Lateral Flow Quantitative Method for the Detection of Mycotoxins

Chapter 19

Lateral Flow Quantitative Method for the Detection of Mycotoxins

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Steven J . Saul and Mark Tess Charm Sciences, Inc., 659 Andover Street, Lawrence, MA 01843-1032 Lateral flow methods have been developed for the quantitative detection of mycotoxins. The method for aflatoxin detection (5100 ppb) in corn has received GIPSA approval.The general procedure is as follows: (i) ground sample is extracted with solvent; (ii) 100 μL portion of extract is added to 1 mL dilution buffer; (iii) 300 μL portion of the diluted extract is added to a lateral flow test strip; (iv) test strip is incubated for 10 min at 45 °C and (v) reflectance reader provides a numerical result from 0 to 150 ppb by comparing the binding intensities of 2 test lines and control line. A quantitative method for zearalenone (501000 ppb) in corn also has received GIPSA approval. Initial inhouse data are available on quantitative lateral flow methods for the detection of deoxynivalenol in wheat and ochratoxin A in wine and grape juice.

Mycotoxins are naturally occurring substances produced by mold that are harmful to human and animal health (7-3) Action levels have been set by the United States Food and Drug Administration (FDA) for aflatoxin in grains and feed and it is mandatory to test for aflatoxin in all corn exported from the United States. The United States Department of Agricultural Grain Inspection, Packers and Stockyards Administration (GIPSA) has established criteria for rapid test methods for aflatoxin, deoxynivalenol (DON), and zearalenone for quantitative testing, and also criteria for approving qualitative mycotoxin test kits. The lateral flow format is considered a rapid user-friendly method which has traditionally been used as a qualitative method to give a positive or negative result (4). There are several lateral flow qualitative methods which have received approval from GIPSA (5). The Charm ROSA Aflatoxin (Quantitative) and Charm ROSA Zearalenone (Quantitative) are the first lateral flow quantitative tests approved by GIPSA. 314

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Siantar et al.; Food Contaminants ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


315 The Charm ROSA (Rapid One Step Assay) system was initially developed for testing antibiotics in milk (6). The test system includes an incubator, reader and the lateral flow test strip. The methods require detection at specified tolerances/safe levels for a family of analytes, such as beta-lactams. The Charm ROSA beta-lactam test uses a biological receptor where the sensitivity of a betalactam to the receptor does not necessarily match the defined tolerance/safe level. Therefore in this test method the sensitivity of some of the analytes was adjusted to target detection at their tolerance/safe level (6). For example, the sensitivity of cephapirin is adjusted from 4 ppb towards its tolerance/safe level of 20 ppb by adding a monoclonal antibody specific for cephapirin. The ability to adjust test sensitivity to the tolerance level prevents the unnecessary destruction of milk while ensuring that the milk supply does not have a betalactam above the FDA established tolerance/safe levels. The Charm ROSA quantitative tests use a test zone, a control zone, and antibody-labeled gold beads. The test zone, generally consisting of two test lines, captures antibody-labeled beads that are not bound by the target analyte. The control zone captures both bound and unbound antibody-labeled beads. In a negative sample, colored beads bind strongly to the test zone allowing only a small fraction of beads to bind to the control line. In a sample containing the target analyte there is less binding of beads to the test zone and subsequently more binding to the control line. A calibration curve is generated using a reader to compare the relative intensities of test and control lines as a function of analyte concentration. The reader includes a calibration curve to generate a concentration of analyte in the assayed strip. The quantitative tests for aflatoxin, deoxynivalenol, and zearalenone use two test lines and one control line. The ochratoxin A test for wine is a quantitative test that has one test line and one control since the defined detection range is only from 0 to 10 ppb.

