Sequential ELISA to profile multiple cytokines from small volumes.

Due to the low cost and relative effectiveness the enzyme-linked immunosorbent assay (ELISA) is widely used to measure the concentration of inflammatory cytokines in plasma and other sources. Blood volume represents a limiting factor in those mouse models requiring repeated sample collection at multiple time intervals to monitor the trajectory of inflammatory processes. The small blood volumes in such scenarios restrict the array of cytokines that can be measured using the traditional ELISA. The implementation of the sequential ELISA protocol presented here can dramatically increase the number of measured cytokines, since the plasma samples are not discarded after the initial assay but re-used to measure additional selected inflammatory proteins in consecutive tests. From the original 20 mul of blood volume collected, up to fifteen cytokines can be successfully assayed in five consecutive cycles. With more unstable cytokines analyzed in the initial cycles, no inter-assay interference and/or deterioration of samples occurs. The sequential ELISA technique based on commercially-available antibody pairs can be an attractive alternative to more advanced, costly methods. Given the simplistic validation procedure, the proposed sequential ELISA protocol has a wide potential for further modifications to include other inflammation-related targets.