Determination of tumor necrosis factor-alpha (TNF-alpha) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay.
OBJECTIVE
To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-alpha (TNF-alpha).


METHODS
The method is based on the use of two monoclonal antibodies against TNF-alpha, one "capture" antibody and one labeled with biotin, in a "sandwich type" assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves phosphate from diflunisal phosphate (DIFP) to produce diflunisal (DIF). The detection system is based on the combination of enzymatic amplification introduced by ALP and the formation of a highly fluorescent terbium complex that can be monitored by time resolved or conventional fluorimetry.


RESULTS
By using 50 microL of sample, the dynamic range of the assay extends up to 2000 ng/L of TNF-alpha, with a detection limit of 1 ng/L, within-run CVs ranging from 3 to 15% and recoveries of 97 +/- 2%. By using 100 microL of sample the dynamic range of the assay extends up to 1000 ng/L of TNF-alpha with a detection limit of 0.2 ng/L, recoveries of 94 +/- 13%, within-run CVs ranging from 2 to 6.5% and between-run CVs ranging from 5 to 15%, in a total incubation time of 3h. No interference by the presence of other cytokines (IL-1beta IL-2, IL-4, IL-6, IFN-gamma) or by rheumatoid factors has been observed. The results obtained by the proposed method and by a commercially available kit (Medgenix TNF-alpha EASIA) correlated well (n = 26, r = 0.934).


CONCLUSIONS
The proposed method is highly sensitive, simple and rapid and can reliably measure TNF-alpha in the ng range in biological specimens.
Petrovas
C
C
Department of Chemistry, University of Athens, Greece.
Daskas
S M
SM
Lianidou
E S
ES
D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008163:Luminescent Measurements; D008674:Metals, Rare Earth / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D008674:Metals, Rare Earth; D014409:Tumor Necrosis Factor-alpha