Materials and Methods All lateral flow test kits and equipment (45 °C ROSA-M incubator and ROSA-M Reader) were manufactured at Charm Sciences, Inc. (Lawrence, MA). Certified grain samples were purchased from Trilogy Laboratories (Washington, MO). Mycotoxin standards were purchased from Trilogy Laboratories or from Sigma (St. Louis, MO). Methanol was of analytical grade or higher and purchased from Sigma. Minisart RC 15 filters were purchased from Sartorius (Hannover, Germany). Extractions: For aflatoxin and zearalenone testing, samples were extracted using methanol-water (70:30, v/v). For official GIPSA testing, 50 g of a ground sample and 100 mL methanol-water (70:30, v/v) are blended or shaken. The sample is allowed to settle. If particles are present the extract is filtered or centrifuged. For testing aflatoxin in soybeans and distillers dried grain, 150 mL


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ppb Figure 1. ROSA Ochratoxin (Quantitative) test results for red wine spiked with ochratoxin A standard. Each data point is the average of 8 test results with error bars representing one standard deviation about the mean.

of methanol is used to extract the 50 g sample. For deoxynivalenol testing, 50 g of a ground sample and 250 mL of water are blended or shaken. No extraction is required for testing wine or grape juice for ochratoxin A Diluted Extract Preparation: For the aflatoxin, deoxynivalenol, and zearalenone tests a 100 |nL portion of the extract is added to a 1 mL of dilution buffer. For aflatoxin testing in corn flour, corn meal, corn/soy blend, flaking corn grits, popcorn, and wheat a precipitate can form, which requires the mixture to be drawn into a syringe and passed through a Minisart RC 15 syringe filter. Zearalenone testing also requires the diluted extract to be syringefiltered.For ochratoxin A testing, 300 \iL of the liquid sample is added to 2 mL of the ochratoxin dilution buffer. Assay Procedure: Label test strip with sample identification and open incubator lid. Place test strip in incubator with flat side up. Peel back tape and slowly pipet 300 ^ L of the diluted extract into the sample compartment. Reseal tape, repeat for additional samples, and then incubate for 10 min. Remove strip and read in the ROSA-M Reader on the appropriate assay channel and matrix.

Results The USDA/GIPSA program notice FGIS-PN-04-15 provides the performance criteria for quantitative aflatoxin tests (7). The program notice requires assays be run on naturally-contaminated corn samples by the test method and by LC. The results for each level must be within the defined range


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for the kit to pass. Results from an independent laboratory testing the ROSA Aflatoxin (Quantitative) lateral flow assay and LC are shown in Table I. The results are an average of 21 independent corn samples with naturallycontaminated aflatoxin levels at around 0, 5, 10, 20, and 100 ppb. Additional matrices such as corn flour, corn meal germ, corn gluten meal, corn screenings, corn soy blend, cracked corn, distillers dried grains, flaking corn, corn grits, milled rice, popcorn, rough rice, sorghum, soybeans, and wheat were also tested using fortified samples and passed specifications.

Table I. ROSA Quantitative Lateral Flow Test Comparisons to H P L C Mycotoxin

Aflatoxin

Zearalenone

DON

LCppb(%CV)

ROSA ppb (%CV)

< 1 (NA) 5.7 (9%) 9.2 (12%) 21 (11%) 119(7%) 0(NA) 105 (10%) 265 (6%) 1028 (4%) < 100 (NA) 700(14%) 1100 (9%) 1930(4%) 5100 (8%)

1.1 (29%) 6.0 (26%) 10.0(18%) 24 (20%) 113(18%) 0(NA) 121 (10%) 252 (9%) 951(11%) 0(NA) 628(15%) 1144(14%) 2031 (11%) 5256 (9%)

NOTE: NA is not applicable COMMODITY: Naturally-contaminated com was use to compare performance of aflatoxin and zearalenone test methods and naturally-contaminated wheat for DON test methods.

The detection range required for zearalenone is from 0 to 1000 ppb (5). Table I shows the comparison of the ROSA Zearalenone (Quantitative) assay results and LC with naturally-contaminated zearalenone in corn at about 0, 100, 250, and 1000 ppb. The results for the zearalenone tests are well within kit specifications. The reader measures the line intensities of the test lines (Tl and T2) and control line (C), determines the summed difference in intensity of each test line relative to the control line as a reader result, and calculates a concentration using the calibration curve of the test assay (9). As the analyte concentration in the sample increases there is a corresponding decrease in the



