hygiena KIT5001 961AFLM01C – 96 Helica Aflatoxin M1 ELISA Instruction Manual

hygiena KIT5001 961AFLM01C – 96 Helica Aflatoxin M1 ELISA

Helica® Aflatoxin M1 ELISA
For the quantitative detection of Aflatoxin M1 in milk, milk powder and cheese.

Introduction – Aflatoxins
Aflatoxins are toxic metabolites produced by a variety of molds such as Aspergillus flavus and Aspergillus parasiticus. They are carcinogenic and can be present in grains, nuts, cottonseed and other commodities associated with human food or animal feeds. Crops may be contaminated by one or more of the four sub-types of Aflatoxin: B1, B2, G1 and G2. Aflatoxin B1 is the most toxic and frequently detected form. The other types present a significant danger if the concentration is high.

Aflatoxins have been implicated in human health disorders including hepatocellular carcinoma, aflatoxicosis, Reye’s syndrome and chronic hepatitis. Animals are exposed to aflatoxins by consuming feeds that are contaminated by aflatoxin-producing fungal strains during growth, harvest or storage. When cows are fed contaminated feed, Aflatoxin B1 is converted by hydroxylation to Aflatoxin M1, which is subsequently secreted in the milk of lactating cows. Aflatoxin M1 is quite stable during normal milk processing methods such as pasteurization and if present in raw milk, it may persist into final products for human consumption.

Most controlling government agencies worldwide have regulations regarding the amount of aflatoxins allowable in human and animal foodstuffs. Many countries have declared limits for the presence of Aflatoxin M1 in milk and milk products. In the EU, the limit for the presence of M1 in milk and reconstituted milk powders has been set at 50 pg/mL or 50 parts per trillion (50 ppt).

Intended Use

Hygiena’s Helica® Mycotoxin ELISA kits are user-friendly, cost-effective kits for the detection of mycotoxins in a wide range of commodities including animal feeds, grains, corn and animal urine, designed to protect humans and animals from dangerous side effects of mycotoxins.

The Helica Aflatoxin M1 ELISA assay is a competitive enzyme-linked immunoassay intended for the quantitative detection of Aflatoxin M1 in milk, milk powder and cheese. Data obtained from assays should not be used for human diagnostic or human treatment purposes. Assays are not approved by the United States Food and Drug Administration or any other US or non-US regulatory agency for use in human diagnostics or treatment. Assays should not be used as the sole basis for assessing the safety of products for release to consumers. The information generated is only to be used in conjunction with the user’s regular quality assurance program. Not approved for clinical diagnosis. Use for research and development, quality assurance and quality control under the supervision of technically qualified persons.

Principle of the Method

The Helica Aflatoxin M1 ELISA assay is a solid-phase competitive enzyme immunoassay. An antibody with a high affinity for Aflatoxin M1 is coated onto polystyrene microwells. Standard or sample is added to the appropriate well and if Aflatoxin M1 is present, it will bind to the coated antibody. Subsequently, aflatoxin bound to horseradish peroxidase (HRP) is added and binds to the antibody not already occupied by Aflatoxin M1 present in the sample or standard. After this incubation period, the contents of the wells are decanted, washed and an HRP substrate is added, which develops a blue color in the presence of an enzyme.

The intensity of the color is directly proportional to the amount of bound conjugate and inversely proportional to the amount of Aflatoxin M1 in the standard or sample. Therefore, as the concentration of Aflatoxin M1 in the sample or standard increases, the intensity of the blue color will decrease. An acidic stop solution is added which changes the chromogen color from blue to yellow. The microwells are measured optically by a microplate reader with an absorbance filter of 450 nm (OD450). The optical densities of the samples are compared to the ODs of the kit standards and an interpolated result is determined.

Kit Contents

a microwell holder coated with a mouse anti-aflatoxin antibody, Ready-to- Use.

6X Vials

| ****

Standards

| 3.0 mL/vial of Aflatoxin M1 at the following concentrations: 0.0, 5.0, 10.0, 25.0, 50.0 and 100.0 pg/mL (ppt) in stabilized skim milk,

Ready-to- Use.

1X Bottle| Conjugate| 12 mL of Aflatoxin conjugated to peroxidase in buffer with preservative,

Ready-to- Use.

1X Bottle| Substrate| 12 mL stabilized tetramethylbenzidine (TMB), Ready-to- Use.
1X Bottle| Stop solution| 12 mL acidic solution, Ready-to-Use.
1X Pouch| PBS-T powder| PBS with 0.05% Tween® 20*, bring to 1 liter with distilled water and

store refrigerated. (Wash buffer)

2X Bottles| Assay diluent| 12 mL of proprietary assay diluent, Ready-to-Use

TWEEN® 20 is a registered trademark of CRODA International Plc.

Materials Required But Not Provided

• Glass tubes
• Centrifuge
• Vortex
• Pipettor with tips: 100 μL and 200 μL
• Timer
• Wash bottle
• Absorbent paper towels
• Microplate reader with 450 nm filter

Storage and Shelf Life

• Store reagents at 2 to 8 °C. Do not freeze.
• Reagents should be used by the expiration date stamped on the individual labels.
• HRP-labeled conjugate and TMB-substrates are photosensitive and are packaged in a light-protective opaque bottle. Store in the dark and return to storage after use.

