Aflatoxin Plate Kit PN 53012B

Transcription

1 Aflatoxin Plate Kit PN 53012B INTENDED USE The Aflatoxin Plate Kit is a competitive ELISA for the quantitative analysis of aflatoxin in nuts, grain and grain products. ASSAY PRINCIPLES The Aflatoxin kit is a competitive enzyme-labeled immunoassay. Aflatoxin is extracted from a ground sample by blending or shaking with methanol/water. The extract is then diluted with water, filtered and then tested in the immunoassay. Aflatoxin-HRP enzyme conjugate is pipetted into the test wells followed by calibrators or sample extracts. Aflatoxin antibody is then pipetted into the test wells to initiate the reaction. During the 10 minute incubation period, aflatoxin from the sample and aflatoxin-hrp enzyme conjugate compete for binding to aflatoxin antibody which, in turn, binds to the test well. Following this 10 minute incubation, the contents of the well are removed and the wells are washed to remove any unbound toxin or enzyme-labeled toxin. A clear substrate is then added to the wells and any bound enzyme-toxin conjugate causes the conversion to a blue color. Following a 10 minute incubation, the reaction is stopped and amount of color in each well is read. The color of unknown samples is compared to the color of the calibrators and the Aflatoxin concentration of the samples is derived. REAGENTS AND MATERIALS PROVIDED The kit in its original packaging can be used until the end of the month indicated on the box label when stored at 2-8ºC. Plate containing 12 test strips of 8 wells each vacuum-packed in aluminized pouch with indicating dessicant. 5 vials each containing 2 ml of Aflatoxin calibrators corresponding to 0, 2.0, 7.5, 25 and 100 µg/l (ppb) of Aflatoxin. (Note: Because of the 1:10 dilution of the grain sample in the extraction step, the calibrators actually contain 1/10th of the stated value. No further correction back to the concentration in the original grain sample is required.) 1 vial containing 8 ml of Aflatoxin-HRP Enzyme Conjugate. 1 vial containing 8 ml of Rabbit anti-aflatoxin antibody. 1 vial containing 14 ml of Substrate. 1 vial containing 14 ml of Stop Solution. (Caution! 1N HCl. Handle with care.) Instructions PRECAUTIONS

2 1. Each reagent is optimized for use in the Aflatoxin Plate Kit. Do not substitute reagents from any other manufacturer into the test kit. Do not combine reagents from other Aflatoxin Plate Kits with different Lot numbers. 2. Dilution or adulteration of reagents or samples not called for in the procedure may result in inaccurate results. 3. Do not use reagents after expiration date. 4. Reagents should be brought to room temperature, 20-28ºC (62-82ºF) prior to use. Avoid prolonged (> 24 hours) storage at room temperature. 5. Aflatoxin is a very toxic substance. Dispose of all liquids in a plastic container containing household bleach (minimum 10%). All labware should be soaked for at least 1 hour in a 30% solution of household bleach. Avoid contact of skin and mucous membranes with reagents and sample extracts by wearing gloves and protective apparel. If exposure of skin and mucous membranes to liquids should occur, immediately flush with water. 6. The Stop Solution is 1N hydrochloric acid. Avoid contact with skin and mucous membranes. Immediately clean up any spills and wash area with copious amounts of water. If contact should occur, immediately flush with copious amounts of water. MATERIALS REQUIRED BUT NOT PROVIDED 1. Laboratory quality distilled or deionized water. 2. Methanol, ACS grade 3. Graduated cylinder, 100 ml or larger. 4. Glassware for sample extraction and extract collection. 5. Filters, Whatman GF/A or equivalent 6. Pipet with disposable tips capable of dispensing 50 µl. 7. Multi-channel pipet; 8 channel capable of dispensing 50 and 100 µl. 8. Paper towels or equivalent absorbent material. 9. Microwell plate or strip reader with 450nm filter. 10. Timer 11. Blender EXTRACTION SOLUTION PREPARATION

3 1. Carefully measure 20 ml of distilled or deionized water for each 100 ml being prepared and transfer to a clean glass container with tight-fitting lid. 2. Carefully measure 80 ml of Methanol for each 100 ml being prepared and add to the container. 3. Cover and swirl to mix completely. Store tightly sealed to minimize evaporation. SAMPLE PREPARATION 1. Grind samples to pass a 20 mesh sieve and thoroughly mix prior to sub-sampling. Samples not being immediately analyzed should be stored refrigerated. 2. Weigh 50 g ground sample and 5.0 g NaCl and transfer to clean blender jar. 3. Add 100 ml of 80% Methanol/water to the jar. 4. Blend for 1 minute in a high-speed blender. 5. Filter a minimum of 10 ml through a glass fiber filter. 6. Dilute 5 ml of extract with 20 ml of water and mix thoroughly. 7. Filter through a glass fiber filter. TEST PROCEDURE (Note: Running calibrators and samples in duplicate will improve assay precision and accuracy.) 1. Allow reagents and sample extracts to reach room temperature prior to running the test. 2. Place the appropriate number of test wells and into a microwell holder. Be sure to re-seal unused wells in the zip-lock bag with dessicant. 3. Dispense 50 µl of Enzyme Conjugate into each test well. 4. Using a pipet with disposable tips, add 50 ul of calibrators and samples to the appropriate test wells. Be sure to use a clean pipet tip for each. 5. Dispense 50 µl of Antibody Solution into each test well. 6. Incubate the test wells for 10 minutes. 7. Dump the contents of the wells into an appropriate waste container. Fill the wells to overflowing with tap water and dump. Repeat 4X for a total of five washes. 8. Following the last wash tap the inverted wells onto absorbent paper to remove the last of the wash solution. 9. Dispense 100 µl of Substrate into each well. 10. Incubate the wells for 10 minutes.

