Determination of recombinant human tumor necrosis factor-α in serum by chemiluminescence imaging

A simple, sensitive and high throughput chemiluminescence (CL) imaging method was described for the determination of recombinant human tumor necrosis factor-α (rh TNF-α). The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high throughput of CL imaging method. In this system, 96 well transparent microtiter plates were used as solid phase materials. The method was based on the use of two monoclonal antibodies against rh TNF-α, one “capture” antibody and one labeled with horseradish peroxidase (HRP), in a “sandwich” ELISA format. A cooled CCD camera has been applied to image the weak chemiluminescence from the ECL. The CL intensity was proportional with the concentration of rh TNF-α in the range of 9.0-312.0 pg ml−1 and the detection limit was 1 pg ml−1. The proposed method has been successfully applied to the determination of rh TNF-α in human serum, the reliability of the assay method was established by parallel determination and by standard-addition method (R.S.D. = 4.7%, recoveries = 94-108%).