Human prothrombin precursor protein (PIVKA-II) elisa assay kit - Database & Sql Blog Articles

Human prothrombin precursor protein (PIVKA-II)

ELISA Assay

Kit

Procedure

This kit is for research use only and not for human or animal consumption.

Experimental Principle

The ELISA kit employs a double-antibody sandwich method to quantify the level of human prothrombin precursor protein (PIVKA-II) in biological samples. The microplate is pre-coated with purified anti-PIVKA-II antibodies, forming a solid-phase antibody. After adding the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, TMB substrate is introduced, leading to a color change from blue to yellow under HRP catalysis and acid termination. The intensity of the color is directly proportional to the PIVKA-II concentration in the sample. The absorbance at 450 nm is measured using a microplate reader, and the concentration is determined by comparing against a standard curve.

Human prothrombin precursor protein (PIVKA-II)

ELISA Assay

Kit

Components

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

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (400ng/L) – 0.5ml × 1 bottle

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

6. Sample Diluent – 6ml × 1 bottle

7. TMB Developer A – 6ml × 1 bottle

8. TMB Developer 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. If testing cannot be done immediately, store at -20°C and avoid repeated freeze-thaw cycles.

2. Avoid using samples containing NaN3, as it may inhibit HRP activity and affect results.

Human prothrombin precursor protein (PIVKA-II)

ELISA Assay

Kit

Procedure

1. Standard Dilution: Start with the provided stock standard. Prepare dilutions according to the following table:

- 200 ng/L: Add 200 μl of standard diluent + 150 μl of stock

- 100 ng/L: Add 150 μl of 200 ng/L standard + 150 μl of diluent

- 50 ng/L: Add 150 μl of 100 ng/L standard + 150 μl of diluent

- 25 ng/L: Add 25 μl of standard diluent + 150 μl of 150 ng/L standard

- 12.5 ng/L: Add 150 μl of 25 ng/L standard + 150 μl of diluent

2. Loading: Set up blank, standard, and sample wells. Add 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample (final 5-fold dilution). Mix gently without touching the well walls.

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

4. Washing: Dilute the 30x wash solution 30 times with distilled water. Perform 5 washes, each lasting 30 seconds.

5. Enzyme Addition: Add 50 μl of enzyme reagent to all wells except blanks.

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 to each well. Incubate at 37°C for 10 minutes.

9. Stop: Add 50 μl of stop solution to each well to halt the reaction.

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

Calculation

Plot the standard curve using standard concentrations vs. OD values. Use linear regression to calculate the sample concentration from its OD value. Multiply by the dilution factor (×5) to determine the actual sample concentration.

Human prothrombin precursor protein (PIVKA-II)

ELISA Assay

Kit

Precautions

1. Allow the kit to reach room temperature (15–30 minutes) before use. Store unopened enzyme reagents in a sealed bag.

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

3. Use a pipette for accurate measurements. Limit loading time to 5 minutes; use a multichannel pipette for large batches.

4. Always prepare a standard curve, ideally in duplicate. If the sample OD exceeds the highest standard, dilute the sample before testing and adjust the final result accordingly.

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

6. Keep the substrate away from light during storage and handling.

7. Follow all instructions carefully. Results must be read using a microplate reader.

8. Treat all samples, waste, and wash solutions as biohazardous materials.

9. Do not mix components from different batches.

Storage Conditions & Expiration

1. Store the kit at 2–8°C.

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