A 3RD GENERATION IMMUNOASSAY TUMOR-NECROSIS-FACTOR-ALPHA

Tumor necrosis factor alpha (TNF alpha) is a polypeptide cytokine prod uced primarily by monocytes and macrophages. It is involved in a wide variety of immune reactions. Measurement of TNF alpha originally depen ded upon bioassays that are of varying reliability and reproducibility . Early immunoassays for TNF alpha required handling of radioisotopes and costly disposal of radioactive waste. Subsequent use of enzymes as reporter molecules in enzyme immunoassay (EIA) has eliminated the bur den of radioisotope handling and its associated costs. However EIA has presented new challenges. Use of thimerosal as a preservative in EIAs may require high disposal costs due to its mercury content. In additi on, many EIAs lack the sensitivity achievable in radioimmunoassay (RIA ). We have developed a simple microplate enzyme-linked immunosorbent a ssay (ELISA) for the detection of TNF alpha in serum, plasma and cultu re supernatants. Our high affinity capture antibody has enabled us to achieve a sensitivity of 1.5 pg/mL. The assay is calibrated to the Wor ld Health Organization (W.H.O.) first international standard for TNF a lpha (87/650) and exhibits excellent precision and reproducibility. Te tramethylbenzidine is used to generate the colored end product of the reaction, and thimerosal has been removed from all components.