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

Plant Protease (PROTEASE)

ELISA

Kit

Principle of Operation

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

Experimental Principle

The ELISA Kit for Plant Protease (PROTEASE) utilizes a double-antibody sandwich method to quantify the concentration of plant protease in biological samples. The microplate is pre-coated with a specific monoclonal antibody against PROTEASE. After incubation, the sample is added, allowing the target protein to bind to the immobilized antibody. A secondary HRP-labeled antibody is then introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following thorough washing, TMB substrate is added, which turns blue under HRP catalysis and changes to yellow when an acidic stop solution is applied. The intensity of the color correlates directly with the amount of PROTEASE present in the sample. The absorbance at 450 nm is measured using a microplate reader, and the sample concentration is determined by comparing it to a standard curve.

Plant Protease (PROTEASE)

ELISA

Kit

Composition

1. 130x Washing Solution – 20ml × 1 bottle

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

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

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

6. Sample Diluent – 6ml × 1 bottle

7. TMB Substrate A – 6ml × 1 bottle

8. TMB Substrate B – 6ml × 1 bottle

9. Standard Dilutions – 1.5ml × 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 the relevant extraction protocols. If immediate testing is not feasible, store the samples at -20°C, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 cannot be used, as this compound inhibits horseradish peroxidase (HRP) activity.

Plant Protease (PROTEASE)

ELISA

Kit

Procedure

1. Standard Dilution: This kit provides one original standard. Prepare serial dilutions according to the instructions provided in the manual.

2. Loading: Set up blank wells (no sample or enzyme reagent), standard wells, and sample wells. Add 50 μl of standard solution and 40 μl of sample diluent to each well, followed by 10 μl of the sample. Ensure proper mixing without touching the well walls.

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

4. Washing: Use 30x diluted washing solution. Wash the plate five times, ensuring complete removal of unbound substances.

5. Enzyme Addition: Add 50 μl of enzyme-labeled reagent to each well except the blank wells.

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

7. Washing: Repeat the washing procedure again.

8. Color Development: Add 50 μl of TMB substrate A and 50 μl of TMB substrate B to each well. Incubate at 37°C for 10 minutes.

9. Termination: Add 50 μl of stop solution to each well to halt the reaction. The color will change from blue to yellow.

10. Measurement: Read the OD values at 450 nm within 15 minutes of adding the stop solution.

Calculation

Plot the standard curve using the concentrations of standards on the x-axis and their corresponding OD values on the y-axis. Determine the unknown sample concentration based on the standard curve. Multiply by the dilution factor to obtain the actual concentration. Alternatively, use linear regression analysis to calculate the concentration mathematically.

Plant Protease (PROTEASE)

ELISA

Kit

Precautions

1. Allow the kit to reach room temperature (15–30 minutes) before use. If the enzyme label is opened, store the remaining components in a sealed bag.

2. If the washing solution crystallizes, warm it gently in a water bath before use. This will not affect the results.

3. Use a pipette for accurate measurements. Avoid contamination and ensure consistent timing during loading.

4. Always prepare a standard curve and run duplicates. If the sample OD is too high, dilute the sample before testing and adjust the final calculation accordingly.

5. Use the sealing film only once to prevent cross-contamination.

6. Protect the substrate from light exposure.

7. Follow all procedures strictly as outlined in the manual.

8. All samples, washes, and waste should be treated as biohazardous materials.

9. Do not mix components from different batches.

Storage Conditions and Expiration Date

1. Store the kit at 2–8°C.

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