318 intensities of the two test lines and a subsequent increase in the binding to the control. For example, at 1027 ppb there is a 75% inhibition in the intensity at T l , a 40% inhibition in the intensity at T2 and a 115% increase in the intensity at the control line. These changes in line intensity correlate to the quantitative result of the ROSA test kit. Some preliminary in-house results for the Charm ROSA DON (Quantitative) test in comparison with HPLC certified samples can be seen in Table I. The detection range required for DON testing is from 0 to 5 ppm. This test uses a water extraction of the sample at a ratio of 5 parts water to one part sample. The Charm ROSA Ochratoxin A (Quantitative) Test for wine and grape juice has a detection range from 0 to 10 ppb. Due to the narrow detection range the test format consists of one test line and a control line. White or red wine samples were spiked with ochratoxin A over a range of 0 to 10 ppb and run on the lateral flow test method. An initial titration curve for spiked red wine is presented in Figure 1, where the result is the difference in intensity between the control line and test line. Each point is the average of 8 test strips. The overall range from 0 to 10 ppb is approximately 2000 intensity points as measured by the reader. The spread from 0 to a 2 ppb spiked red wine sample is 1000 intensity points indicating the ability to detect at the 2 ppb European Union level of concern for ochratoxin A in wine.

Summary Lateral flow quantitative test methods were developed for aflatoxin, zearalenone, deoxynivalenol (DON), and ochratoxin A. Both aflatoxin and zearalenone have received GIPSA certification as quantitative methods for the detection of the mycotoxins. The Charm ROSA lateral flow quantitative method combines the ease of use of a lateral flow method with the precision of a quantitative method over the defined concentration range. After the extraction step and a sample dilution step the diluted sample is added to the strip, incubated, and then read in a calibrated reader. The method uses test and control zones to capture gold beads with a reflectance reader capable of measuring the intensity of the gold beads bound to each line (9). The relative intensity of the control line in comparison to the test line intensities is used to calculate the amount of analyte in the sample. For the corn samples naturally contaminated with zearalenone, the inhibition of intensity at the first test line (Tl) for 104 ppb, 264 ppb, and 1027 ppb is 32%, 51%, and 75%, respectively, and at the second test line (T2) the inhibition is 3%, 15%, and 40%, respectively. The line intensity at the control line also changes in comparison to the zero value and for 104 ppb, 264 ppb, and 1027 ppb naturally-contaminated zearalenone samples the control line intensity increase is 70%, 84%, and 115%, respectively. For ochratoxin A the test uses one test line and one control and determines a quantitative value over a 0 to 10 ppb detection range.


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References 1. Bennett, J. W.; Klich, M . Mycotoxins. Clin. Microbiol. Rev. 2003, 16, 497516. 2. Diekman, M . A; Green, M . L. Mycotoxins and reproduction in domestic livestock. J. Anim. Sci. 1991, 70, 1615-1627. 3. Couloumbe, R. A. Jr. Symposium: Biological action of mycotoxins. Biological action of mycotoxins. J. Dairy Sci. 1993, 76, 880-891. 4. May, K. Home tests to monitor fertility. Amer. J. Obstet. Gynecol. 1991, 165 Suppl., 2000-2002. 5. USDA Grain Inspection, Packers and Stockyards Administration; http://archive.gipsa.usda.gov/tech-servsup/metheqp/testkits.pdf (accessed December 2006). 6. Salter, R. S.; Legg, D.; Ossanna, N.; Boyer, C.; Scheemaker, J.; Markovsky, R.; Saul, S. J. Charm Safe-Level ß-Lactam test for amoxicillin, ampicillin, ceftiofur, cephapirin and pencillin G in raw commingled milk. J. AOAC Int. 2001, 84, 29-36. 7. USDA Grain Inspection, Packers and Stockyards Administration. http://archive.gipsa.usda.gov/reference-library/bulletins/pn04-15.pdf (accessed December 2006). 8. USDA Grain Inspection, Packers and Stockyards Administration. http://archive.gipsa.usda.gov/reference-library/directives/9180-66.pdf (accessed December 2006). 9. Saul, S. J.; Tess, M . E.; Markovsky, R. J. Lateral flow test kit and method for detecting an analyte. WO patent WO06089027A2. 2006.


Lateral Flow Quantitative Method for the Detection of Mycotoxins

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