Diachemix, LLC 223 North Water Street

Suite 500

Milwaukee, WI 53202-5707

U.S.A.

Tel: 1-414-902-6770

Fax: 1-414-902-6771

E-mail: Vvucic@diachemix.com

Website: http://www.diachemix.com

Technical Assistance

Diachemix, LLC 683 E. Center Street, Unit H

Grayslake, IL 60030-1686

U.S.A.

Tel: 1-847-548-2339

Fax: 1-847-548-2984

E-mail: mickj@diachemix.com

Or

graham@diachemix.com

Diachemix, Rapid Definitive Detection, and Sentry are registered trademarks of Diachemix, LLC as conferred by the United States Department of Patents and Trademarks.

DIAGNOSTIC TEST KIT USING

FLUORESCENCE POLARIZATION

TECHNOLOGY FOR THE DETECTION

OF AFLATOXIN IN CORN

AFLATOXIN FPA

QUALITATIVE TEST - CORN

RAPID ● EASY ● ACCURATE ● COST EFFECTIVE

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AFLATOXIN FPA

QUALITATIVE TEST - CORN

BREAKTHROUGH TECHNOLOGY

Diachemix is the world leader in the development of

Fluorescence Polarization (FP) technology for detection

of animal disease antibodies and food pathogens. FP is

a versatile and robust technology that can be applied to

various matrices including serum, whole blood, milk,

juices, and water.

Aflatoxins are a group of structurally related toxic com-

pounds produced by certain strains of the fungi Asper-

gillus flavus and A. parasiticus. Under favorable condi-

tions (temperature, humidity), the fungi grow on foods

and feed, and produce Aflatoxins. Contaminations most

commonly occur in tree nuts, peanuts, and other oil

seeds (including corn and cottonseed). The most com-

mon forms of Aflatoxin are known as B1, B2, G1, and G2,

although B1 is most predominant and the most toxic.

Aflatoxins are associated with a variety of adverse ef-

fects in a number of animal species, including humans,

such as acute necrosis, cirrhosis, and carcinoma of the

liver. In livestock, typical results of Aflatoxin contami-

nation are reduced weight gain, growth inhibition, ane-

mia, decreased fertility, or abortion.

The Aflatoxin Qualitative FPA kit is a rapid, easy, accu-

rate and cost effective diagnostic method that can be

used to detect Aflatoxins in corn at, or above, 20 ppb.

Page 7

2. Prepare two (2) control tubes. Pipette 1 ml (1,000 µl) of antibody solution into the 10

X 75 mm test tube. Add 100 µl of “Control” into the above tube, label the tube (Control) and vortex. Similarly pipette 1 ml (1,000 µl) of “Control” into the above tube, label the tube (Control) and vortex.

3. Similarly prepare all the sample tubes by adding 1 ml (1,000 µl) of antibody solution into each individual 10 X 75 mm test tube, and 100 µl of each “sample” into the above tubes containing antibody solution. Label the tubes (sample #) and vortex.

4. Open the reader chamber lid. Grasp the first tube (containing “Control”) firmly and place the tube in the instrument. Close the reader chamber lid. Press the “Down”

arrow once to select “Blank Int” and then press the “Read” button. Remove the tube from the reader only after instrument reading is complete. After reading the tube, the reader will automatically advance to the next blank intensity reading.

5. Continue by reading the 2nd “Control” tube, and then the “sample” tubes one by one similar to the 1st “Control” tube (step 4).

6. After reading “Blank Int” for all the samples, add 100 µl of Aflatoxin FP tracer to the

first tube (containing “Control”). Vortex thoroughly and incubate at room tempera-ture for 2 minutes.

7. Go to the first reading by entering the number “1” on the reader screen. Place the

above tube back in the reader. Press the “Down” arrow to display ”Sample Int.” and press the “Read” button. The instrument will display the mP value directly. (The reading will appear briefly at the bottom of the display before advancing to the next

sample, and can also be retrieved after all the sample tubes have been read.

8. Add 100 µl of Aflatoxin FP tracer to the 2nd tube (containing “Control”). Vortex and

incubate at room temperature for 2 minutes. Place the above tube back in the reader with respect to its blank reading and press the “Read” button.

9. Add 100 µl of Aflatoxin FP tracer to each sample tube one by one, vortex and read

after incubation for 2 minutes each.

10. When finished with all the samples, Press the number “1” again and note the mP values with respect to their sample numbers by pressing the “Forward” arrow key.

Do not press “Done” key. Data is saved to data manager as raw data. 11. Take an mP value of two “control” values (first two tubes) to set the cutoff value.

In order to turn off the instrument, press “Done”, then press “Yes”, then press “Back” and hold down the “Power Key” (to the right of the number 3 key).

Results & Interpretation

The cutoff value to be used is [“Control mP Average” + 20]. Any sample with a mP value greater than the cutoff value is considered as negative. Any sample with a mP value of lesser or equal to the cutoff is considered positive.

Rerun the Control and recalculate the cutoff following every 100 samples or in the event of a

four (4) degree Fahrenheit temperature change.

