D. D.; Desjardins, A. E. Appl. Environ. Mircobiol. 1992, 58, 984-989. Thiel, P. G.; Marasas, W. F. O.; Sydenham, E . W.; Shephard, G. S.; Gelderblom, W. C. Α.; Nieuwenhuis, J. Appl. Environ. Microbiol. 1991, 57, 1089-1093. Pohland, A. E . In The Toxicology Forum; Caset Assoc. Ltd.: Fairfax, V A , 1994; pp 186-196. Harrison, L. R.; Colvin, Β. M.; Greene, J. T.; Newman, L. E.; Cole, J. R. J. Vet. Diagn. Invest. 1990, 2, 217-221.



Immunochemical Methods for Fumonisin B 367


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Gelderblom, W. C. Α.; Kriek, N. P.J.;Marasas, W. F. O.; Thiel, P. G. Carcinogenesis 1991, 12, 1247-1251. 8. Sydenham, E . W.; Thiel, P. G.; Marasas, W. F. O.; Shephard, G. S.; van Schalkwyk, D. J.; Koch, K. R. J. Agric. Food Chem. 1990, 38, 1900-1903. 9. Thiel, P. G.; Marasas, W. F. O.; Sydenham, E . W.; Shephard, G. S.; Gelderblom, W. C. A. Mycopathologia 1992, 117, 3-9. 10. International Agency for Research on Cancer, Monograph 56, 1993; pp 445-466. 11. Sydenham, E . W.; Shephard, G. S.; Thiel, P. G. J. AOAC Int. 1992, 75, 313-318. 12. Rottinghaus, G. E . ; Coatney, C. E.; Minor, H. C. J. Vet. Diagn. Invest. 1992, 4, 326-329. 13. Plattner, R. D.; Branham, Β. E . J. AOAC Int. 1994, 77, 525-532. 14. Azcona-Olivera, J. I.; Abouzied, M . M . ; Plattner, R. D.; Pestka, J. J. J. Agric. Food Chem. 1992, 40, 531-534. 15. Shelby, R. Α.; Rottinghaus, G. E . ; Minor, H. C. J. Agric. Food Chem. 1994, 42, 2064-2067. 16. Usleber, E . ; Straka, M . ; Terplan, G. J. Agric. Food Chem. 1994, 42, 1392-1396. 17. Trucksess, M . W.; Stack, M . E . ; Allen, S.; Barrion, N. J. AOAC Int. 1995, 78, 705-710. 18. Avrameas, S.; Ternynck, T. Immunochemistry 1969, 56, 1729-1733. 19. Ware, G. M . ; Umrigar, P. P.; Carman, A. S.; Kuan, S. S. Anal. Lett., 1994, 27, 693-715. RECEIVED

December 4, 1995


Evaluation and Application of Immunochemical Methods for Fumonisin

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