4 11. Dispense 100 µl of Stop Solution into each test well. 12. Read and record the absorbance of the wells at 450nm using a strip or plate reader. RESULTS INTERPRETATION 1. Semi-quantitative results can be derived by simple comparison of the sample absorbance s to the absorbance of the calibrator wells: Sample containing less color than a calibrator well have a concentration of Aflatoxin greater than the concentration of the calibrator. Samples containing more color than a calibrator well have a concentration less than the concentration of the calibrator. 2. Quantitative interpretation requires graphing the absorbances of the calibrators (X axis) versus the log of the calibrator concentration (Y axis) on semi-log graph paper. A straight line is drawn through the calibrator points and the sample absorbances are located on the line. The corresponding point on the Y axis is the concentration of the sample. Samples with absorbances greater than the lowest calibrator or less than the highest calibrator must be reported as < 2 ppb or >100 ppb, respectively. Alternatively, Abraxis can supply a spreadsheet template which can be used for data reduction. Please contact Abraxis for further details. The following table is for illustration only. A standard curve must be run with each assay run. Well content OD Mean OD SD % RSD %Bo Concentration (ppb) 0 ppb ppb ppb ppb ppb Sample SPECIFICITY The Abraxis Aflatoxin Plate Kit can t differentiate between the various Aflatoxins, but detects their presence to differing degrees. The following table shows the relative values for 50% Bo and the (%) crossreactivity versus Aflatoxin B1. All concentration are in parts per billion (ppb). Compound 50% Bo (ppb) Cross-Reactivity (%) Aflatoxin B Aflatoxin B Aflatoxin G Aflatoxin G

5 ASSISTANCE For ordering or technical assistance contact: Abraxis LLC 54 Steamwhistle Drive Warminster, Pennsylvania, Phone: (215) * Fax: (215) info@abraxiskits.com WEB: abraxiskits.com GENERAL LIMITED WARRANTY Abraxis LLC warrants the products manufactured by the Company, against defects and workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product s printed expiration date. Abraxis makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose.

6 Aflatoxin Test Strip 20 ppb A Screening Test for Rapid Detection of Aflatoxin in Grain Samples. ABOUT AFLATOXIN Aflatoxin is a potent liver toxin known to cause cancer in animals. In swine, aflatoxin can cause reduced weight gain, reduced Abraxislity to resist diseases, hepatitis and death. The Food and Drug Administration (FDA) has established action levels of 20 parts per billion (ppb) for grain and feed products, and 0.5 ppb for milk. Aflatoxins are produced by the fungus Aspergillus flavus. This fungus causes a disease in preharvest corn known as Aspergillus ear rot. Although Aspergillus flavus Bis a common fungus growing as a saprophyte on dead plant debris, infection and aflatoxin production in pre-harvest corn occur almost exclusively in years when plants are severely stressed by drought. Aflatoxin production in the field is favored by high grain moisture, temperatures in the range of º F, severe drought stress, nitrogen deficiency, and significant insect damage. Except in hot, dry years, aflatoxin in the Midwest is almost exclusively associated with improper storage of grain or feed. Aflatoxin production in stored grain should not occur if grain is sufficiently dried to 13-14% moisture and maintained at that level. Grain, feed, or milk containing aflatoxin at or above these levels cannot be sold for food or feed in interstate sales. Mixing aflatoxin contaminated grain with sound grain for sale is illegal. In the U.S. corn and other grain with less than 20 ppb aflatoxin can be sold as normal grain. Recommended limits in feed are: i 0.5 ppb for milk 20 ppb for dairy animals 100 ppb for breeding cattle, breeding swine, and mature poultry 300 ppb for finishing cattle and swine. INTENDED USE This Rapid Aflatoxin Test is designed solely for use in preliminary screening of grain samples. It is a competitive inhibition immunoassay for the qualitative detection of aflatoxin in samples such as corn, corn meal and rice. The test produces a discernible positive result in grain samples containing aflatoxin in quantities of approx. 5 ppb or higher. The Rapid Aflatoxin Test provides only a preliminary qualitative analytical test result. Other methods must be used to obtain a more confirmed analytical result. Professional judgment should be applied to any test results, particularly when preliminary positive results are used. HPLC or GCMS are recommended as methods of choice for confirmation of positive results obtained with the Rapid Aflatoxin Test. INTRODUCTION The Rapid Aflatoxin Test is a qualitative one-step competitive inhibition immunoassay for the detection of aflatoxin. It detects the presence of aflatoxin at 5 ppb or higher in grain samples by utilizing highly specific reactions between antibodies and aflatoxin in grain samples. The need for this product is illustrated by recent literature discussing rapid, on-site tests for aflatoxin. 2,3,4 TEST PRINCIPLE The toxin conjugate competes for antibody binding sites with toxins that may be present in the grain sample. The test device consists of a membrane strip to which a conjugate of the toxin of interest is attached. A colloidal gold labeled antibody is located at one end of the membrane. A control line, produced by a different antibody/antigen reaction, is also present on the membrane strip. The control line is not influenced by the presence or absence of mycotoxins in the grain sample, and therefore, it should be present in all reactions. In the absence of toxin in the grain sample, the colloidal gold labeled antibody complex moves with the grain sample by capillary action to contact the immobilized aflatoxin conjugate. An antibodyantigen reaction occurs forming a visible line in the test area. The formation of two visible lines indicates a negative test result. This means the test did not detect the toxin at or below the cut-off point established for the toxin. If aflatoxin is present in the grain sample, it competes with the immobilized toxin conjugate in the test area for the antibody binding sites on the colloidal gold labeled antibody complex. If a sufficient amount of toxin analyte is present, it will fill all of the available binding sites, thus preventing attachment of the labeled antibody to the toxin conjugate. If a colored line is not visible in the Test Line region, or Test Line is lighter than negative controls, aflatoxin is present at levels of concern. Available Aflatoxin Controls may be used to approximate the quantity of toxin present in grain samples. MATERIALS PROVIDED 1. Aflatoxin Test Strips 2. Package insert sheet MATERIALS REQUIRED BUT NOT SUPPLIED: ml screw cap vial for extracting specimen 4. Graduated cylinder for preparing Extraction Solution (70% Methanol/DI Water) 5. Methanol 6. Deionized Water ml microcentrifuge tubes 8. Pipets to deliver 250 ul 9. Timer WARNINGS AND PRECAUTIONS 1. This test is for screening use by professionals only. 2. Prior to use, ensure that the product has not expired by verifying that the date of use is prior to the expiration date on the label. 3. The desiccant vial containing the test strips must remain completely closed except for opening to remove test strips. When re-closing, snap lid firmly. 4. Avoid cross-contamination of grain samples by using a new container for each specimen. STORAGE The Rapid Aflatoxin Test should be stored at room temperature (15 to 30 C) or refrigerated (2 to 8 C). The test strip and grain extract should be at room temperature before use. SAMPLE COLLECTION AND HANDLING 1. Prepare a solution of 70% methanol in PBS by mixing Methanol and PBS in a ratio of 70: Add 10 gm grain to 20 ml 70% methanol/ PBS ph Shake mixture for 3 minutes and allow to settle for 2 minutes. 4. Mix 1 ml of extract from above with 1 ml PBS ph 7.4. ASSAY PROCEDURE 1. Equilibrate test strip and grain extract to room temperature before conducting any testing.