Precautions and Waste Disposal

General Precautions

• Bring all reagents to room temperature (19 to 25 °C) before use.
• Do not interchange reagents between kits of different lot numbers.
• Do not use solutions if cloudy or precipitate is present.
• Do not return unused reagents to their original bottles. The assay procedure details volumes required.
• Adhere to all time and temperature conditions stated in the procedure.
• During the sample extraction, avoid cross-contamination.
• Devices, such as a blender, must be cleaned after each sample preparation.
• Samples tested should have a pH of 7.0 (± 1.0). Excessive alkaline or acidic conditions may affect the test results.

Safety Precautions
Mycotoxins (aflatoxins, trichothecenes and others) are well-known human carcinogens and are considered highly toxic. Because mycotoxins can cause human illness, appropriate safety precautions must be taken and personal protective equipment worn when handling samples, reagents, glassware and other supplies and equipment that potentially could be contaminated with mycotoxins.

• Before using this assay, please review the Safety Data Sheet(s) available at www.hygiena.com.
• Refer to your site practices for safe handling of materials.
• It is strongly advised that protective gloves, a lab coat and safety glasses be worn at all times while handling mycotoxin kits and their respective components.
• Consider all materials, containers and devices that are exposed to samples or standards to be contaminated with mycotoxins.
• Never pipette reagents or samples by mouth.
• Standards are flammable. Caution should be taken in the use and storage of these reagents.
• The stop solution contains sulfuric acid, which is corrosive. Please refer to the SDS. Do not allow to contact skin or eyes. If exposed, flush with water.

Disposal
Decontaminate materials and dispose of waste per your site practices and as required by local regulations. Do not dispose of these materials down the drain. Please note that there is a potential for mycotoxin contamination in or on any of the kit components provided.

• Dispose of all materials, containers and devices in the appropriate receptacle after use.
• Before conducting the assay, prepare a waste container to act as a receptacle for your kit waste.
• Eject contaminated pipette tips and discard all other related materials into this container.
• Once the assay is completed, the container should be treated with a sufficient amount of 5 – 6% sodium hypochlorite (NaOCl) to saturate the contents of the container (approximately 1/10th the volume of the container).
• The NaOCl will denature the mycotoxins and neutralize the waste, making it safe for disposal. Invert the container several times to thoroughly coat all waste.
• In the case of an accidental toxin spill, treat the spill surface with 5 – 6% NaOCl for a minimum of 10 minutes, followed by 5% aqueous acetone. Wipe dry with absorbent paper towels.

Preparation of Samples

Note
The sample must be collected according to the appropriate established sampling techniques.

Raw Milk

1. The standards are presented in homogenized skim milk and skim milk (milk plasma) is the appropriate sample for the assay.
2. An aliquot of unprocessed raw fatty milk should be placed at a refrigerated temperature overnight to allow the fat globules to rise to the surface in a natural “creaming” effect. Centrifugation at this point is not necessary.
3. Alternatively, if the sample is at ambient temperature or has been mixed in transit, place an aliquot at refrigerated temperature for 1 – 2 hours and centrifuge at 2,000 x g for 5 minutes to induce separation of the upper fatty layer.
4. Remove the upper fatty layer by aspiration and use the lower plasma in the assay.

Homogenized Milk

1. Homogenized skim milk should be used directly in the assay.
2. Due to the stabilization of the fat globules induced by the homogenizing process, they are difficult to eliminate even by high-speed centrifugation to create a milk plasma.
3. Therefore, use homogenized fatty milk directly in the assay (see recovery data on page 8).

Milk Powder

Reconstitute milk powders according to the manufacturer’s instructions and treat the reconstituted product as above.

Cheese

1. One gram of finely grated or otherwise macerated cheese is mixed with 5 mL of absolute methanol in a capped tube and mixed for 5 minutes.
2. The tube is clarified by centrifugation (5,000 x g for 5 minutes) and the supernatant is removed.
3. 0.5 mL of this supernatant is transferred to a glass tube and the methanol is evaporated by a stream of air (better recovery with nitrogen gas).
4. This procedure results in the deposition of a semi-solid viscous material on the inside of the tube. Add 0.5 mL of the provided blank skim milk to the tube and vortex vigorously for 1 minute.
5. Allow the tube to stand for a further 5 minutes and use 2 X 200 μL of this milk extract in the assay.

Assay Procedure
Note: It is recommended that a multi-channel pipettor be utilized to perform the assay.

1. Bring all the reagents to room temperature before use. Prepare wash buffer by reconstituting the PBS-T powder packet by washing out the contents with a gentle stream of distilled or deionized water into a Liter container.

2. Fill to 1 Liter with distilled or deionized water and store refrigerated when not in use.

3. Place one mixing well in a microwell holder for each Standard and Sample to be tested. Place an equal number of antibody-coated microtiter wells in another microwell holder.

