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

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Insect Cytochrome P450 (CYP450) ELISA Kit

Overview

This kit is designed for research use only and provides a reliable method to measure the concentration of insect cytochrome P450 in biological samples.

Experimental Principle

The kit employs a double-antibody sandwich immunoassay. A microplate is pre-coated with purified anti-CYP450 antibodies. The sample or standard is added, followed by HRP-conjugated secondary antibodies. This forms an antibody-antigen-enzyme complex. After washing, TMB substrate is added, which changes color under the catalytic action of HRP. The final yellow color intensity is directly proportional to the CYP450 concentration in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration is determined by comparing it to a standard curve.

Kit Composition

1. 130× Washing Solution – 20 ml × 1 bottle
2. Stop Solution – 6 ml × 1 bottle
3. Enzyme Standard Reagent – 6 ml × 1 bottle
4. Standard (64 IU/L) – 0.5 ml × 1 bottle
5. Enzyme-Labeled Coating Plate – 12 wells × 8
6. Sample Diluent – 6 ml × 1 bottle
7. TMB Substrate A – 6 ml × 1 bottle
8. TMB Substrate B – 6 ml × 1 bottle
9. Standard Dilutions – 1.5 ml × 1 bottle
10. Instructions – 1 copy
11. Sealing Film – 2 sheets
12. Seal Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
2. Avoid using samples containing NaN3, as it may inhibit HRP activity.

Procedure

1. Standard Dilution: Prepare standards according to the provided dilution chart. For example, add 150 μl of standard diluent to 150 μl of original standard to make 32 IU/L. Continue diluting to prepare 16, 8, 4, 2, and 1 IU/L standards.
2. Loading: Add 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample into each well (final dilution factor of 5). Mix gently without touching the well walls.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Use 30× diluted washing solution. Wash 5 times, then pat dry.
5. Enzyme Addition: Add 50 μl of enzyme-labeled reagent to each well except blank wells.
6. Incubation: Repeat step 3.
7. Washing: Repeat step 5.
8. Color Development: Add 50 μl of TMB A and 50 μl of TMB B, incubate at 37°C for 10 minutes.
9. Stop Reaction: Add 50 μl of stop solution to each well to halt the reaction.
10. Measurement: Measure OD at 450 nm within 15 minutes of adding the stop solution.

Calculation

Plot the standard curve using OD values versus concentrations. Determine the sample concentration from the curve, then multiply by the dilution factor. Alternatively, use linear regression to calculate the exact value.

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, warm it gently in a water bath before use.
3. Use a micropipette for accuracy. Limit loading time to 5 minutes per plate.
4. Always run a standard curve with duplicate wells. If sample OD exceeds the highest standard, dilute the sample before testing.
5. Use a new sealing film for each experiment to prevent contamination.
6. Protect the TMB substrate from light.
7. Follow all instructions strictly for accurate results.
8. Treat all waste materials as biohazardous.

Storage Conditions

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