U.S. DEPARTMENT OF AGRICULTURE

Grain Inspection, Packers and Stockyards Administration

Federal Grain Inspection Services

CERTIFICATE NO. FGIS 2003-101

CERTIFICATE OF CONFORMANCE

Quantitative Aflatoxin Test Kit for Corn, Milled Rice, Popcorn, Corn Meal, Cracked Corn, Wheat, Soybeans, Corn Germ Meal, Corn Gluten Meal, Corn Soy Blend, and Sorghum.

For: R-Biopharm, Inc.Submitted by:

Method: RIDASCREEN® FAST AflatoxinR-Biopharm Inc.

7950 Old US 27 South Marshall, MI 49068

Tel: (877) 789-3033

Contact: Mr. Kurt Johnson

Standard Features and Options

SampleExtractionTemperatureAflatoxin

Model PreparationMethodRangeRange

RIDASCREEN® Clean, Ground Sample50 g sample18-30 ºC 5-300 ppb

FASTso that 95% passes70% Methanol(64-86 ºF)

Aflatoxinthrough a 20 mesh

sieve.

Test kits must be operated according to the instructions in Attachment 2.

This quantitative aflatoxin test kit underwent an initial Type Evaluation under the authority of Section 7B (c) of the United States Grain Standards Act, as amended, and was found to meet all design and test performance criteria as defined in “Design Criteria and Test Performance Specifications for Quantitative Aflatoxin Test Kits” June 2002 Revision.

Evaluation tests that were passed are summarized in Attachment 1. For further information, contact:

USDA, Grain Inspection, Packers and Stockyards Administration

Technical Services Division

Analytical, Reference, and Testing Services Branch

10383 N. Ambassador Drive

Kansas City, Missouri 64153-1394 Tel: (816) 891-0464

______Date: ______

Director

Technical Services Division

Note: The mention of firm name or trade products does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned.

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 1Manufacturer:R-Biopharm Inc.

7950 Old US 27 South Marshall, MI 49068

TEST 1: TIME REQUIRED FOR COMPLETION OF ANALYSIS

Data submitted by the manufacturer indicated that the analysis time required for one sample was less than the maximum limit of 30 minutes.

TEST 2: CAPABILITY FOR ANALYZING FOR AFLATOXINS

Data submitted by the manufacturer showed that the test kit was capable of detecting aflatoxins at 5 ppb.

TEST 3: IDENTIFIED COMMODITIES

Data submitted by the manufacturer showed that the test kit met the established accuracy and precision specifications at 10, 40, and 160 ppb combined aflatoxins for each of the commodities listed below:

CORN, MILLED RICE, POPCORN, CORN MEAL, CRACKED CORN, WHEAT, SOYBEANS, CORN GERM MEAL, CORN GLUTEN MEAL, CORN SOY BLEND , AND SORGHUM.

TEST 4: AVOIDANCE OF TOXIC OR HAZARDOUS SUBSTANCES

Data submitted by the manufacturer showed that this test kit meets safety criteria.

TEST 5: ACCURACY AND PRECISION OF TEST KIT WITH CORN SAMPLES

Data submitted by test kit manufacturer showed that this test kit met all of the accuracy and precision specifications for ground corn spiked at 0, 10, 20, 30, 40, 80, 160 and 320 ppb. Personnel from the Analytical, Reference, and Testing Services Branch (ARTS), Technical Services Division, Grain Inspection, Packers and Stockyards Administration (GIPSA) verified performance at 20, 80, and 320 ppb.

TEST 6: ACCURACY OF TEST KIT ON NATURALLY CONTAMINATED CORN SAMPLES

Data submitted by test kit manufacturer on two naturally contaminated corn samples containing approximately 20 ppb and 100 ppb aflatoxin met the performance criteria when test kit results were compared with results from a standard reference method. GIPSA personnel using a naturally contaminated corn sample containing approximately100 ppb aflatoxin verified these data.

TEST 7: LIMIT OF DETECTION OF TEST KIT FOR EACH COMMODITY TESTED

Data supplied by the manufacturer showed that this test kit had a limit of detection of less than or equal to 5- ppb aflatoxin for each of the commodities listed under Test 3.

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 1Page 2

TEST 8: SENSITIVITY OF ELECTRONIC EQUIPMENT TO ELECTROMAGNETIC FIELDS

The Biotek EL 301 Microwell Reader; the Awareness Technology, Inc. and the Stat-Fax Model 303 PLUS; offered by the manufacturer have been approved by GIPSA for use in the official inspection system. No additional certification was required. This equipment meets all of the electromagnetic field sensitivity specifications.

TEST 9: TEMPERATURE SENSITIVITY

Data submitted by the manufacturer showed that this test kit at temperatures ranging from 18 to 30 ºC gave results within acceptable limits.

TEST 10: STABILITY

Data submitted by the manufacturer supported storage and stability claims.

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 2Manufacturer:R-Biopharm Inc.

