The optimisation of a murine TNF-alpha ELISA and the application of the method to other murine cytokines.

Abstract

Cytokines occur in biological systems at low levels of concentration, therefore assays developed to measure them must be very sensitive. Enzyme linked immunosorbent assays (ELISA's) developed using manufacturers recommended end points can detect cytokines to picogram levels but the lower parts of their standard curves can be unreliable. In this study the relative merits of different substrate systems - 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2 forms of tetramethyl benzidine (TMB), were investigated with regard to assay sensitivity. Further, a signal amplification method involving biotinylated tyramine has been used to increase the absorbance signal and thus the assay sensitivity and to achieve a sigmoidal standard curve. The amplified assay approach has been applied successfully to achieve more sensitive detection of TNF-alpha and improve the sensitivity of assays for a wide range of other cytokines. The optimised amplification method is the same for all the cytokine ELISA's performed in this work and this enables them to be performed