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Notes and Precautions

1. The qualitative Aflatoxin fluorescence polarization assay (FPA) is intended

for identifying total Aflatoxin in corn at above or below the 20 ppb level. 2. Use fresh extracted samples for analysis. (Extracts that are a few days old

may develop particulates.) 3. Keep all reagents away from heat or flames.

4. Avoid practices that may contaminate the antibody solution, Aflatoxin FP tracer or test reagents.

5. Aflatoxins are toxic. Avoid contact with skin. Use un-powdered gloves and lab coat when working with Aflatoxins.

6. Use clean glassware for each sample to avoid contamination among sam-ples.

7. Do not use scratched or defective test tubes.

8. Do not handle the lower portion of the glass test tube. Fingerprints can distort the FP value.

9. Due to the high affinity of antibody, accurate and precise pipetting is very important in order to avoid any inconsistency.

10. Centrifuge or filter a sample if particulates are present. Particulates may distort FP values.

11. Keep the Sentry ® 100 reading chamber lid closed unless inserting or re-moving a tube.

12. Make sure the test tube is inserted to the bottom stop of the reading cham-ber.

13. Make sure Sentry ® 100 reader is calibrated.

14. DO NOT PRESS THE “DONE” KEY ON THE READER UNLESS IN-

STRUCTED TO DO SO IN THE PROTOCOL.

Preliminary Steps Reagent and Sample Preparation

1. Extract 20 g of ground samples with 100 ml of 70% methanol by blending

for 1 minute, or shaking for 2 minutes, and then filtering with a fine qualita-tive filter paper (1µm particle retention).

2. Make sure the kit contents are equilibrated to room temperature.

Instrument Adjustment

Instruments must be set up and calibrated according to manufacturer’s specifi-cations. For detailed information, please consult instrument manual and contact

Technical Support.

Sentry®100 Single Tube Testing Procedure

1. Turn on the Sentry®100 reader. Wait for the screen to display “Run Assay”. Press the

“Select” button. The reader will display the next screen. Press “Down” arrow once and then “Forward” arrow once. Advance to the next screen by pressing the

“Down” arrow repeatedly. When “Run Assay” is highlighted, press the “Select” key to go to “Run Assay Batch” mode.

Screen display is represented with blue type.

Button to be pressed is represented with green type.

Required kit ingredient is represented with red type.

Page 3

AFLATOXIN FPA IS:

RAPID

• Unrivalled speed, with test completion typically in 5 minutes (only 2 minute incubation)

• Requires no washing steps

EASY

• One-step solution

• Performed in a laboratory or point-of-care field setting

• Few reagents required

ACCURATE

• Leading performance, yielding qualitative results

• Sensitivity at or above 20 ppb

• Detects all forms of Aflatoxin

COST EFFECTIVE

• Rapid results means more tests can be run in a shorter period of time, requiring fewer laboratory technicians

APPROVED

• United States Department of Agriculture (USDA) approved

⇒ Grain Inspection, Packers and Stockyards Administration (GIPSA)

certified test method

Page 4

AFLATOXIN TEST KIT (AFLATOXIN FPA QUALITATIVE TEST - CORN)

50 ● 100 ● 1,000 Test Format

General Description & Intended Use

The AFLATOXIN TEST KIT is a

qualitative test using Fluores-

cence Polarization technology

designed to determine the

presence of AFLATOXIN in corn.

The test kit includes a specific

antibody solution that reacts to

the presence of AFLATOXIN

and permits an FP reader to

detect the toxin at a level equal

to or greater than 20 ppb.

Kit Contents

Components 50 Tests 100 Tests 1,000 Tests

Component 1: Aflatoxin Control 1 mL 2 mL 4 X 5 mL

Component 2: Aflatoxin Antibody

Solution 60 mL 110 mL 2 X 510 mL

Component 3: Aflatoxin Tracer 6 mL 12 mL 110 mL

All components contain 0.1% sodium azide as a preservative

Page 5

Materials Required But Not Provided Other required materials not provided are: FPA instrument, 10x75 mm borosili-cate glass test tubes for single tube FPA instrument (highly recommended: VWR item number 7729-568; see http://www.vwr.com), pipettor, pipette tips, and a

tube vortex mixer.

Storage & Stability FPA reagents must be stored in a refrigerator (4-8˚ C) and brought to room tem-perature (18-25˚ C) before usage. Normal shelf life is one (1) year when stored in a refrigerator. When stored at room temperature, shelf life is reduced to one

(1) month. Aflatoxin Antibody Solution (from the kit) should preferably by stored at room temperature (18-25˚ C). Do not use test kit past the expiration date printed on the box. A Certificate of Analysis (COA) is available upon re-quest.

Warnings FPA technology works on clear and cloudy homogeneous liquids. Avoid prac-tices that may contaminate the test kit components (control, antibody, or tracer).

Polarization readings change by 3 mP (millipolarization units) for a temperature change of 4˚ C. Therefore, the Control should be run at regular intervals as mentioned in the protocol.

All materials in the kit should be treated as any other laboratory chemicals. Avoid ingestion, eye contact, and other potential detrimental exposure. A Mate-rial Safety Data Sheet (MSDS) is available upon request. Do not use expired or

contaminated components, or components from other kits. Do not mix compo-nents from different manufactured lots.