Development of a two-color laser fluorescence detector. On-line detection of internal standards and unknowns by...

A two-laser, two-color detector has been developed for the simultaneous detection of naturally occurring and recombinant (internal standards) cytokines within the same biological sample. The internal standards were labeled with Bimane and detected with a 408 nm laser while the natural cytokines were labeled with AlexaFluor633 and detected with a 633 nm laser. The two resulting electropherograms were plotted as overlaid traces and quantification of the natural materials determined by comparison with the standards. Using this system, recovery of all four cytokine standards was greater than 94% in both saline and cytokine-depleted plasma. These recoveries could be achieved with intra- and inter-assay coefficients of variance (c.v.) of less than 4.5 and 5.6, respectively. Application of this system to the examination of clinical samples demonstrated that measurement of the four pro-inflammatory cytokines could distinguish between normals, sub-clinical and clinical inflammation. An advantage of this approach is that direct calculation of unknowns by comparison to identical internal standards can shorten analytical time by eliminating the need for additional standard or calibration runs.