Rabbit interleukin-1β (IL-1β) ELISA kit operation technical steps - Database & Sql Blog Articles

Rabbit IL-1β

(

IL-1β

ELISA

Kit

Operational technical steps

This reagent is for research only.

ELISA kit experimental sample:

For the determination of rabbit serum, plasma, and related liquid samples.

Experimental principle:

Rabbit IL-1β (IL-1β) ELISA Kit determines the level of interleukin-1β in rabbits using a double antibody sandwich assay. A microporous plate is coated with purified rabbit interleukin-1β antibody to create a solid-phase antibody. The sample containing interleukin-1β is added to the wells, where it binds to the immobilized antibody. An HRP-labeled goat anti-rabbit antibody is then introduced, forming an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, TMB substrate is added, which turns blue under HRP catalysis and then yellow upon acid addition. The color intensity correlates directly with the IL-1β concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the IL-1β concentration is calculated from a standard curve.

Rabbit IL-1β (IL-1β) ELISA Kit composition:

The kit is available in 48-well or 96-well configurations.

Includes: 1 instruction manual, 2 sealing films, 1 sealed bag, enzyme label package stored at 2–8°C, standard solution (90 ng/L, 0.5 ml), standard dilution (1.5 ml), enzyme standard reagent (3 ml), sample dilution (3 ml), Reagent A (3 ml), Reagent B (3 ml), stop solution (3 ml), 20× washing solution (20 ml).

Sample preparation and requirements:

1. Serum: Blood is allowed to clot at room temperature for 10–20 minutes, then centrifuged at 2000–3000 rpm for 20 minutes. Supernatant is collected carefully. If precipitate forms during storage, re-centrifuge.

2. Plasma: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully. If precipitate forms, re-centrifuge.

3. Urine: Collected in sterile tubes, centrifuged at 2000–3000 rpm for 20 minutes. Supernatant is collected carefully. If precipitate occurs, re-centrifuge.

4. Cell culture supernatant: Collected in sterile tubes, centrifuged at 2000–3000 rpm for 20 minutes. For intracellular components, cells are lysed by freezing-thawing and centrifuged again.

5. Tissue specimens: Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect supernatant. Store remaining portion at 2–8°C.

6. Samples should be processed immediately after collection. If not tested right away, store at –20°C, avoiding repeated freeze-thaw cycles.

7. Avoid samples containing NaN3, as it inhibits HRP activity.

Steps:

1. Dilute standards and load into 10 wells. Prepare serial dilutions to achieve concentrations of 60, 40, 20, 10, and 5 ng/L.

2. Load samples into designated wells, adding 40 μl of sample diluent and 10 μl of sample (final 5× dilution).

3. Incubate at 37°C for 30 minutes.

4. Dilute washing solution 20× with distilled water.

5. Wash wells 5 times, pat dry.

6. Add 50 μl of enzyme reagent to each well except blank.

7. Incubate again at 37°C for 30 minutes.

8. Repeat washing steps.

9. Add 50 μl of TMB developer, incubate at 37°C for 15 minutes.

10. Add 50 μl of stop solution to terminate reaction.

11. Measure OD at 450 nm within 15 minutes after stopping the reaction.

Precautions:

1. Allow kit to reach room temperature before use. Seal unused enzyme reagent in a bag.

2. Crystallization in concentrated washing solution is normal; dissolve by heating if needed.

3. Use accurate pipettes, avoid cross-contamination, and complete loading within 5 minutes.

4. Always prepare a standard curve with duplicates. If sample OD exceeds that of the first standard, dilute the sample and adjust calculation accordingly.

5. Use a new sealing film per experiment to prevent contamination.

6. Keep substrates away from light.

7. Follow the manual strictly. Results must be based on microplate reader readings.

8. Treat all samples and waste as biohazardous materials.

9. Do not mix reagents from different batches.

10. In case of discrepancy, the English manual takes precedence.

Calculation:

Plot a standard curve using standard concentrations and corresponding OD values. Determine sample concentration from the curve, multiply by dilution factor. Alternatively, calculate using linear regression equation from the standard curve.

Performance:

1. Correlation coefficient R ≥ 0.990.

2. Intra-batch and inter-batch variation < 9% and 11%, respectively.

Storage conditions and expiration date:

1. Store at 2–8°C.

2. Shelf life: 6 months.