The Attogene Aflatoxin M1 ELISA Kit (EL2053-01) is a competitive aflatoxin M1 ELISA kit for quantitative measurement of aflatoxin M1 in raw milk and powdered milk. The standard curve runs from 5 to 300 ppt (0.005 to 0.3 ppb), the detection limit in raw milk is 0.025 ppb, and total incubation time is 75 minutes on a 96-well strip plate read at 450 nm.
Aflatoxin M1 is the hydroxylated metabolite of aflatoxin B1. It appears in milk when dairy animals eat feed contaminated with aflatoxin B1, which is why milk is screened for it at very low concentrations.
Key Features
- Low detection limits: 0.025 ppb in raw milk and 0.08 ppb in powdered milk.
- Quantitative results: six ready-to-use standards (0, 0.005, 0.015, 0.03, 0.09 and 0.3 ppb) for a full standard curve.
- Fast workflow: two 30-minute incubations and a 15-minute substrate step.
- Flexible format: 96-well microtiter plate in 12 strips of 8 wells, so you can run only the strips you need.
- Reproducible: accuracy of 80 ± 10% and a CV below 10%.
What’s in the Kit
Each aflatoxin M1 ELISA kit includes:
- 96-well microtiter plate coated with aflatoxin M1 (12 strips × 8 wells)
- Aflatoxin M1 standards, 6 vials × 0.8 mL: 0, 0.005, 0.015, 0.03, 0.09 and 0.3 ppb
- Antibody #1, 11 mL
- 100X HRP-conjugated Antibody #2, 0.25 mL
- Antibody #2 Diluent, 20 mL
- 20X Wash Solution, 28 mL
- TMB Substrate Solution, 12 mL
- Stop Solution, 14 mL
You will also need: an ELISA plate reader (450 nm/630 nm) such as the Chromate 4300 microplate reader, deionized water, a vortex mixer, a centrifuge, a timer, a wash bottle, 50 mL and 2 mL polystyrene centrifuge tubes, micropipettes (20–200 µL and 100–1000 µL) with tips, and an 8-channel pipette for 50, 100 and 150 µL.
Who It’s For
- Dairy processors screening incoming raw milk before it is pooled.
- Milk powder and ingredient producers checking finished lots.
- Food safety and contract testing labs running routine aflatoxin M1 surveillance.
- Researchers studying the carry-over of aflatoxin from feed into milk.
Sample Preparation
- Raw milk: test within 24 hours of milking and keep at 2–8 °C. Use directly, or centrifuge at 4000 g for 10 minutes, remove the fat layer and use 50 µL of the skim milk.
- Powdered milk: dissolve 1 g in 10 mL of deionized water by vortexing. Centrifuge for 10 minutes if needed, remove the fat layer and use 50 µL. The dilution factor is 10.
How to Use the Aflatoxin M1 ELISA Kit
- Bring all reagents to 20–25 °C for at least 30 minutes and mix gently. Dilute the 20X Wash Solution and prepare the 1X HRP-conjugated Antibody #2 fresh.
- Add 50 µL of each standard or prepared sample to the wells. Run standards and samples in duplicate.
- Add 100 µL of Antibody #1, shake gently for 30 seconds, cover and incubate for 30 minutes at room temperature.
- Empty the wells, wash 3 times with 250 µL of 1X Wash Solution and blot the plate on absorbent paper.
- Add 150 µL of 1X HRP-conjugated Antibody #2, shake for 30 seconds, cover and incubate for 30 minutes at room temperature.
- Wash 3 times as before and blot dry.
- Add 100 µL of TMB Substrate, mix, cover and incubate for 15 minutes at 25 °C.
- Add 100 µL of Stop Solution, mix and read absorbance at 450 nm within 5 minutes.
Full instructions and precautions are in the Aflatoxin M1 ELISA Kit manual (PDF).
How the Assay Works and Reading Results
Aflatoxin M1 in the sample competes with aflatoxin M1 coated on the well for binding to Antibody #1. An HRP-conjugated secondary antibody and TMB substrate then produce a blue color before the reaction is stopped and read. More aflatoxin M1 in the sample means less color.
Divide the mean absorbance of each standard and sample by the mean absorbance of the zero standard and multiply by 100. Plot these values for the standards against the log of concentration, read sample concentrations from the curve, and multiply by the dilution factor. A zero-standard absorbance below 0.5 suggests one or more reagents have deteriorated.
For context, the US FDA action level for aflatoxin M1 in milk is 0.5 ppb, and the EU maximum level for raw milk is 0.05 µg/kg. The raw milk detection limit of 0.025 ppb sits below both.
Frequently Asked Questions
What samples can I test with this aflatoxin M1 ELISA kit?
The manual covers raw milk and powdered milk. For other dairy matrices, contact Attogene before testing.
What if a sample reads above the top standard?
Dilute raw milk samples above 0.3 ppb with deionized water, rerun them, and multiply the result by the dilution factor.
Can I extend the substrate step if color is weak?
Yes. The TMB incubation can be extended to 35 minutes if color is too faint, but never beyond 40 minutes.
How should the kit be stored?
Store the aflatoxin M1 ELISA kit at 2–8 °C, protected from light, and do not freeze. Shelf life is 12 months or until the expiration date, whichever comes first.
Do you offer ELISA kits for other mycotoxins?
Yes. See the Aflatoxin B1 ELISA Kit and Total Aflatoxin ELISA Kit for grain and feed, the Ochratoxin A ELISA Kit, the Deoxynivalenol ELISA Quick Test Kit, the T2 ELISA Kit, the Ractopamine ELISA Kit for veterinary drug residues, or the full Food Safety ELISA Kit Menu.
For research use only. Not for use in diagnostic procedures. Questions? Call 512-333-1330 or email sales@attogene.com.