7 2. Transfer 500 ul of diluted grain extract (from step 3 above) to a small test tube. 3. Remove test strip from the desiccant vial and dip into diluted extract with arrows pointing downward. 4. Allow the test to develop for 10 minutes and read the result as explained below under Interpretation of Results. INTERPRETATION OF RESULTS Negative test results may be visible within 2-3 minutes. It is recommended that results be reviewed after 10 minutes when strips are completely developed. Figure 1: Positive Result Aflatoxin greater than 20 ppm. Lower aflatoxin levels produce test line of increasing intensity. SENSITIVITY The ABRAXIS Rapid Aflatoxin Test will detect aflatoxin at 5 ppb or lower. At this level the test line exhibits moderate intensity. At greater than 20 ppb the test line is not visible. When compared with samples of known aflatoxin contamination, it is possible to obtain a semi-quantitative result. CONTROLS It is good laboratory practice to use positive and negative controls to ensure proper test performance. Grain samples containing known quantities of Aflatoxin should be run on each lot of test strips to provide a reference for line intensity to be expected. BIBLIOGRAPHY Control Line Test Line Interpretation No control line present Control line present Control line present Control line present Test Line TEST INTERPRETATION No test line present Distinct test line present Moderate intensity test line present No test line present Control Line Figure 2: Negative Result Aflatoxin less than 20 ppm Invalid result 0 ppb Between 0 and 20 ppb >20 ppb 1 Woloshuk, Charles P.; Corn Diseases; Mycotoxins and Mycotoxin Test Kits. BP-47 Department of Botany and Plant Pathology, Purdue University; West Lafayette, IN Delmulle BS, De Saeger SM, Sibanda L, Barna-Vetro I, Van Peteghem CH. Development of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. J Agric Food Chem May 4;53(9): Xiulan S, Xiaolian Z, Jian T, Zhou J, Chu FS. Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1. Int J Food Microbiol Mar 15;99(2): Stubblefield RD, Greer JI, Shotwell OL, Aikens AM. Rapid immunochemical screening method for aflatoxin B1 in human and animal urine. J Assoc Off Anal Chem May- Jun;74(3): LIMITATIONS OF PROCEDURE The assay is designed for use with grain extracts. The Rapid Aflatoxin Test provides only a preliminary qualitative test result. Use another more quantitative analytical method to obtain a confirmed quantitative analytical result. Apply professional judgment to any test result, particularly when preliminary positive results are observed. 20 ppb Aflatoxin Test Strip Ver

8 Importance of Aflatoxin M1 Determination Aflatoxins are highly toxic mycotoxins produced by a variety of molds such as Aspergillus flavus, A. parasiticus and A. nomius. These toxins are known carcinogens and can be present in grains, nuts, cottonseed and other foods consumed by humans or in animal feed. Crops may be contaminated by some of these toxins during growth, harvest or storage. The toxins most frequently detected are Aflatoxin B1, B2, G1, and G2. When animals are fed contaminated feed, Aflatoxin B1 is converted to M1 by hydroxylation, which is subsequently secreted into the milk of lactating animals. Human breast milk can also contain Aflatoxin M1 if a lactating woman has consumed food contaminated with Aflatoxin B1. Aflatoxin M1 is very stable throuhout milk processing methods such as pasteurazation. To protect humans, regulatory agencies around the world have imposed regulatory limits regarding the amount of Aflatoxins that are allowable in human and animal foods. In Europe, the Aflatoxin M1 tolerance levels (ML) are as follows: Milk (raw milk, milk used in the production of milk based products and heat treated milk): 0.05 ppb Infant formula and infant milk: ppb Dietary foods intended for infants: ppb The Aflatoxin M1 ELISA allows the determination of 42 samples in duplicate determination. Less than 1 ml of sample extract is required. The test can be performed in less than 2 hours. Performance Data Test sensitivity: The detection limit for Aflatoxin M1 is 11 pg/ml (mean of 6 blank determinations minus 3 standard deviations). The middle of the test (50% B/B0) is at approximately 100 pg/ml. Determinations closer to the middle of the calibration curve give the most accurate results. Test reproducibility: Coefficients of variation (CVs) for standards: <10%; CVs for samples: <15%. Specificity: The cross-reactivity of the Abraxis Aflatoxin M1 Kit for various other Aflatoxins (B1, B2, G1, and G2) in milk were not determined as these mycotoxins are not excreted in milk. Recoveries: Recoveries of Aflatoxin M1 from spiked whole milk are as follows: General Limited Warranty: Abraxis LLC warrants the products manufactured by the Company, against defects and workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product s printed expiration date. Abraxis makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose For ordering or technical assistance contact: Abraxis LLC 54 Steamwhistle Drive Warminster, PA Tel.: (215) Fax: (215) info@abraxiskits.com WEB: R Aflatoxin M1 ELISA (Microtiter Plate) Enzyme-Linked Immunosorbent Assay for the Determination of Aflatoxin M1 in Contaminated Samples Product No M 1. General Description The Aflatoxin M1 ELISA is an immunoassay for the quantitative and sensitive screening of Aflatoxin M1. This test is suitable for the quantitative and/or qualitative screening of Aflatoxin M1 in milk and milk products (please refer to the appropriate technical bulletins for extraction/dilution procedures). Samples requiring regulatory action should be confirmed by HPLC, GC/MS, or other conventional methods. 2. Safety Instructions The standard solutions of the test kit contain small amounts of Aflatoxin M1. In addition, the substrate solution contains tetramethylbenzidine and the stop solution contains diluted sulfuric acid. Avoid contact of stopping solution with skin and mucous membranes. If these reagents come in contact with the skin, wash with water. 3. Storage and Stability The Aflatoxin M1 ELISA should be stored in the refrigerator (4 8 C). The solutions must be allowed to reach room temperature (20-25 C) before use. Reagents may be used until the expiration date on the box. 4. Test Principle The test is a forward ELISA and it is based on the recognition of Aflatoxin M1 by antibodies. The calibrators and sample extract(s) are pipetted into test wells coated with Aflatoxin M1 antibody to initiate the reaction. After a 30 minute incubation and washing of the wells, Aflatoxin-M1 HRP conjugate is added, followed by a 60 minute incubation period. The HRP conjugate binds to unbound sites on the Aflatoxin M1 antibody. Following this 60 minute incubation, the contents of the well are removed and the wells are washed to remove any unbound Aflatoxin M1 HRP conjugate. After washing with the diluted wash solution, a clear substrate is added to the wells and any bound enzyme conjugate causes the conversion of the colorless substrate to a blue color. Following a 20 minute incubation, the reaction is stopped and the amount of color in each well is read using an ELISA reader. The color of the unknown samples is compared to the color of the calibrators and the Aflatoxin M1 concentration of the samples is derived. The concentrations of the samples are determined by interpolation using the standard curve constructed with each run. 5. Limitations of the Aflatoxin M1 ELISA, Possible Test Interference Although many organic and inorganic compounds commonly found in samples have been tested and found not to interfere with this test, due to the high variability of compounds that might be found in samples, test interferences caused by matrix effects can t be completely excluded. Milk fats will cause interference in the test, therefore milk samples should be defatted as instructed in the sample preparation step (Section H) before testing in the ELISA. Mistakes in handling the test also can cause errors. Possible sources for such errors can be: inadequate storage conditions of the test kit, wrong pipetting sequence or inaccurate volumes of the reagents, too long or too short incubation times during the immune and/or substrate reaction, extreme outside temperatures (lower than 10 0 C or higher than 30 0 C) during the test performance.

