Mouse type I collagen carboxyl cross-linking peptide (ICTP) kit operating principle - Database & Sql Blog Articles

Mouse type I collagen carboxyl cross-linking peptide (ICTP) Kit

Principle of operation

This kit is for research use only and is designed to detect the level of mouse type I collagen carboxyl cross-linking peptide (ICTP) in biological samples using a double antibody sandwich ELISA method.

The process involves coating a microplate with a purified monoclonal anti-ICTP antibody. The sample, which may contain ICTP, is then added to the plate, allowing the antigen to bind to the immobilized antibody. An HRP-labeled secondary antibody is introduced, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, producing a color change that correlates with the concentration of ICTP in the sample. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader.

The results are calculated based on a standard curve generated from known concentrations of ICTP, ensuring accurate quantification of the target molecule in the tested samples.

Kit Composition

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

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (8μg/L) – 0.5ml × 1 bottle

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

6. Sample Diluent – 6ml × 1 bottle

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

8. TMB Color Development Agent 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 used immediately, store at -20°C to prevent degradation. Avoid repeated freeze-thaw cycles.

2. Do not use samples containing NaN3, as it may inhibit horseradish peroxidase (HRP) activity and interfere with the assay.

Kit Steps

1. Standard Dilution: Prepare serial dilutions of the original standard according to the provided instructions.

2. Loading: Add 50 μl of each standard and 50 μl of diluted sample to the appropriate wells. Ensure accurate pipetting and gentle mixing.

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

4. Washing: Wash the plate 5 times with diluted washing solution to remove unbound components.

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

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

7. Washing: Perform the wash step again to remove excess enzyme-labeled antibody.

8. Color Development: Add 50 μl of TMB A and B solutions, incubate at 37°C for 15 minutes.

9. Termination: Add 50 μl of stop solution to each well to terminate 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 a standard curve using the OD values of the standards. Use this curve to determine the ICTP concentration in the unknown samples. Multiply the result by the dilution factor to obtain the actual concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store any unused enzyme-labeled reagents in a sealed bag.

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

3. Use a calibrated pipette and ensure accuracy throughout the procedure. Limit loading time to 5 minutes for best results.

4. Always run a standard curve and consider diluting high-concentration samples if necessary.

5. Use a new sealing film for each experiment to avoid contamination.

6. Keep all substrates away from light to maintain stability.

7. Follow the manufacturer's instructions precisely and rely solely on microplate reader readings for final results.

8. Treat all waste materials as biohazardous and dispose accordingly.

9. Do not mix components from different batches.

10. In case of discrepancies, refer to the English manual for official guidance.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

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