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

Porcine Intestinal Peptidase (Erepsin) ELISA Kit

This kit is for research use only. It should not be used for diagnostic or therapeutic purposes.

Experimental Principle

The Porcine Intestinal Peptidase (Erepsin) ELISA Kit utilizes a double-antibody sandwich assay to detect the presence of porcine intestinal peptidase in biological samples. The process involves coating a microplate with a specific antibody, followed by incubation with the sample and an HRP-labeled secondary antibody. After washing, the substrate TMB is added, which changes color based on the enzyme activity. The final color intensity is directly proportional to the concentration of the target protein in the sample.

Kit Components

• 130x Washing Solution – 20ml × 1 bottle
• Stop Solution – 6ml × 1 bottle
• Enzyme Standard Reagent – 6ml × 1 bottle
• Standard (2000U/L) – 0.5ml × 1 bottle
• Enzyme-Labeled Coating Plate – 12 wells × 8
• Sample Diluent – 6ml × 1 bottle
• TMB Color Developing Agent A – 6ml × 1 bottle
• TMB Color Developing Agent B – 6ml × 1 bottle
• Instructions – 1 copy
• Sealing Film – 2 sheets
• Sealed 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 horseradish peroxidase (HRP) activity.

Kit Steps

1. Standard Dilution: Prepare standards according to the provided dilution chart.
2. Sample Loading: Load 50μl of standard, 40μl of sample diluent, and 10μl of sample into each well. Final dilution is 5-fold.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash the plate 5 times with diluted washing solution.
5. Add Enzyme Labeled Reagent: Add 50μl of enzyme reagent to all wells except blank controls.
6. Incubation: Repeat incubation at 37°C for 30 minutes.
7. Color Development: Add 50μl of TMB A and B, incubate at 37°C for 10 minutes.
8. Stop Reaction: Add 50μl of stop solution to each well. The color will turn from blue to yellow.
9. Measurement: Read absorbance at 450nm within 15 minutes of adding the stop solution.

Calculation

Plot the standard curve using OD values and concentrations. Use linear regression to calculate the unknown sample concentration, then multiply by the dilution factor.

Precautions

• Allow the kit to reach room temperature before use. Store unopened enzyme reagents in sealed bags.
• If washing solution crystallizes, dissolve it in a water bath before use.
• Use accurate pipettes and avoid cross-contamination. For large batches, use a multichannel pipette.
• Always prepare a standard curve and run duplicates. If sample OD exceeds the first standard, dilute accordingly.
• Discard all waste materials as biohazardous.
• Do not mix reagents from different batches.

Storage Conditions

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