9 The Abraxis Aflatoxin M1 ELISA kit provides screening results. As with any analytical technique (GC, HPLC, etc.), samples requiring some regulatory action should be confirmed by an alternative method.

10 A. Reagents and Materials Provided 1. Microtiter plate (12 X 8 strips) coated with polyclonal anti-aflatoxin M1 antibody, in an resealable aluminum pouch 2. Calibrators/Standards (6): 0, 15, 25, 50, 100, 250 pg/ml (ppt) of Aflatotoxin M1, 1 ml each 3. Aflatoxin M1-HRP Conjugate, 12 ml 4. Wash Solution (5X) Concentrate, 100 ml. Must be diluted 1:5 with deionized water before use 5. Substrate (Color) Solution (TMB), 12 ml 6. Stop Solution, 6 ml (Handle with care) B. Test Preparation Micro-pipetting equipment and pipette tips for pipetting the standards and the samples are necessary. We recommend using a multi-channel pipette or a stepping pipette for adding the enzyme conjugate, the substrate solution and the stop solution in order to equalize the incubation periods on the entire microtiter plate. Please only use the reagents and standards from one package lot in one test, as they have been adjusted in combination. 1. Adjust the microtiter plate and the reagents to room temperature before use. 2. Remove the number of microtiter plate strips required from the aluminum pouch. The remaining strips are stored in the aluminum pouch and zip-locked closed. Store the remaining kit in the refrigerator (4-8 C). 3. The standard solutions, enzyme conjugate, substrate and stop solution are ready to use and do not require any further dilutions. 4. Dilute the wash buffer at a ratio of 1:5. If using the entire bottle (100 ml), add to 400 ml of deionized or distilled water. 5. The stop solution must be handled with care as it contains diluted H2SO4. C. Assay Procedure 1. Add 100 µl of the standard solutions or the sample extracts (Section H) into the wells of the test strips according to the working scheme given. We recommend using duplicates or triplicates. 2. Incubate for 30 minutes at room temperature. 3. Wash the strips three times using the diluted washing buffer solution. Please use at least a volume of 250 µl of washing buffer for each well and each washing step. Remaining buffer in the wells should be removed by patting the plate dry on a stack of paper towels. 4. Add 100 µl of enzyme conjugate solution to the individual wells successively using a multi- channel pipette or a stepping pipette. 5. Incubate the strips for 60 min at room temperature. 6. Wash the strips three times using the diluted washing buffer solution. Please use at least a volume of 250 µl of washing buffer for each well and each washing step. Remaining buffer in the wells should be removed by patting the plate dry on a stack of paper towels. 7. Add 100 µl of substrate (color) solution to the wells. Incubate the strips for 20 min at room temperature. Protect the strips from direct sunlight. 8. Add 50 µl of stop solution to the wells in the same sequence as for the substrate solution. 9. Read the absorbance at 450 nm using a microplate ELISA photometer within 15 minutes after the addition of stopping solution. D. Evaluation The evaluation of the ELISA can be performed using commercial ELISA evaluation programs such as Logit/Log or 4- Parameter (preferred). For a manual evaluation, calculate the mean absorbance value for each of the standards. Calculate the %B/B0 for each standard by dividing the mean absorbance value for each standard by the Zero Standard (Standard 0) mean absorbance. Construct a standard curve by plotting the %B/B0 for each standard on the vertical linear (y) axis versus the corresponding Aflatoxin M1 concentration on the horizontal logarithmic (x) axis on graph paper. %B/B0 for samples will then yield levels in ppt or pg/ml of Aflatoxin M1 by interpolation using the standard curve. Results can also be obtained by using a spreadsheet macro available from Abraxis upon request. The concentrations of the samples are determined using the standard curve run with each test. Samples showing a lower concentrations of Aflatoxin M1 compared to standard 1 (15 pg/ml or ppt) must be reported as containing < 15 ppt Aflatoxin M1. Samples showing a higher concentration than standard 5 (250 pg/ml) must be diluted further to obtain accurate results. Semi-quantitative results can be derived by simple comparison of the sample absorbances to the absorbance of the calibrators. Sample containing less color than a calibrator will have a concentration of Aflatoxin M1 greater than the concentration of the calibrator. Samples containing more color than a calibrator will have a concentration less than the concentration of the calibrator. E. Additional Materials (not delivered with the test kit) 1. Micro-pipettes with disposable plastic tips ( µl) 2. Multi-channel pipette ( µl) or stepper pipette with plastic tips ( µl) 3. Microtiter plate washer (optional) 4. Microtiter plate reader (wave length 450 nm) 5. Shaker for microtiter plates (optional) 6. Deionized or distilled water 7. Paper towels or equivalent absorbent material 8. Timer 9. Microcentrifuge capable of spinning at 12,000 14,000 RPM 10. Microcentrifuge tubes F. Working Scheme The microtiter plate consists of 12 strips of 8 wells, which can be used individually for the test. The standards must be run with each test. Never use the values of standards which have been determined in a test performed previously. Std 0-Std 5: Standards (0; 15; 25; 50; 100; 250 ppt) Sam1, Sam2, etc.: Samples G. Standard Curve (These values are used for demonstration purposes; do not use these values for your determinations) Abraxis Aflatoxin M1 ELISA B/Bo pg/ml (parts per trillion) H. Milk Sample Extraction 1. Pipette 1 ml of milk into a centrifuge tube, centrifuge at 12,000 14,000 RPM for 5 minutes. 2. Using a pipette, carefully remove the middle layer (the milk fat is the top layer). 3. Run 100 ul of the middle layer directly in the assay (step 1 on Assay Procedure). 4. Highly contaminated samples (samples outside the standard curve range) should be diluted in order to get the value in the middle of the curve and re-analyzed. I. Powder Milk Extraction (non-fat) 1. Weigh 1 gm of milk powder, add 10 ml of DI water, shake to dissolve. Analyze directly in the ELISA (step 1 Assay Procedure). Assay results will need to be multiplied by 10. J. Powder Milk (regular) 1. Weigh 1 gm of milk powder and dilute with 10 ml of DI water, shake to dissolve. 2. Centrifuge at 12,000 14,000 RPM for 5 minutes. 3. Follow same steps as H Assay results will need to be multiplied by 10.

