hygiena KIT5003 961AFLM01C-ULTRA Aflatoxin M1 Elisa Instruction Manual

: June 16, 2024
: Hygiena

hygiena KIT5003 961AFLM01C-ULTRA Aflatoxin M1 Elisa Instruction Manual

Product Information

Product Name: Helica Aflatoxin M1 Low Matrix High Sensitivity ELISA

assay

Product Model: MYC-INS-5000-1.0

Manufacturer: Hygiena

Website: www.hygiena.com

Specifications

For the quantitative detection of Aflatoxin M1 in milk, milk powder, and cheese
Solid-phase, competitive enzyme immunoassay
Measurement wavelength: 450 nm (OD450)
Kit standards with concentrations: 0.0, 5.0, 10.0, 25.0, 50.0, and 100.0 pg/mL (ppt)
Storage: Store in an opaque bottle, in the dark

Materials Required But Not Provided

No additional materials are required for this kit.

Storage and Shelf Life

The components of the kit should be stored in the provided opaque bottle. It is recommended to store the kit in a dark place and return it to storage after use.

Precautions and Waste Disposal

Safety Precautions: Mycotoxins, including aflatoxins, are known to be highly toxic and carcinogenic. When handling samples, reagents, glassware, and other supplies potentially contaminated with mycotoxins, appropriate safety precautions must be taken, and personal protective equipment should be worn to prevent human illness.

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; 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 on 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 Low Matrix High Sensitivity ELISA assay is a solid- phase, competitive enzyme immunoassay for the quantitative determination of aflatoxin in milk, milk powder and cheese. The assay has not been approved by the FDA for diagnostic purposes.
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. Helica 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 supervision of technically qualified persons.

Principle of the Method

The Helica Aflatoxin M1 Low Matrix High Sensitivity 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

Package/ Number	Component	Description
1X Pouch	Antibody- coated microwell plate	96 wells (12 eight-well strips) in

a microwell holder coated with a mouse anti-aflatoxin monoclonal 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. (Mixing wells)
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 andstore refrigerated. (Wash buffer)
1X Bottle| Milk diluent| 12 mL skim milk for preparation of cheese extract, Ready-to-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

Raw Milk

The standards are presented in homogenized skim milk and skim milk (milk plasma) is the appropriate sample for the assay.
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.
Alternatively, if the sample is at ambient temperature or has been mixed in transit, place an aliquot at refrigerated temperature for 12 hours and centrifuge at 2,000 x g for 5 minutes to induce separation of the upper fatty layer.
Remove the upper fatty layer by aspiration and use the lower plasma in the assay.

Homogenized Milk

Homogenized skim milk should be used directly in the assay.
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. 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

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. The tube solution is clarified by centrifugation (5,000 x g for 5 minutes) and the supernatant is removed. 2. 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). 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. Allow the tube to stand for 5 minutes more and use 2 X 200 L of this milk extract in the assay.

Assay Procedure

Note: It is recommended that a multi-channel pipettor be utilized to perform the assay.
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 1-Liter container. Fill to 1 Liter with distilled or deionized water and store refrigerated when not in use.
Place one mixing well in a microwell holder for each standard and sample. Return unused wells to the foil pouch with desiccant and reseal.
Mix each reagent by swirling the reagent bottle prior to use.
Using a new pipette tip for each, dispense 200 L aliquots of standards and samples into the appropriate
microwells in duplicate.
Cover the plate with sealing tape to avoid evaporation and protect from excess UV light.
Incubate at ambient temperature (19 to 25 °C) for 2 hrs.
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 for a total of three (3) washes.
Tap the microwells (face down) on a layer of absorbent paper towels to remove residual wash buffer.
Using a new pipette tip for each, add 100 L of the conjugate to each well. Re-seal the plate and incubate
at ambient temperature for 15 minutes.
Repeat steps 7 and 8.
Measure the required volume of substrate solution (1 mL/strip or 120 µL/well) and place in a separate
container. Using a new pipette tip for each, add 100 L of enzyme-substrate (TMB) to each well and incubate for 15 minutes. Cover to avoid direct light.
Measure the required volume of stop solution (1 mL/strip or 120 µL/well) and place in a separate container. Add 100 L stop solution to each well at the same pace and in the same sequence as the substrate solution was added. The blue color will change to yellow.
Read the optical density (OD) of each microwell with a microplate reader at 450 nm using an air blank or a differential filter of 630 nm.

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.

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

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.

Interpretation of Results

Construct a dose-response curve using either 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 ppt 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

Standard curve

Data from the 2-hour ambient study was collected and a standard curve was generated. Results are shown in the graph below.

Recovery and Reproducibility

Recovery of 50 ppt spiked into milk is as follows:

Type of Milk	% Recovery	CV (%)
Skim milk, 50 ppt	100	4.2
1% fat, homogenized, 50 ppt	93	4.4
Full fat, homogenized, 50 ppt	92	2.2

Recovery in Cheese

**** For testing, 100 pg of Aflatoxin M1 was spiked into 1g 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 ppt

Precision

Intra-assay

Sample (ppt)| 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 (ppt)| 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.

FAQ (Frequently Asked Questions)

Q: Are any additional materials required for this kit?
A: No, all the necessary materials are included in the kit.
Q: What is the storage recommendation for the kit components?
A: It is recommended to store the kit components in the provided opaque bottle, in a dark place.
Q: Can this assay be used for diagnostic purposes?
A: No, this assay has not been approved by the FDA for diagnostic purposes.
Q: What is the purpose of this assay?
A: The Helica Aflatoxin M1 Low Matrix High Sensitivity ELISA assay is designed to quantitatively determine the presence of aflatoxin in milk, milk powder, and cheese. Q: Can this assay be used for diagnostic purposes?