2-Arachidylglycerol (2-AG) ELISA Kit

Sample Collection and Storage

The following are general guidelines for sample collection and storage. Sodium azide must not be used as a preservative during all sample collection and storage.

1. Cell Culture Supernatant: Centrifuge at 4000 rpm for 20 minutes to remove cell particles and aggregates. Store the supernatant below -20°C and avoid repeated freezing and thawing.

2. Serum: Use pyrogen- and endotoxin-free tubes, avoiding any cell stimulation during the procedure. Centrifuge at 4000 rpm for 20 minutes, carefully separate the serum, and store below -20°C. Avoid repeated freezing and thawing.

3. Plasma: Use heparin, EDTA, or sodium citrate as an anticoagulant. Centrifuge at 4000 rpm for 20 minutes, remove the supernatant, and store the plasma below -20°C to avoid repeated freezing and thawing.

After sample collection, if testing cannot be completed in one go, please aliquot and freeze the sample in a single-use aliquot to avoid repeated freezing and thawing. Thaw at room temperature before use to ensure that the sample is evenly and fully thawed.

4. Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01 M, pH=7.4) to remove residual blood. Weigh and mince the tissue. Add the minced tissue to the corresponding volume of PBS (generally a 1:9 weight-to-volume ratio, for example, 1 g of tissue sample corresponds to 9 mL of PBS. The specific volume can be adjusted appropriately according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be ultrasonically disrupted or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and collect the supernatant for testing. 5. Cell Extract: Gently wash adherent cells with cold PBS, then digest with trypsin and collect the cells by centrifugation at 1000 × g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with cold PBS. Resuspend in 150-200 μL of PBS per 1 × 106 cells and disrupt the cells by repeated freeze-thaw cycles (if the cell count is very low, reduce the volume of PBS). Centrifuge the extract at 1500 × g for 10 minutes, and collect the supernatant for testing. 6. Other biological fluids: Centrifuge at 1000×g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing. Reagent Preparation 1. Before use, rewarm all components for at least 60 minutes to ensure they are fully warmed to room temperature. 2. Concentrated Wash Solution: Crystals may form when the concentrated wash solution is removed from the refrigerator. This is normal. Heat in a water bath to completely dissolve the crystals. Dilute the concentrated wash solution with distilled water at a ratio of 1:20: i.e., add 19 parts distilled water to 1 part concentrated wash solution. 3. Substrate: Before use, thoroughly mix substrate solutions A and B at a 1:1 ratio by volume. Use within 15 minutes of mixing.

Operational Procedure

Allow all reagents and components to return to room temperature. Duplicate wells are recommended for standards, controls, and samples. 1. Prepare the working solutions of all kit components as described in the previous instructions.

2. Remove the required strips from the aluminum foil pouch. Seal the remaining strips in a ziplock bag and return them to the refrigerator.

Set up standard wells, blank wells, and sample wells. Add 50 μL of the standard of varying concentrations to the standard wells, leave the blank wells untouched, and add 50 μL of the test sample to the sample wells.

3. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antigen to the standard and sample wells, except for the blank wells.

4. Cover the reaction plate with sealing film and incubate in a 37°C water bath or incubator for 60 min. 5. Remove the sealing film, discard the liquid, and pat dry on absorbent paper. Fill each well with wash solution, let it sit for 20 seconds, shake off the wash solution, and pat dry on absorbent paper. Repeat this process five times. If using an automated plate washer, wash the plate according to the machine's operating procedures. Adding a 30-second soaking period can improve assay accuracy. After washing, thoroughly pat the plate dry on a clean, lint-free paper before adding substrate. 6. Thoroughly mix substrates A and B at a 1:1 ratio by volume and add 100 μL of the substrate mixture to all wells. Cover the plate with sealing film and incubate at 37°C in a water bath or incubator for 15 minutes. 7. Add 50 μL of stop solution to all wells and read the absorbance (OD) of each well on a microplate reader. Calculation of Results 1. Using the standard concentration as the horizontal axis and the corresponding absorbance (OD value) as the vertical axis, use computer software and four-parameter logistic curve fitting (4-PL) to create a standard curve equation. Calculate the sample concentration using the sample absorbance (OD value) using this equation. 2. If the sample is diluted, the concentration measured using the above method must be multiplied by the dilution factor to obtain the final sample concentration.

Standard Curve

Dose-Response Curve Linearity

The correlation coefficient, r, of the calibrator dose-response curve is greater than or equal to 0.9900.

Precision

Intra-assay precision: Three sets of samples with known high, medium, and low concentrations were evaluated for precision twenty times within the same plate. The intra-assay coefficient of variation (CV%) was less than 10%.

Inter-assay precision: Three sets of samples with known high, medium, and low concentrations were evaluated for precision twenty times within different plates. The inter-assay coefficient of variation (CV%) was less than 15%. Recovery was assessed five times within the same plate for three sets of known high, medium, and low concentration samples, with recoveries ranging from 85% to 115%. Specificity: This kit recognizes both natural and recombinant 2-arachidonoylglycerol (2-AG), with no cross-reactivity with structural analogs. Technical Tips: 1. Avoid foaming when mixing protein solutions. 2. When adding calibrators and samples, change pipette tips for each calibrator concentration and sample. Common components should be pipetted using a cantilever to avoid cross-contamination. 3. Appropriate incubation times and adequate wash steps are essential for accurate results. 4. When using an automated plate washer, adding a 30-second soak step can improve assay accuracy.

5. The substrate solution is a colorless liquid. If it turns blue during storage, it means that the substrate solution has expired and should not be used.

6. The order of adding the stop solution should be the same as the order of adding the substrate solution. After adding the stop solution, the blue substrate product will instantly turn yellow.

7. During the experiment, the remaining strips should be immediately returned to the self-sealing bag and sealed (low temperature drying) for storage.

8. All liquid components should be shaken thoroughly before use. Incubation operations should be carried out strictly according to the time, addition volume and addition order indicated in the instructions.

The concentrations of the standard solutions are: 8, 4, 2, 1, 0.5, and 0 ng/mL.

1. Testing must comply with laboratory management regulations and strictly prevent cross contamination. All samples, wash solutions and various wastes should be disposed of as infectious agents.

2. The liquid components of the test kit contain proclin-300 preservative, which may cause skin allergic reactions. Avoid inhalation of smoke and skin contact.

3. Do not mix test kits or components from other manufacturers.

4. Use the sample diluent provided with the test kit.

5. If the sample value is higher than the highest standard concentration, please dilute the sample appropriately and re-measure.

6. Human anti-mouse and other heterophilic antibodies present in the test sample will interfere with the test results. Please rule out this factor before testing.

7. Test results obtained by other methods are not directly comparable to the results of this kit.
8. Wear protective gloves and wash hands thoroughly after the experiment.