11 Aflatoxin M1 Plate, Detailed ELISA Procedure 1. Addition of Standards, Samples Add 50 ul of the standard solutions, samples or sample extracts into the wells of the test strips according to the working scheme given. We recommend using duplicates or triplicates. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a rapid circular motion on the benchtop. Be careful not to spill contents. Incubate the strips for 30 minutes at room temperature. 2. Washing of Plates After incubation, remove the covering and vigorously shake the contents of the wells into a sink. Wash the strips three times with a multi-channel pipette or wash bottle using the diluted 1X washing buffer solution. Please use at least 250 ul of washing buffer for each well and each washing step. Remaining buffer in the wells should be removed by patting the plate dry on a stack of paper towels. 5. Addition of Substrate/Color Solution Add 100 ul of substrate/color solution to the individual wells successively using a multichannel pipette or a stepping pipette. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a rapid circular motion on the benchtop. Be careful not to spill contents. Incubate the strips for 20 minutes at room temperature away from direct sunlight. 6. Addition of Stopping Solution Add 50 ul of stop solution to the wells, in the same sequence as for the substrate solution, using a multi-channel pipette or a stepping pipette. 3. Addition of Enzyme Conjugate Add 100 ul of the Aflatoxin M1 enzyme conjugate to the individual wells successively using a multi-channel pipette or a stepping pipette. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a rapid circular motion on the benchtop. Be careful not to spill contents. Incubate the strips for 60 minutes at room temperature. 7. Measurement of Color Read the absorbance at 450 nm using a microplate ELISA reader within 15 minutes. Calculate results. 4. Washing of Plates After incubation, remove the covering and vigorously shake the contents of the wells into a sink. Wash the strips three times with a multi-channel pipette or wash bottle using the diluted 1X washing buffer solution. Please use at least 250 ul of washing buffer for each well and each washing step. Remaining buffer in the wells should be removed by patting the plate dry on a stack of paper towels. For Ordering or Technical Assistance Contact: ABRAXIS, LLC 54 Steamwhistle Drive, Warminster, PA Phone: Fax: Aflatoxin M1 Plate Kit Part # 53012M

12 Aflatoxin M1 Plate, Concise ELISA Procedure 1. Addition of Standards, Samples Add 100 ul of standard solutions, sample or sample extract. Cover and mix for 30 seconds by moving strip holder in a circular motion on benchtop. Incubate 30 minutes at room temperature away from direct sunlight. 5. Addition of Substrate/Color Solution Add 100 ul of substrate/color solution. Cover and mix for 30 seconds by moving strip holder in a circular motion on benchtop. Incubate 20 minutes at room temperature away from direct sunlight. 4. Washing of Plates Wash the wells three times with 250 ul of diluted 1X washing buffer. 6. Addition of Stopping Solution Add 50 ul of stop solution. 2. Addition of Enzyme Conjugate Add 100 ul of enzyme conjugate. Cover and mix for 30 seconds by moving strip holder in a circular motion on benchtop. Incubate 60 minutes at room temperature away from direct sunlight. 7. Measurement of Color Measure color at 450 nm within 15 minutes. Calculate results. 4. Washing of Plates Wash the wells three times with 250 ul of diluted 1X washing buffer. For Ordering or Technical Assistance Contact: ABRAXIS, LLC 54 Steamwhistle Drive, Warminster, PA Phone: Fax: Aflatoxin M1 Plate Kit Part # 53012M

13 Fumonisin Plate Kit PN 53014B INTENDED USE The Fumonisin Plate Kit is a competitive ELISA for the quantitative analysis of fumonisin in corn, corn meal, corn germ meal, corn gluten meal and corn/soy blend. ASSAY PRINCIPLES The Fumonisin kit is a competitive enzyme-labeled immunoassay. Fumonisin is extracted from a ground sample by shaking with methanol/water. The extract is then filtered and the extract is tested in the immunoassay. Fumonisin-HRP enzyme conjugate is pipetted into the test wells followed by calibrators or sample extracts. Fumonisin antibody is then pipetted into the test wells to initiate the reaction. During the 10 minute incubation period, fumonisin from the sample and fumonisin-hrp enzyme conjugate compete for binding to fumonisin antibody which, in turn, binds to the test well. Following this 10 minute incubation, the contents of the well are removed and the wells are washed to remove any unbound enzyme-labeled toxin. A clear substrate is then added to the wells and any bound enzyme-toxin conjugate causes the conversion to a blue color. Following a 5 minute incubation, the reaction is stopped and amount of color in each well is read. The color of unknown samples is compared to the color of the calibrators and the Fumonisin concentration of the samples is derived. SPECIFICITY The antibody utilized in the Abraxis Fumonisin Plate Kit is specific for fumonisins. The calibrators are formulated with fumonisin B 1 The following table shows the relative reactivity for other forms: Compound Cross-Reactivity fumonisin B2 44% fumonisin B3 104% REAGENTS AND MATERIALS PROVIDED The kit in its original packaging can be used until the end of the month indicated on the box label when stored at 2-8ºC.

