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

Cytokines occur in biological systems at low levels of concentration, there fore assays developed to measure them must be very sensitive. Enzyme linked immunosorbent assays (ELISA's) developed using manufacturers recommended e nd points can detect cytokines to picogram levels but the lower parts of th eir standard curves can be unreliable. In this study the relative merits of different substrate systems - 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfo nic acid) (ABTS) and 2 forms of tetramethyl benzidine (TMB), were investiga ted with regard to assay sensitivity. Further, a signal amplification metho d involving biotinylated tyramine has been used to increase the absorbance signal and thus the assay sensitivity and to achieve a sigmoidal standard c urve. The amplified assay approach has been applied successfully to achieve more sensitive detection of TNF-alpha and improve the sensitivity of assay s for a wide range of other cytokines. The optimised amplification method i s the same for all the cytokine ELISA's performed in this work and this ena bles them to be performed simultaneously to allow multiple cytokine analysi s of one sample.