Development of a novel dual fluorescent ELISA (FELISA) for the simultaneous quantification of C3 and C3a in biological fluids

Le résumé fourni par la source

Enzyme linked immunosorbent assay (ELISA), enable rapid quantification of specific proteins within biological samples. ‘Sandwich' ELISAs utilise a pair of antibodies in combination to detect specific proteins. A ‘capture antibody' is used to bind the target protein in the sample and, after washing, a second‚ ‘detection antibody', specific for a different epitope in the target protein is applied. The detection antibody can be directly HRP-tagged or detected with an anti-IgG-HRP. Bound HRP is measured by adding an appropriate chromogenic substrate. In a fluorescence ELISA (FELISA) the detection antibody is fluorophore conjugated, providing greater dynamic range and sensitivity, thus beneficial for high-throughput applications. FELISA offer a further advantage, facilitating the detection of more than one analyte simultaneously by using different detection antibodies labelled with different fluorophores. Here we describe a FELISA that simultaneously measures intact C3 and the C3a fragment in biological samples. Black 96 well MaxiSorp plates were coated with a monoclonal anti-C3/C3a antibody, blocked, washed and sample or standards (C3a or intact C3) added in duplicate. After incubation and washing, monoclonal anti-C3a labelled with Alexafluor-488 and monoclonal anti-C3 (β-chain) labelled with Alexafluor-647 were added simultaneously, plates incubated, washed and fluorescence measured in a BMG Clariostar platereader. Concentrations of intact C3 and C3a concentrations in samples were automatically read from the relevant standard curve and C3a:C3 ratios automatically calculated. For both analytes, the FELISA showed improved sensitivity and increased working range compared to separate in-house ELISAs. Values obtained for C3a and intact C3 in plasma and serum samples using the FELISA closely matched those from the ELISA. Time taken to perform the assay was reduced from 8 to 3 h. Elevated C3a levels and C3a/C3 ratios were apparent in serum compared to plasma in matched samples. The increased sensitivity enabled quantification of C3a and C3a/C3 ratios in cerebrospinal fluid. We report a dual fluorescent ELISA (FELISA) enabling the simultaneous quantification of C3 and C3a and the C3a/C3 ratio in biological samples. This assay is readily adaptable to measurement of other C3 fragments or multiplexing for measurement of other complement components and activation products.

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