Human tumor necrosis factor beta (TNF-β) elisa kit operation - Database & Sql Blog Articles

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Human tumor necrosis factor beta (TNF-β) is a key cytokine involved in inflammatory responses and immune regulation. This ELISA kit provides a reliable method for quantifying TNF-β levels in biological samples, making it an essential tool for research and diagnostic purposes.

ELISA Kit – Designed for the quantitative detection of human TNF-β in serum, plasma, and other biological fluids.

Operating Principle

The kit employs a sandwich ELISA technique. A monoclonal antibody specific to TNF-β is pre-coated onto a microplate. After adding the sample, TNF-β binds to the immobilized antibody. A secondary HRP-conjugated antibody then recognizes the captured TNF-β, forming a complex. The addition of TMB substrate leads to a color change that correlates with the amount of TNF-β present. The reaction is stopped, and the absorbance is measured at 450 nm using a microplate reader. The concentration of TNF-β in the sample is determined by comparing the OD value to a standard curve.

Kit Composition

• 130x concentrated washing solution: 20ml × 1 bottle
• Stop solution: 6ml × 1 bottle
• Enzyme standard reagent: 6ml × 1 bottle
• Standard (640ng/L): 0.5ml × 1 bottle
• Enzyme-labeled coating plate: 12 wells × 8
• Sample diluent: 6ml × 1 bottle
• TMB Color Reagent A: 6ml × 1 bottle
• TMB Color Reagent B: 6ml × 1 bottle
• Standard dilutions: 1.5ml × 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. Avoid repeated freeze-thaw cycles.

2. Samples containing NaN3 cannot be used, as it inhibits HRP activity and may lead to false results.

Procedure

1. Standard Dilution: Prepare a series of standards from the original stock according to the provided dilution chart.
2. Loading: Add 50μl of standard or sample to the appropriate wells. For samples, add 40μl of diluent and 10μl of sample (final 5-fold dilution).
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash the plate 5 times with diluted washing buffer.
5. Add Enzyme: Add 50μl of enzyme-labeled reagent to each well except blank controls.
6. Incubation: Incubate again at 37°C for 30 minutes.
7. Color Development: Add 50μl of TMB A and B, incubate at 37°C for 15 minutes.
8. Stop Reaction: Add 50μl stop solution to each well. The color will turn yellow.
9. Measurement: Read the absorbance at 450nm within 15 minutes of stopping the reaction.

Calculation

Plot the standard curve using OD values versus concentrations. Determine the sample concentration from the curve and multiply by the dilution factor. Alternatively, use linear regression analysis to calculate the exact concentration.

Precautions

1. Bring the kit to room temperature before use and allow it to equilibrate for 15–30 minutes.
2. If the enzyme reagent is opened, store it in a sealed bag to prevent contamination.
3. Use a pipette for accurate measurements and avoid cross-contamination.
4. Always prepare a standard curve and consider diluting high-concentration samples if necessary.
5. Discard the sealing film after single use to prevent cross-contamination.
6. Keep the substrate away from light during storage and use.
7. Follow the operation manual strictly and ensure all results are confirmed with a microplate reader.
8. Dispose of all waste materials as biohazardous substances.
9. Avoid mixing components from different batches.

Storage Conditions

Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.

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