Porcine Apoptosis Inducing Factor (AIF) ELISA Kit Instructions - Database & Sql Blog Articles

Porcine Apoptosis-Inducing Factor (AIF)

ELISA Test

Kit

Instruction Manual

This kit is for research use only.

Experimental Principle

The level of porcine apoptosis-inducing factor (AIF) in the specimen was determined using a double antibody sandwich ELISA method. The microwell plate was pre-coated with a purified monoclonal antibody against AIF, creating a solid-phase antibody. After adding the sample, the AIF in the sample binds to the immobilized antibody. Then, HRP-labeled secondary antibody is added, forming an antibody-antigen-enzyme complex. Following thorough washing, TMB substrate is introduced, which changes color under the catalytic action of HRP. The reaction is stopped with a stop solution, and the final color intensity is measured at 450 nm. The absorbance value is directly proportional to the concentration of AIF in the sample. A standard curve is used to calculate the actual concentration of AIF in the samples.

Composition

1. 30× Washing Solution – 20 ml × 1 bottle

2. Enzyme Standard Reagent – 6 ml × 1 bottle

3. Enzyme-Labeled Coating Plate – 12 wells × 8 strips

4. Sample Diluent – 6 ml × 1 bottle

5. Reagent A – 6 ml × 1 bottle

6. Color Developer B – 6 ml × 1 bottle

7. Stop Solution – 6 ml × 1 bottle

8. Standard (800 pg/ml) – 0.5 ml × 1 bottle

9. Standard Dilutions – 1.5 ml × 1 bottle

10. Instruction Manual – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing.

2. Samples containing NaN3 should not be used, as it may inhibit HRP activity.

Procedure

1. Standard Dilution: Prepare dilutions from the original standard according to the provided chart.

2. Loading: Add 50 µl of standard and 40 µl of sample diluent to each well, followed by 10 µl of sample (final 5× dilution).

3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.

4. Washing: Use 20× diluted washing solution. Wash 5 times and pat dry.

5. Add Enzyme: Add 50 µl of enzyme reagent to each well except blank.

6. Incubation: Repeat step 3.

7. Washing: Repeat step 4.

8. Color Development: Add 50 µl of TMB developer and incubate at 37°C for 15 minutes.

9. Stop Reaction: Add 50 µl of stop solution to each well.

10. Measurement: Read OD values at 450 nm within 15 minutes of stopping the reaction.

Calculation

Create a standard curve using known concentrations and their corresponding OD values. Use linear regression to calculate the unknown sample concentration. Multiply by the dilution factor to obtain the actual concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme reagents in a sealed bag.

2. If the washing solution crystallizes, gently warm it in a water bath before use.

3. Use a pipette for accurate measurements. Keep loading time under 5 minutes if possible.

4. Always prepare a standard curve and consider diluting high-concentration samples if needed.

5. Use one sealing film per experiment to prevent cross-contamination.

6. Keep substrates away from light.

7. Follow the manual strictly and rely on microplate reader results.

8. Treat all samples and waste as biohazardous materials.

9. Do not mix components from different batches.

10. In case of discrepancies, the English manual takes precedence.

Storage: 2–8°C

Shelf Life: 6 months