7950 Old US 27 South Marshall, MI 49068

Test Procedures

Sample Preparation

  1. Weigh 50 g of ground sample and add it to a suitable container with 250 mL of 70% methanol.
  1. Blend for 2 minutes.
  1. Filter the extract through a filtering syringe (JM1000) or equivalent.
  1. Dilute 1 ml of the filtrate with 1 ml of distilled or deionized water.
  1. Proceed to test procedure.

Test Procedure

  1. Insert a sufficient number of wells into the microwell holder for all standards and samples to be run.

Note: Do not run more than 3 strips (19 samples) per set of control standards.

  1. Pipet 50 uL of standard and prepared sample to separate wells; use a new pipette tip for each standard and sample.
  1. Add 50 uL of enzyme conjugate to each well (red capped bottle).
  1. Add 50 uL of anti-aflatoxin antibody to each well (black capped bottle). Mix thoroughly and incubate for 5 minutes (± 0.5 minutes) at room temperature.
  1. Empty the liquid from the wells. Tap the microwell holder upside down on a clean paper towel or other absorbent material to remove all remaining liquid from the wells. Using a wash bottle, fill the wells with distilled or deionized water. Empty the wells again and remove all remaining liquid. Repeat the washing step two more times.
  1. Add 100 uL of substrate/chromagen to each well (white dropper bottle). Mix thoroughly and incubate for 5 minutes (± 0.5 minutes) at room temperature. Cover wells with a paper towel to protect from light.
  1. Add 100 uL of stop solution to each well (yellow or orange dropper bottle). Mix well and measure absorbance at 450 nm using the Biotek EL 301 Microwell Reader or the Awareness Technology, Inc. Stat-Fax Model 303 PLUS. Read within 10 minutes.

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 2Page 2

Results

The absorbance values obtained for the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100. The zero standard is thus made equal to 100 % and the absorbance values are quoted in percentages.

Values calculated for the standards are entered in a system of coordinates on semi-logarithmic graph paper against the aflatoxin concentration in [mg/kg]. The aflatoxin concentration in mg/kg corresponding to the extinction of each sample can be read from the calibration curve.

Alternatively, commercial software can be used for data reduction. The manufacturer recommends logit/log for single determinations, and cubic spline curve fitting is recommended for double or multiple determinations.

Reagents / Materials Provided in Test Kits:

  1. 1 microtiter plate.
  2. 48 antibody coated microwells.
  3. 5 aflatoxin standard solutions of 1.3 mL each; 0, 4, 10, 20, and 50 ppb aflatoxins.
  4. 1 red-capped bottle of 3 mL peroxidase conjugated aflatoxin solution.
  5. 1 black-capped bottle of 3 mL anti-aflatoxin antibody.
  6. 1 white dropper bottle of 6 mL Substrate/Chromagen.
  1. 1 yellow or orange dropper bottle of Stop reagent.

Materials Required But Not Provided:

  1. Extraction Materials:
  1. ACS Grade methanol.
  2. Deionized or distilled water.
  3. 250 mL graduated cylinder.
  4. 125 mL container.
  5. Filtering syringe (JM1000), or equivalent

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 2Page 3

  1. Sample collection tubes.
  1. Waring® high-speed blender with a one liter jar, or equivalent.
  2. Sample grinder.
  3. Balance.
  4. Biotek EL 301 Microwell Reader or an Awareness Technology, Inc. Stat-Fax Model 303 PLUS with 450-nm filter.
  1. Eppendorf Repipettor, or equivalent and 2.5 ml syringes
  2. 50 uL, and 1000-uL pipettor and pipette tips.
  3. Paper towels, Kaydry paper or equivalent absorbent material.
  4. Waste receptacle.
  5. Timer: 3-channel minimum.
  1. Waterproof marker, Sharpie or equivalent.
  2. Wash bottle.
  3. Deionized or distilled water.

Type Evaluation

Certificate No. FGIS 2003-101

ATTACHMENT 3

Manufacturer:R-Biopharm Inc.

7950 Old US 27 South Marshall, MI 49068

Manufacturer’s Notification Responsibilities

Manufacturers with test kits on the list of equipment approved for official inspection must notify the Director, Technical Services Division, USDA-GIPSA, 10383 N. Ambassador Drive, Kansas City, MO 64153-1394, in writing when any changes or alterations are made to the approved test kit, any reagents or equipment used in the test kit, or to any part of the analytical method. Failure to notify GIPSA of these changes will serve as grounds for immediate removal of the test kit from the approved list. Changes in packaging or other marketing information about the test kit are exempt from this requirement.

If the manufacturer wishes for an altered test kit to remain on the approved list without having to repeat all of the tests listed under Performance Specifications, she/he must submit a testing waiver request. A complete explanation of the change(s) made and how test results are affected must accompany this waiver. Data must be submitted which support the claim that the test kit still meets all Performance Standards that were potentially affected by the change that was made.

If a test waiver is not submitted, the test kit will be removed from the approved list until retesting has confirmed compliance with ALL listed Performance Standards.