Insect cytochrome P450 (CYP450) enzyme-linked immunoassay kit - Database & Sql Blog Articles

Insect Cytochrome P450 (CYP450) ELISA Kit

Overview

This kit is designed for the quantitative detection of insect cytochrome P450 (CYP450) in biological samples. It is intended for research use only and not for diagnostic or therapeutic purposes. The kit utilizes a sandwich ELISA technique, which allows for accurate and reliable measurement of CYP450 levels in various sample types.

Principle of Operation

The assay is based on the principle of antibody-antigen interaction. A microplate is pre-coated with a specific monoclonal antibody against CYP450. After adding the sample, the CYP450 present in the sample binds to the immobilized antibody. A secondary antibody conjugated with horseradish peroxidase (HRP) is then added, forming a complex. The enzyme catalyzes a color change in the substrate TMB, resulting in a visible color reaction that correlates with the concentration of CYP450 in the sample. The absorbance is measured at 450 nm using a microplate reader, and the results are calculated by comparing the OD values to a standard curve.

Kit Components

• 130x Wash Solution – 20 ml × 1 bottle
• Stop Solution – 6 ml × 1 bottle
• Enzyme Standard Reagent – 6 ml × 1 bottle
• Standard (64 IU/L) – 0.5 ml × 1 bottle
• Enzyme-Labeled Coating Plate – 12 wells × 8
• Sample Diluent – 6 ml × 1 bottle
• TMB Color Reagent A – 6 ml × 1 bottle
• TMB Color Reagent B – 6 ml × 1 bottle
• Instructions – 1 copy
• Sealing Film – 2 sheets
• Sealed Bag – 1
• Standard Dilutions – 1.5 ml × 1 bottle

Sample Preparation

For optimal results, samples should be processed immediately after collection. If not used right away, store them at -20°C. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3 as it may inhibit HRP activity.

Step-by-Step Procedure

1. Dilute the standard according to the provided chart to create a range of concentrations.
2. Add 50 µL of standard solution to the designated wells, 40 µL of sample diluent, and 10 µL of the sample. Ensure the final dilution factor is 5.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing solution.
5. Add 50 µL of enzyme-labeled reagent to each well (except blank wells).
6. Incubate again at 37°C for 30 minutes.
7. Wash the plate again.
8. Add 50 µL of TMB A and 50 µL of TMB B to each well. Incubate at 37°C for 10 minutes.
9. Add 50 µL of stop solution to each well to halt the reaction.
10. Measure the absorbance at 450 nm within 15 minutes.

Data Analysis

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

Safety and Precautions

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

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

3. Use a micropipette for accuracy. Keep all steps consistent to avoid errors.

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

5. Use a new sealing film for each experiment to prevent cross-contamination.

6. Protect the substrate from light.

7. Follow all instructions strictly and verify results using a microplate reader.

8. Treat all waste materials as biohazardous.

9. Do not mix components from different batches.

Storage and Shelf Life

Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.

Notes

This kit is suitable for research purposes only. Always refer to the latest version of the instructions for detailed procedures and safety information.