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Related Experiment Videos

A third generation immunoassay for tumor necrosis factor alpha

B Millett1, A M Sullivan, M Morimoto

  • 1T Cell Diagnostics, Inc., Woburn, MA.

Biotechniques
|December 1, 1994
PubMed
Summary

A new enzyme-linked immunosorbent assay (ELISA) accurately detects tumor necrosis factor alpha (TNF alpha) in biological samples. This sensitive method avoids radioisotopes and mercury, offering a reliable tool for immune response research.

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Area of Science:

  • Immunology
  • Biochemistry
  • Assay Development

Background:

  • Tumor necrosis factor alpha (TNF alpha) is a key cytokine in immune responses, primarily produced by monocytes and macrophages.
  • Traditional TNF alpha bioassays lack reliability, while early immunoassays involved hazardous radioisotopes or costly enzyme immunoassay (EIA) preservatives like thimerosal.
  • Existing EIAs often lack the sensitivity of radioimmunoassay (RIA) and may incur high disposal costs due to mercury content.

Purpose of the Study:

  • To develop a simple, sensitive, and reproducible microplate enzyme-linked immunosorbent assay (ELISA) for detecting TNF alpha.
  • To overcome the limitations of previous TNF alpha detection methods, including radioisotopes and problematic preservatives.
  • To provide a reliable assay for TNF alpha measurement in serum, plasma, and culture supernatants.

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Main Methods:

  • Development of a novel microplate enzyme-linked immunosorbent assay (ELISA) utilizing a high-affinity capture antibody.
  • Quantification of TNF alpha in serum, plasma, and culture supernatants.
  • Calibration of the assay against the World Health Organization (W.H.O.) first international standard for TNF alpha (87/650).

Main Results:

  • The developed ELISA achieves a high sensitivity of 1.5 pg/mL for TNF alpha detection.
  • The assay demonstrates excellent precision and reproducibility across different sample types.
  • Thimerosal has been eliminated from assay components, addressing disposal cost concerns.

Conclusions:

  • A simple and highly sensitive ELISA has been successfully developed for TNF alpha detection.
  • This assay offers a reliable, cost-effective, and safe alternative to existing methods for measuring TNF alpha.
  • The assay is suitable for various biological matrices and meets international standardization requirements.