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

Human Prothrombin Precursor Protein (PIVKA-II)

ELISA Determination

Kit

Steps

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

Experimental Principle

The Human PIVKA-II ELISA Kit utilizes a double-antibody sandwich immunoassay to quantitatively measure the concentration of PIVKA-II in biological samples. The microtiter plate is pre-coated with a specific antibody against PIVKA-II, allowing the target antigen to bind. After incubation, an HRP-conjugated secondary antibody is added, forming an immune complex. Following thorough washing, TMB substrate is introduced, which changes color under the catalytic action of HRP. The reaction is stopped by adding an acidic stop solution, resulting in a yellow color that correlates with the PIVKA-II concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the sample concentration is calculated from a standard curve.

Human Prothrombin Precursor Protein (PIVKA-II)

ELISA Determination

Kit

Composition

1. 130x Washing 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 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. If not tested immediately, store at -20°C and avoid repeated freeze-thaw cycles.

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

Human Prothrombin Precursor Protein (PIVKA-II)

ELISA Determination

Kit

Steps

1. Standard Dilution: Prepare a dilution series using the provided standard. For example, start with 200 μl of standard diluent and add 150 μl of the original standard to create a 200 ng/L standard. Continue diluting accordingly.

2. Loading: Set up blank, standard, and sample wells. Add 50 μl of standard and 40 μl of sample diluent, followed by 10 μl of sample (final 5x dilution). Mix gently and avoid 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 5 times, ensuring each well is filled and drained thoroughly.

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

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

7. Washing: Repeat the washing procedure as above.

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

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

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

Calculation

Plot a standard curve using known concentrations and their corresponding OD values. Determine the sample concentration from the curve or use linear regression to calculate the value. Multiply by the dilution factor to obtain the actual sample concentration.

Human Prothrombin Precursor Protein (PIVKA-II)

ELISA Determination

Kit

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme reagents in a sealed bag.

2. If the washing solution crystallizes, warm it gently in a water bath before use.

3. Use a pipette for accuracy. Control loading time to avoid errors. For large batches, consider using a multichannel pipette.

4. Always include a standard curve. If the sample OD exceeds the highest standard, dilute the sample before testing and adjust calculations accordingly.

5. Discard sealing films after one use to prevent cross-contamination.

6. Keep substrates away from light.

7. Follow all instructions carefully for accurate results.

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 is 6 months from the date of manufacture.