4. Return unused wells to the foil pouch with desiccant and reseal.

5. Mix each reagent by swirling the reagent bottle prior to use. VORTEX STANDARDS BEFORE USE.

6. Since precipitate can form over long-term storage, please vortex prior to testing for best results.Using a fresh pipette tip for each, dispense 200 μL aliquots of standards and samples into the appropriate wells in duplicate.

7. Cover the plate with sealing tape to avoid evaporation and protect from excess UV light.
   Note : Operator must record the location of each Standard and Sample throughout test.

8. Incubate at ambient temperature (19 to 25 °C) for 2 hrs.

9. Decant the contents from microwells into a discard basin. Wash the microwells by filling each with PBS-T wash buffer, then decanting the wash into a discard basin. Repeat wash for a total of 3 washes.

10. Tap the microwells (face down) on a layer of absorbent towels to remove residual buffer.

11. Add 100 μL of the HRP-conjugate to each well. Re-seal the plate and incubate at ambient temperature (19 to 25 °C) for 15 min.

12. Repeat steps 6 and 7.

13. Measure the required volume of substrate solution (1 mL/strip or 120 μL/well) and place in a separate container. Add 100 μL of enzyme substrate (TMB) to each microwell.

14. Incubate at room temperature for 15 minutes. Cover to avoid direct light.

15. Measure the required volume of stop solution (1 mL/strip or 120 μL/well) and place in a separate container.

16. Add 100 μL of stop solution in the same sequence and at the same pace as the substrate reagent was added. The blue color will change to yellow.

17. Read the optical density (OD) of each microwell with a microtiter plate reader using a 450 nm filter (using an air blank or a differential filter of 630 nm). Record the optical density (OD) of each microwell.

18. Setting the zero standard as 100% binding (Bo), calculate % binding (%B) for each standard and sample as a percentage of the zero binding (%B/Bo).

Alternative Incubation Procedure
The first incubation period, with standards and sample, may be performed overnight at a refrigerated temperature (4 to 6 °C) with noticeable improvement in inhibition of zero binding for samples of 5 ppt (pg/mL) and above. If this option is chosen, the subsequent incubations with conjugate and TMB should be performed at ambient temperature. Limit of detection (LOD) is defined as 2 standard deviations below the mean OD of multiple determinations of zero binding (n=18, CV <2%) is 2 ppt (pg/mL) in both cases.

Incubation Temperature

Standard (pg/ml)| %B/B 0 , Overnight (4-6 °C)| %B/B 0 , 2 Hours Ambient
0| 100.0| 100.0
5| 83.8| 89.0
10| 67.5| 80.1
25| 37.4| 55.5
50| 19.8| 36.3
100| 12.5| 21.2

Interpretation of Results
Construct a dose-response curve using the OD values expressed as a percentage of the OD of the zero standard against the Aflatoxin M1 content of the standard. Unknowns are measured by interpolation from the standard curve. The mean value of the absorbance values obtained for the standards and the samples are divided by the absorbance value of the zero standard and multiplied by 100. The zero standard is thus made equal to 100% and the absorbance values of other standards and samples are quoted in percentages of this value. absorbance standard (or sample) / absorbance zero standard x 100 = % absorbance

The values calculated for the standards are entered in a system of coordinates on semilogarithmic graph paper against the Aflatoxin M1 concentration in pg/mL. The Aflatoxin M1 concentration in pg/mL corresponding to the absorbance of each sample can be read from the calibration curve. In order to obtain the Aflatoxin M1 concentration in pg/mL actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor. This is 1 for milk samples and 5 for cheese samples.

Assay Characteristics
Recovery and Reproducibility
Recovery of 50 ppt spiked into milk is as follows:

Recovery in Cheese
For testing, 100 pg of Aflatoxin M1 was spiked into 1 g of finely grated parmesan cheese and allowed to remain in contact for one hour at ambient temperature. Following the extraction procedure as described, the average recovery of Aflatoxin M1 was 60.5% with a CV of 5.5% for 8 separate spiking/extractions.

Performance Data

Sensitivity

Number of Tests (n)| Mean OD| Std Dev| CV (%)| Sensitivity
---|---|---|---|---
18| 1.857| 0.023| 1.2| 2 pg/mL

Precision

Intra-assay

Sample (pg/mL)| Number of Tests (n)| Mean OD| %B/B 0| CV (%)
0| 8| 1.245| 100| 1.4
5| 8| 1.092| 87.7| 2.0
10| 8| 0.945| 75.9| 3.0
25| 8| 0.719| 57.8| 2.1
50| 8| 0.449| 36.1| 2.7
100| 8| 0.246| 19.8| 2.0
Intra-assay

(Over a seven-month period, testing multiple lots)

Sample (pg/mL)| Number of Tests (n)| %B/B 0| CV (%)
5| 8| 86.6| 1.5
10| 8| 76.2| 3.3
25| 8| 55.5| 4.7
50| 8| 36.3| 4.8
100| 8| 22.0| 10.8

Technical Assistance
For questions or comments, please contact your local distributor. You can also email techsupport@hygiena.com, visit our Contact Us page for regional phone numbers or request technical support at https://www.hygiena.com/hygiena/technical-support-request.html.

References

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