1、Purpose:

The immunoaffinity column can selectively adsorb aflatoxin A from traditional Chinese medicine sample solutions, thereby achieving highly targeted purification of the samples. The purified sample solution can be directly used for HPLC analysis or LC-MS/MS detection after nitrogen blowing reconstitution.

2、Overview:

Ohratoxin A (OTA) is a toxic metabolite produced by certain strains of Aspergillus and Penicillium. It is a fungal toxin with strong nephrotoxicity and hepatotoxicity, and has teratogenic, mutagenic, and carcinogenic effects.

3、Principle:

The basis of determination is antigen antibody reaction, where the antibody is connected to the column. After extraction and filtration, the sample slowly passes through the ochratoxin A immunoaffinity column, where endotoxin binds to the antibody. Afterwards, the immunoaffinity column is washed to remove any unrelated substances that have not been bound. Wash ochratoxin A with methanol and inject it into the analytical instrument for detection.

4、Packaging composition:

Each box contains various specifications of ochratoxin A immunoaffinity columns and one instruction manual.

5、Required materials but not provided in the box:

5.1 Equipment and consumables

----HPLC/LC-MS/MS

----Pneumatic control frame

----Air pump

----Balance scale

----High speed homogenizer (speed ≥ 10000/min) or shaker

----Crusher

----Sample sieve

----Measuring cylinder: 100mL/10mL

----Syringe: 20mL

----Pipette or pipette: 1mL

----Homogeneous cup (or 250mL conical flask with stopper)

-----Sample bottle

-----Measuring bottle

5.2 Reagents

----Methanol (CH)₃OH): Analytical grade for extraction process/chromatographic grade for elution process

----Sodium chloride (NaCl): analytical grade

----Hydrochloric acid (HCl): analytical grade

----Sodium hydroxide (NaOH): analytical grade

----Sodium dihydrogen phosphate (NaH)₂PO₄）: Analytical Pure

----Potassium dihydrogen phosphate (KH)₂PO₄）: Analytical Pure

----KCl: analytical grade

----Tween-20 (C₅₈H₁₁₄O₂₆）: Analytical Pure

----Water (H)₂O) Distilled water or deionized water

6、Notes:

----Before use, the immunoaffinity column needs to be returned to room temperature (22-25 ℃).

----Affinity columns should be stored at 2-8 ℃ and should not be frozen.

----Please use the immunoaffinity column within the valid date.

----The liquid inside the affinity column must be emptied before performing the sample loading operation.

----Sampling volume: The sampling volume can be increased or decreased as needed, and the amount of extraction solution should be adjusted accordingly.

----Column capacity: The column capacity is 200ng. When the content of the toxin to be detected in the sample divided by the dilution factor is higher than the column capacity, it is necessary to appropriately reduce the volume of the sample solution and retest.

----PH: The pH of the loading solution on the affinity column should be between 6 and 8. If it deviates from this range, hydrochloric acid or sodium hydroxide should be used to adjust the pH.

----Keeping the solvent consistent with the mobile phase during machine testing can eliminate the influence of solvent effects.

----Ochratoxin can cause cancer and should be operated with protective tools such as gloves and masks.

----Soak the used containers and standard solution in sodium hypochlorite solution (5% V/V) overnight.

7、Preparation of test solution:

7.1 Extraction solution

70% methanol water solution (V methanol: V water=70:30): Take 700mL of methanol and add deionized water to make up to 1L.

7.2 Phosphate buffered saline (PBS)

Weigh 8.00g of sodium chloride, 1.20g of disodium hydrogen phosphate (or 2.92g of disodium hydrogen phosphate dodecahydrate), 0.20g of potassium dihydrogen phosphate, and 0.20g of KCL, dissolve in 900mL of water, adjust the pH to 7.0 ± 0.1 with hydrochloric acid, and dilute with water to 1000mL.

7.3 Phosphate Tween buffer (PBST)

Dilute 1mL of Tween-20 with PBS buffer to 1000mL.

8Sample processing：

----3g ± 0.01g sample (solid sample needs to be crushed and sieved through a 2mm sieve), 3g sodium chloride is added to a homogenizer bottle, and 60mL of extraction solution is added (see 7.1);

----High speed homogenization (≥ 10000r/min) for 2 minutes;

----Centrifuge for 10 minutes (8000r/min);

----Take 5mL of supernatant and place it in a 50mL volumetric flask. Dilute to the mark with PBS buffer, mix well, and centrifuge for 15 minutes (8000r/min);

----Take 20mL of supernatant and purify it using an immunoaffinity column.

Dilution ratio: 10

9、Operating procedure:

9.1 Sample loading

----Remove the immune affinity column and directly penetrate the lower end of the syringe barrel through the affinity column

Connect the syringe barrel to the affinity column using a square stopper and place it on the pneumatic control rack for fixation;

----After emptying the affinity column, take the sample solution from step 8 and inject it into the syringe barrel;

----Make the liquid flow out at a rate of 1-2 drops per second;

9.2 Washing

----Wait for the liquid to drain;

----Wash the immunoaffinity column with 10mL of BST buffer (see 7.3) and 10mL of water at a flow rate of 2-3 drops per second;

9.3 Elution

----After the liquid is drained, remove the syringe, blow dry the liquid in the column, wash with 1.5mL of methanol, collect the eluent in a 2mL injection bottle, dilute with water to the mark, and mix well;

----Filter with a 0.22 μ m microporous filter and transfer to a sample bottle for HPLC analysis.

*Before each sample, the previous liquid must be drained.

10、Result interpretation:

The content of ochratoxin A=detection concentration x dilution factor

11Storage conditions and expiration date：

Storage conditions: 2-8 ℃.

Validity period: The product is valid for 18 months.