14 Plate containing 12 test strips of 8 wells each vacuum-packed in aluminized pouch with indicating dessicant. 5 vials each containing 2 ml of Fumonisin calibrators corresponding to 0, 0.3, 1.0, 3.0 and 6.0 µg/ml (ppm) of Fumonisin. (Note: Because of the 1:5 dilution of the grain sample in the extraction step, the calibrators actually contain 1/5th of the stated value. No further correction back to the concentration in the original grain sample is required.) 1 vial containing 8 ml of Fumonisin-HRP Enzyme Conjugate. 1 vial containing 8 ml of Rabbit anti-fumonisin antibody. 1 vial containing 14 ml of Substrate. 1 vial containing 14 ml of Stop Solution. (Caution! 1N HCl. Handle with care.) Instructions PRECAUTIONS 1. Each reagent is optimized for use in the Abraxis Fumonisin Plate Kit. Do not substitute reagents from any other manufacturer into the test kit. Do not combine reagents from other Abraxis Fumonisin Plate Kits with different Lot numbers. 2. Dilution or adulteration of reagents or samples not called for in the procedure may result in inaccurate results. 3. Do not use reagents after expiration date. 4. Reagents should be brought to room temperature, 20-28ºC (62-82ºF) prior to use. Avoid prolonged (> 24 hours) storage at room temperature. 5. Fumonisin is a very toxic substance. Dispose of all liquids in a plastic container containing household bleach (minimum 10%). All labware should be soaked for at least 1 hour in a 30% solution of household bleach. Avoid contact of skin and mucous membranes with reagents and sample extracts by wearing gloves and protective apparel. If exposure of skin and mucous membranes to liquids should occur, immediately flush with water. 6. The Stop Solution is 1N hydrochloric acid. Avoid contact with skin and mucous membranes. Immediately clean up any spills and wash area with copious amounts of water. If contact should occur, immediately flush with copious amounts of water.

15 MATERIALS REQUIRED BUT NOT PROVIDED 1. Laboratory quality distilled or deionized water. 2. Methanol, ACS grade 3. Graduated cylinder, 100 ml or larger. 4. Glassware for sample extraction and extract collection. 5. Filter, Whatman GF/A or equivalent 6. Pipet with disposable tips capable of dispensing 50 µl. 7. Multi-channel pipet; 8 channel capable of dispensing 50 and 100 µl. 8. Paper towels or equivalent absorbent material. 9. Microwell plate or strip reader with 450nm filter. 10. Timer EXTRACTION SOLUTION PREPARATION 1. Carefully measure 30 ml of distilled or deionized water for each 100 ml being prepared and transfer to a clean glass container with tight-fitting lid. 2. Carefully measure 70 ml of Methanol for each 100 ml being prepared and add to the container. 3. Cover and swirl to mix completely. Store tightly sealed to minimize evaporation. SAMPLE PREPARATION 1. Grind samples to pass a 20 mesh sieve and thoroughly mix prior to sub-sampling. Samples not being immediately analyzed should be stored refrigerated. 2. Weigh 20 grams of ground sample and combine with 100 ml of 70% methanol in a clean container with tight fitting lid. 3. Vigorously shake the container for 3 minutes. 4. Allow sample to stand for 2-3 minutes to allow some settling of the slurry.

16 5. Filter a minimum of 15 ml of the extract through Whatman GF/A filters and collect the extract into a clean container. TEST PROCEDURE (Note: Running calibrators and samples in duplicate will improve assay precision and accuracy.) 1. Allow reagents and sample extracts to reach room temperature prior to running the test. 2. Place the appropriate number of test wells and into a microwell holder. Be sure to reseal unused wells in the zip-lock bag with dessicant. 3. Dispense 50 µl of Enzyme Conjugate into each test well. 4. Using a pipet with disposable tips, add 50 μl of calibrators and samples to the appropriate test wells. Be sure to use a clean pipet tip for each. 5. Dispense 50 µl of Antibody Solution into each test well. 6. Incubate the test wells for 10 minutes. 7. Dump the contents of the wells into an appropriate waste container. Fill the wells to overflowing with tap water and dump. Repeat 4X for a total of five washes. 8. Following the last wash tap the inverted wells onto absorbent paper to remove the last of the wash solution. 9. Dispense 100 µl of Substrate into each well. 10. Incubate the wells for 5 minutes. 11. Dispense 100 µl of Stop Solution into each test well. 12. Read and record the absorbance of the wells at 450nm using a strip or plate reader. RESULTS INTERPRETATION 1. Semi-quantitative results can be derived by simple comparison of the sample absorbances to the absorbance of the calibrator wells: Sample containing less color than a calibrator well have a concentration of Fumonisin greater than the concentration of the calibrator. Samples containing more color than a calibrator well have a concentration less than the concentration of the calibrator.

17 2. Quantitative interpretation requires graphing the absorbances of the calibrators (X axis) versus the log of the calibrator concentration (Y axis) on semi-log graph paper. A straight line is drawn through the calibrator points and the sample absorbances are located on the line. The corresponding point on the Y axis is the concentration of the sample. Samples with absorbances greater than the lowest calibrator or less than the highest calibrator must be reported as < 0.3 ppm or >6 ppm, respectively. TECHNICAL ASSISTANCE For questions regarding this kit or for additional information about Abraxis products, call (215) ASSISTANCE For ordering or technical assistance contact: Abraxis LLC 54 Steamwhistle Drive Warminster, Pennsylvania, Phone: (215) * Fax: (215) info@abraxiskits.com WEB: abraxiskits.com GENERAL LIMITED WARRANTY Abraxis LLC warrants the products manufactured by the Company, against defects and workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product s printed expiration date. Abraxis makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose.

