Pig intestinal peptidase (Erepsin) ELISA kit instructions - Database & Sql Blog Articles

Porcine Intestinal Peptidase (Erepsin) ELISA Kit

This kit is intended for research use only and not for diagnostic or therapeutic purposes.

Experimental Principle

The Porcine Intestinal Peptidase (Erepsin) ELISA Kit utilizes a double-antibody sandwich immunoassay to quantify the presence of Erepsin in biological samples. The process involves coating a microplate with a specific antibody that binds to Erepsin. After incubation, the sample is added, allowing any Erepsin present to bind to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, a substrate (TMB) is added, which changes color in the presence of HRP. The reaction is stopped, and the absorbance is measured at 450 nm using a microplate reader. The intensity of the color is directly proportional to the concentration of Erepsin in the sample.

Kit Components

• 130× Washing Solution – 20 ml × 1 bottle
• Stop Solution – 6 ml × 1 bottle
• Enzyme Standard Reagent – 6 ml × 1 bottle
• Standard (2000 U/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 Requirements

• Immediately extract specimens after collection and perform the experiment as soon as possible. If testing cannot be done immediately, store samples at -20°C. Avoid repeated freezing and thawing.
• Do not use samples containing NaN3, as it may inhibit the activity of horseradish peroxidase (HRP).

Kit Procedure

1. Standard Dilution: Prepare a series of standard dilutions based on the provided instructions. For example, add 150 μL of standard diluent to 150 μL of the original standard to create a 1000 U/L solution. Continue serial dilution according to the chart provided.
2. Sample Loading: Add 50 μL of standard, 40 μL of sample diluent, and 10 μL of sample to each well. Ensure proper mixing without touching the well walls.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash the plate 5 times with diluted washing solution (30× concentrated solution diluted 30× with distilled water).
5. Add Enzyme-Labeled Reagent: Add 50 μL of enzyme-labeled reagent to each well except the blank control.
6. Second Incubation: Incubate again at 37°C for 30 minutes.
7. Color Development: Add 50 μL of TMB A and 50 μL of TMB B to each well. Incubate at 37°C for 10 minutes.
8. Stop Reaction: Add 50 μL of stop solution to each well. The color will change from blue to yellow.
9. Measurement: Read the absorbance at 450 nm within 15 minutes of adding the stop solution.

Calculation

Create a standard curve by plotting the known concentrations of the standards against their corresponding OD values. Use this curve to determine the concentration of the unknown sample. Multiply the calculated value by the dilution factor to obtain the actual sample concentration.

Precautions

• Allow the kit to reach room temperature before use. Store unopened enzyme-labeled reagents in a sealed bag.
• If the washing solution crystallizes, warm it gently in a water bath before use.
• Use a pipette for accuracy. Keep all steps within 5 minutes if possible.
• Always include a standard curve and duplicate wells for accuracy.
• Discard used sealing films after single use to prevent cross-contamination.
• Keep the TMB substrate away from light.
• Follow the manual strictly and verify results using a microplate reader.
• Treat all waste materials as biohazardous.
• Do not mix components from different batches.

Storage Conditions & Expiration Date

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