Canine matrix lysin (ST2) ELLISA kit professional experimental test - Database & Sql Blog Articles

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Canine Matrix Lysin (ST2) ELISA Kit

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

Experimental Principle:

The Canine Matrix Lysin (ST2) ELISA Kit uses a double-antibody sandwich assay to determine the level of ST2 in biological samples. A microplate is pre-coated with a specific antibody against canine ST2, which captures the antigen from the sample. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. After washing, the substrate TMB is added, which changes color in the presence of HRP. The intensity of the color is directly proportional to the concentration of ST2 in the sample. The absorbance is measured at 450 nm using a microplate reader, and the concentration is calculated based on a standard curve.

Kit Components:

• 120x Washing Solution – 20ml × 1 bottle
• Stop Solution – 3ml × 1 bottle
• Enzyme Standard Reagent – 3ml × 1 bottle
• Standard (80ng/L) – 0.5ml × 1 bottle
• Enzyme-Labeled Coating Plate – 12 wells × 4
• Sample Diluent – 3ml × 1 bottle
• Developer A – 3ml × 1 bottle
• Developer B – 3ml × 1 bottle
• Instructions – 1 copy
• Sealing Film – 2 sheets
• Sealed Bag – 1

Sample Requirements:

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

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

Testing Procedure:

1. Standard Dilution: Prepare a dilution series of the original standard according to the provided instructions.
2. Loading: Add 50μL of standard or sample to the microplate, along with 40μL of diluent and 10μL of sample (final dilution 5x).
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash the plate 5 times with diluted washing solution, ensuring thorough removal of unbound substances.
5. Add Enzyme: Add 50μL of enzyme-labeled reagent to each well except the blank control.
6. Incubation: Incubate again at 37°C for 30 minutes.
7. Color Development: Add 50μL of developer A and B, mix gently, and incubate at 37°C for 15 minutes.
8. Stop Reaction: Add 50μL of stop solution to terminate the reaction.
9. Measurement: Read the OD values at 450nm within 15 minutes of stopping the reaction.

Calculation:

Plot a standard curve using the OD values of the standards. Use linear regression to calculate the concentration of ST2 in the sample by substituting its OD value into the equation. Multiply by the dilution factor to obtain the actual concentration.

Precautions:

1. Allow the kit to reach room temperature before use. Store unused enzyme-labeled reagents in a sealed bag.
2. If the washing solution crystallizes, warm it gently before use. It will not affect the results.
3. Use accurate pipettes and ensure consistent timing during sample loading. For large numbers of samples, consider using an automated pipette.
4. Always run a standard curve and duplicate wells. If the sample OD exceeds the first standard, dilute the sample before testing.
5. Use the sealing film only once to prevent cross-contamination.
6. Keep the substrate away from light.
7. Follow the manual strictly and rely on microplate reader readings for final results.
8. Treat all waste materials as biohazardous.
9. Do not mix components from different batches.
10. In case of discrepancies, the English manual takes precedence.

Storage Conditions and Expiration:

1. Store the kit at 2–8°C.

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

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