18 Fumonisin Test Strip 5 ppm A Screening Test for Rapid Detection of Fumonisin in Grain Samples. ABOUT FUMONISIN Fumonisins are environmental toxins produced mainly by the molds Fusarium monoliforme, F. verticelloides, F. proliferatum and other Fusarium species that grow on agricultural commodities in the field or during storage. These mycotoxins have been found worldwide, primarily in corn. More than 10 types of fumonisin have been isolated and characterized. Of these, fumonisin B1, B2 and B3 are the major fumonisins produced. The most prevalent of these mycotoxins in contaminated corn is fumonisin B1, which is believed to the most toxic. 1,2 INTENDED USE The Abraxis Fumonisin Test is designed solely for use in preliminary screening of grain samples. It is a competitive inhibition immunoassay for the qualitative detection of fumonisin in samples such as corn, corn meal and rice. The test produces a positive result in grain samples containing fumonisin in quantities of 5 ppm or higher. The Abraxis Fumonisin Test provides only a preliminary qualitative analytical test result. Other methods must be used to obtain a more confirmed analytical result. Professional judgment should be applied to any test results, particularly when preliminary positive results are used. HPLC or GCMS are recommended as methods of choice for confirmation of positive results obtained with the Abraxis Fumonisin Test. INTRODUCTION The Abraxis Fumonisin Test is a qualitative one-step competitive inhibition immunoassay for the detection of fumonisin. It detects the presence of fumonisin by utilizing highly specific reactions between antibodies and fumonisin in grain samples. PRINCIPLE The specifically labeled drug (drug conjugate) competes for antibody binding sites with toxins that may be present in the grain sample. The test device consists of a membrane strip to which a conjugate of the toxin of interest is attached. A colloidal gold labeled antibody is located at one end of the membrane. A control line, produced by a different antibody/antigen reaction, is also present on the membrane strip. The control line is not influenced by the presence or absence of mycotoxins in the grain sample, and therefore, it should be present in all reactions. In the absence of toxin in the grain sample, the colloidal gold labeled antibody complex moves with the grain sample by capillary action to contact the immobilized drug conjugate. An antibody-antigen reaction occurs forming a visible line in the test area. The formation of two visible lines indicates a negative test result. This means the test did not detect the toxin at or below the cutoff point established for the toxin. MATERIALS PROVIDED 1. Fumonisin Test Strips 2. Package insert sheet MATERIALS REQUIRED BUT NOT SUPPLIED: or 50 ml screw cap vial for extracting specimen 2. Phosphate Buffered Saline (PBS) ml microcentrifuge tubes 4. Pipets to deliver 500 ul 5. Timer WARNINGS AND PRECAUTIONS 1. This test is for screening use by professionals only. 2. Prior to use, ensure that the product has not expired by verifying that the date of use is prior to the expiration date on the label. 3. The desiccant vial containing the test strips must remain completely closed except for opening to remove test strips. When re-closing, snap lid firmly. 4. Avoid cross-contamination of grain samples by using a new container for each specimen. STORAGE The Abraxis Rapid Test should be stored at room temperature (15 to 30 C) or refrigerated (2 to 8 C). The test strips and grain extract should be at room temperature before using. SAMPLE COLLECTION AND EXTRACTION 1. Collect the sample according to GIPSA 2 recommended procedure. 2. Add 10 ml PBS to a suitable screw cap container. Weigh 1 gm grain sample and add to buffer in container, tighten cap and shake vigorously for 3 minutes to extract fumonisin from grain sample. 3. Allow sediment to settle for 5 minutes. It is important to allow the recommended settling time. Excess sediment in the extracts can interfere with the flow of liquid and this may affect test results. 4. Test line intensity may be lighter when extracts are not fresh. Same day testing is recommended. ASSAY PROCEDURE 1. Be certain the test strip and grain extract have equilibrated to room temperature before conducting any testing. Temperature variation can affect test results. 2. Transfer 500 ul of grain extract to a micro centrifuge tube. 3. Insert test strip (with arrows down) into extract and allow test to develop. 4. Read results after 10 minutes. (Negative results may be visible within approx. 3 minutes.) When fumonisin is present in the grain sample, it competes with the immobilized toxin conjugate in the test area for the antibody binding sites on the colloidal gold labeled antibody complex. If a sufficient amount of toxin analyte is present, it will fill all of the available binding sites, thus preventing attachment of the labeled antibody to the toxin conjugate. The formation of one visible line indicates a positive result.

19 INTERPRETATION OF RESULTS Read results after five minutes. Figure 1: Positive Result Fumonisin level greater than 5 ppm. Lower Fumonisin levels produce test line of increasing intensity. Test line is not visible in specimens with Fumonisin content greater than 5 ppm. Test line is visible with Fumonisin content less than 5 ppm. TEST INTERPRETATION Control Line Test Line Interpretation No control line No test line Invalid result present present Control line present Control line present Control line present Test Line Distinct test line present No test line present Light intensity test line present Control Line Figure 2: Negative Result Fumonisin level less than 5 ppm 0 ppm 5 ppm and higher Between 0 and 5 ppm QUALITY CONTROL It is good laboratory practice to use positive and negative controls to ensure proper test performance. Quality control materials are commercially available. It is recommended that each shipment of product be tested upon receipt. LIMITATIONS OF PROCEDURE The assay is designed for use with grain extracts only. The Abraxis Fumonisin Test provides only a preliminary qualitative test result. Use another more specific quantitative analytical method to obtain a confirmed analytical result. Results obtained with the Abraxis Fumonisin Test cannot be considered conclusive evidence that fumonisin is present in quantities greater than the stated threshold. Specimens exhibiting positive results must be submitted to a qualified laboratory for analysis by GCMS or HPLC for definitive detection of fumonisin. SENSITIVITY The detection limit for fumonisin was established as 5 ppm as follows: Known concentrations of fumonisin were added to normal, toxin-free grain samples. Ten determinations were made at each serial dilution of the analyte. Sensitivity is defined as that concentration which produced positive responses in all 10 replicates. REPRODUCIBILITY Reproducibility studies were performed using extracts of grain samples that were previously assayed by HPLC. PPM Fumonisin No. samples Results Precision 0 30 Negative 100% Positive 100% BIBLIOGRAPHY 1. Thiel, P.G., Marasas W.F.O., Sydenham, E.W., Shephard, G.S. and Gelderblom, W.C.A The implications of naturally occurring fumonisins in corn for human and animal health. Mycopathologia 117: Musser, S.M. and Plattner, R.D Fumonisinb compositions in cultures of Fusarium monoliforme, Fusarium proliferatum, and Furarium nygami. Journal of Agricultural and Food Chemistry 45:

