Plant protease (PROTEASE) ELISA kit operating principle - Database & Sql Blog Articles

Plant Protease (PROTEASE)

ELISA

Kit

Principle of Operation

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

Experimental Principle

The Plant Protease ELISA Kit utilizes a double-antibody sandwich method to quantify the concentration of plant protease in biological samples. The microtiter plate is pre-coated with a specific monoclonal antibody against plant protease. After sample incubation, the protease binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following a thorough wash, the TMB substrate is introduced, which changes color under HRP catalysis—from blue to yellow upon acid termination. The intensity of the color is directly proportional to the protease concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the sample concentration is calculated based on a standard curve.

Plant Protease (PROTEASE)

ELISA

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 Product (960 U/L) – 0.5 ml × 1 bottle

5. Enzyme-Labeled Coating Plate – 12 wells × 8

6. Sample Diluent – 6 ml × 1 bottle

7. TMB Color Development Agent A – 6 ml × 1 bottle

8. TMB Color Development Agent B – 6 ml × 1 bottle

9. Standard Dilutions – 1.5 ml × 1 bottle

10. Instructions – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection, following established protocols. If testing cannot be performed immediately, store samples at -20°C, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 are not suitable for this assay, as it may inhibit HRP activity.

Plant Protease (PROTEASE)

ELISA

Kit

Procedure

1. Standard Dilution: Prepare a serial dilution from the original standard according to the provided instructions.

2. Loading: Add 50 μL of standard solution and 40 μL of sample diluent into each well, followed by 10 μL of sample. Ensure gentle mixing without touching the well walls.

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

4. Washing: Use 30× diluted washing buffer and wash 5 times, ensuring complete removal of unbound reagents.

5. Enzyme Addition: Add 50 μL of enzyme-labeled reagent to all wells except blank controls.

6. Incubation: Repeat the 37°C incubation for 30 minutes.

7. Washing: Perform the same washing steps as before.

8. Color Development: Add 50 μL of TMB A and B solutions, incubate at 37°C for 10 minutes.

9. Termination: Add 50 μL of stop solution to each well to halt the reaction.

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

Calculation

Plot a standard curve using known concentrations and corresponding OD values. Determine the unknown sample concentration by interpolation or linear regression. Multiply by the dilution factor to obtain the actual sample concentration.

Plant Protease (PROTEASE)

ELISA

Kit

Precautions

1. Allow the kit to reach room temperature (15–30 min) before use. Store opened enzyme labels in a sealed bag.

2. Wash solution may crystallize; heat gently if needed. This will not affect results.

3. Use calibrated pipettes and ensure accurate measurements. Limit loading time to 5 minutes.

4. Always prepare a standard curve and run duplicates. If sample OD exceeds the highest standard, perform a preliminary dilution before testing.

5. Use one sealing film per test to prevent contamination.

6. Protect the substrate from light exposure.

7. Follow the operating manual strictly. Results must be confirmed using a microplate reader.

8. Treat all waste materials as biohazardous.

9. Do not mix components from different batches.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.