Determination of human tumor necrosis factor alpha by a highly sensitive enzyme immunoassay

Area of Science:

• Immunology
• Biochemistry

Background:

• Tumor necrosis factor alpha (TNF-alpha) is a key polypeptide mediator in immune reactions, primarily produced by monocytes and macrophages.
• Accurate quantification of TNF-alpha is crucial for understanding various immune-related conditions.

Purpose of the Study:

• To develop and validate a simple, sensitive microplate enzyme-linked immunosorbent assay (ELISA) for detecting human TNF-alpha (hTNF-alpha).
• To establish a reliable method for quantifying hTNF-alpha in biological samples like serum, plasma, and cell culture supernatants.

Main Methods:

• A microplate ELISA utilizing a biotin-streptavidin amplification system with horseradish peroxidase.
• Assay calibration against the World Health Organization (WHO) standard for hTNF-alpha (87/650).
• Validation of sensitivity, precision (within-run and between-run coefficients of variation), and correlation with a commercial ELISA kit.

Main Results:

• The developed ELISA achieved a high sensitivity of 0.1 pg hTNF-alpha/ml.
• Excellent precision was demonstrated with within-run coefficients of variation (3.7–5.9%) and between-run coefficients of variation (8.0–9.9%).
• The assay showed strong correlation (r = 0.956) with a commercially available hTNF-alpha ELISA kit.

Conclusions:

• The described microplate ELISA is a simple, sensitive, and reliable method for quantifying hTNF-alpha.
• This assay provides a valuable tool for research in immunology and related clinical applications.
• The high sensitivity and precision make it suitable for detecting low levels of hTNF-alpha in various biological matrices.