20 Importance of Total Ochratoxin Determination Ochratoxins are known contaminants of human food and animal feed produced by certain ubiquitously occurring Aspergillus and Penicillium species. The toxins are frequently detected in cereals, coffee, beer, wine, pet food, etc. Ochratoxins have been shown to be nephratoxic, teratogenic and carcinogenic in laboratory animals. Ochratoxin A (OTA) is the most prominent toxin, followed by ochratoxin B (OTB) and ochratoxin C (OTC). OTB has been reported to be less toxic than OTA, however, more recent data brings this into question. OTA and OTB can occur simultaneously, as they are produced by the same species of fungus and to date several studies have demonstrated synergistic effects of these toxins in vitro and in vivo. Based on those findings, it can be assumed that OTB may pose a risk to humans. Therefore, OTB is as least as important as OTA and to minimize risk it might be concluded that routine detection of OTB and OTA in food and beverages would be a prerequisite for improved determination of ochratoxin risk. To protect consumers from mycotoxin related risks, the European Union (EU) has established regulatory limits for OTA, (10 ppb in dried vine fruits and instant coffee, 5 ppb in cereals and roasted coffee, 2 ppb in wine. No regulatory limits exist currently for OTB or OTC. The Abraxis Total Ochratoxins ELISA allows the determination of 42 samples in duplicate determination. Only a few milliliters of sample are required. The test can be performed in 90 minutes. Performance Data Test sensitivity: The limit of detection for Ochratoxin A in buffer calculated as 90% B/Bo is equal to <0.03 ng/ml. The concentration of residue necessary to cause 50% inhibition (50% B/B0) is approximately 0.12 ng/ml. Determinations closer to the middle of the calibration range of the test yield the most accurate results. The following is the sensitivity in different matrixes: 2.5 ppb in green/roasted coffee; 1.25 ppb in grains; 0.5 ppb in beer; 1.75 ppb in wine; 5.0 ppb in dried pet food. Ochratoxins ELISA, Microtiter Plate Enzyme-Linked Immunosorbent Assay for the Determination of Total Ochratoxins (A/B/C) in Contaminated Samples Product No BA 1. General Description The Ochratoxins ELISA is an immunoassay for the detection of total Ochatroxins (A/B/C). This test is suitable for the quantitative and/or qualitative detection of Ochratoxins in contaminated samples. Positive samples should be confirmed by HPLC, GC/MS, or other conventional methods. 2. Safety Instructions The standard solutions in this test kit contain small amounts of Ochratoxin A in solution. In addition, the substrate solution contains tetramethylbenzidine and the stop solution contains diluted sulfuric acid. Avoid contact of standard and stopping solutions with skin and mucous membranes. If these reagents come in contact with the skin, wash with water. 3. Storage and Stability The Ochratoxins ELISA Kit should to be stored in the refrigerator (4 8 C) prior to use. The solutions must be allowed to reach room temperature (20-25 C) before use. Reagents may be used until the expiration date on the box. Some reagents need to be stored frozen after reconstitution (Test Preparation, section C). 4. Test Principle The test is a direct competitive ELISA based on the recognition of Ochratoxins by specific antibodies. Ochratoxins, when present in a sample and a Ochratoxin-enzyme conjugate compete for the binding sites of anti-ochratoxins antibodies which are immobilized on the wells of the microtiter plate. After a washing step and addition of the substrate solution, a color signal is produced. The intensity of the blue color is inversely proportional to the concentration of Ochratoxins present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA reader. The concentrations of the samples are determined by interpolation using the standard curve constructed with each run. Test reproducibility: Coefficients of variation (CVs) for standards: <10%; CVs for samples: <15%. Selectivity: This ELISA recognizes Ochratoxin A and related compounds with varying degrees: Cross-reactivities: Ochratoxin A 100% Ochratoxin B 231% Ochratoxin C 118% Fumonisin-B1 <0.2% Fumonisin-B2 <0.2% Deoxynivalenol <0.2% Zearalonone <0.2% Samples: To eliminate matrix effects in samples, a sample extraction/clean-up and dilution is required. See Preparation of Samples section. For additional extraction procedures for various other matrices please contact Abraxis LLC. General Limited Warranty: Abraxis LLC warrants the products manufactured by the Company against defects and workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product s printed expiration date. Abraxis makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose. For ordering or technical assistance contact: Abraxis LLC 54 Steamwhistle Drive Warminster, PA Tel.: (215) Fax: (215) info@abraxiskits.com WEB: Limitations of the Ochratoxins ELISA, Possible Test Interference Numerous organic and inorganic compounds commonly found in samples have been tested and found not to interfere with this test. However, due to the high variability of compounds that might be found in samples, test interferences caused by matrix effects can not be completely excluded. Mistakes in handling the test can also cause errors. Possible sources for such errors can be: Inadequate storage conditions of the test kit (or reagents), incorrect pipetting sequence or inaccurate volumes of the reagents, too long or too short incubation times during the immune and/or substrate reaction, extreme temperatures during the test performance (lower than 10 C or higher than 30 C). The Abraxis Ochratoxins ELISA kit provides screening results. As with any analytical technique (GC, HPLC, etc.), positive samples requiring some action should be confirmed by an alternative method. Working Instructions A. Materials Provided 1. Microtiter plate coated anti-ochratoxins antibody, in a resealable foil pouch with desiccant. 2. Ochratoxin Standards (6): 0, 0.025, 0.05, 0.10, 0.20, and 0.40 ng/ml. 3. HRP Conjugate Assay Buffer, 6 ml. 4. Dilution Buffer I, 25 ml. Used to dilute samples. 5. Ochratoxin-HRP Conjugate, 6 ml. 6. Dilution Buffer II, 25 ml. 7. Wash Solution (5X) Concentrate, 100 ml. 8. Color (Substrate) Solution (TMB), 16 ml. 9. Stop Solution, 12 ml.