Aflatoxin M1 Immunoaffinity Column
Cat.# M1IAC
Intended use
for the purification and concentration of AFM1 residues in milk and milk products.. After this step, the AFM1 can be later measured by TLC, HPLC ,GC ,LC-MS/MS, EIA.
Capacity: above 100ng of AFM1
Storage Conditions: IAC should be stored at 2-8℃. Do not freeze.
Use
Clean up of sample extracts for further analysis.
summary
The Aflatoxin M1 belongs Aflatoxin a class of structurally similar compounds.
Mammals intake Aflatoxin B1 contaminated feed or food, through hydroxylation converted Aflatoxin M1.AFM1 hazards mainly in carcinogenicity and mutagenicity, has damaging effects on human and animal liver tissue, can lead to liver cancer and even death , Aflatoxin hence levels of Aflatoxins in the diet an important consideration for human health, most countries have established regulatory limits on Aflatoxin in milk.
Use Principles
The IAC is based on the reaction between antigen and antibody. This AFM1 IAC contains gel binding with Aflatoxin antibody. The AFM1 in the samples will be extracted, filtered, diluted and passed through the AFM1 IAC. AFM1 in the extract will be retained in the IAC by the AFM1 specific antibody, the impurity in the extract will pass through. After rinsing to completely remove the unwanted matrix, the AFM1 is eluted by Methanol by de-associating the AFM1 from the antibody. This elute will be ready for instrumental analysis.
Materials Provided
25 disposable IAC per box. 1ml each
Instructions
Precautions
Aflatoxins are toxic substance. Please wear gloves to go experimental operation
Used lab wares and Aflatoxin solution preferably be soaked with 5% sodium hypochlorite solution soak over night.
Do not use the reagents after the expiration date.
IAC stored at 2-8℃,no not freeze
Reagents should be brought to room temperature (22-25℃) prior to use.
Operation Procedure
6.1 Sample Extraction
6.1.1 Milk
Weigh 50 gram of sample to a centrifugal tube with stopper. And heated to 35-37℃ in a water bath.
4000 rpm centrifuge 15 min, discard the upper layer’s butterfat.
All samples are purified through the IAC.
6.1.2 Milk power and Infant powdered formula
Weigh 5 gram of sample, add 50℃ distilled water to it, and Mix vigorously.
4000 rpm centrifuge 15min; discard the upper layer’s butterfat.
All samples are purified through the IAC.
6.1.3 Fermented milk or lactic acid beverages
Weigh 50g sample; adjust the pH value to 6.8-7.2 using 0.5mol / L of NaOH.
4000 rpm centrifuge 15min; discard the upper layer’s butterfat.
All samples are purified through the IAC.
6.1.4 Butter and Cheese(2.5 gram of sample extract onto the IAC)
Weigh 5 gram of sample and mix with 25 ml of 60% Methanol in water.
4000 rpm centrifuge 10min
Pipette all supernate to separating funnel, add 30ml Petroleum ether to it, Mix vigorously and standing for layering..
Slowly release the lower solution flow into 250ml beaker, add water up to 100ml. Allowed to stand for 5-10 minutes （or use a hair dryer blowing 2-3 minutes),so that the residual petroleum ether completely evaporate in ventilated environment.
Filter the mixture with glass microfiber filter to clarify.
Pipette 50ml filtrate through the IAC
6.2 Column Chromatography
Connect 10 ml of syringe barrel reservoir to the column.
Load the filtered extract and control the flow rate at 1-2 drops per second. Discard the pass through liquid
Use 10 ml of 110% methanol/water to rinse the column and discard the rinse.
Press the air into the column to blow out all the liquid
Use 1 ml of methanol:acetonitrile(V：V=2:3) to elute the sample (flow at one drop/2-3 seconds),
collect the sample for further testing.
Operation Procedure Chart
Liquid chromatography conditions
Mobile phase：acetonitrile -water（25+75）
Flow rate：1mL/min
Capacity：50ul -500ul
Column: C18 length 25cm inner diameter 4.6cm
Injector draw AFM1 standard working solution into the liquid chromatograph, the response value (peak height or peak area) of the standard solution was measured in the above-described chromatographic conditions aflatoxins M1 stand solution by high performance liquid chromatogram.
