Determination of human tumour necrosis factor-α (TNF-α) by time-resolved immunofluorometric assay

Abstract

We have developed a ‘sandwich’-type time-resolved immunofluorometric assay (IFMA) for tumour necrosis factor alpha (TNF-α) using two monoclonal antibodies (mAb) and the streptavidin/biotin (SAB) system. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin coated wells to which we added biotinylated mAb for 3h. After washing, the serum sample was added and incubated for 2h followed by washing. Another monoclonal europium-labelled tracer antibody was added and incubated for 1 h, the wells were washed and the fluorescence of Eu measured. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labelled antibody by hydrophobic interaction chromatography was found to be essential to improve sensitivity. With a sample volume of 50μl the detection limit was 6ngl⁻¹ and the analytical range large, i.e. 10000-fold. The median concentration in serum from healthy subjects was 12ngl⁻¹ and the reference range <39ngl⁻¹. The mean analytical recovery in plasma was 76% and in serum 83%. Separation of serum by gel filtration and assay of TNF-α in fractions showed that the assay also measured the high molecular weight (MW) form of TNF-α, apparently corresponding to its complex with soluble receptors. Advantages of our SAB-IFMA were high sensitivity and low consumption of mAb. The assay performance of the SAB-IFMA was compared to two commercially available enzyme immunoassays also using the SAB system.

Key Words:

• cytokine
• fluorometry
• sandwich assay
• monoclonal antibodies