Purified dimeric S100 proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis and their metal-binding and immunologic properties. The exact masses of S100 proteins were determined by electrospray ionization mass spectrometry (SCIEX API

exchange chromatography (1, 3). Purified dimeric S100 proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their metal-binding and immunologic properties. The exact masses of S100 proteins were determined by electrospray ionization mass spectrometry (SCIEX API 365; Perkin-Elmer). Native proteins (in contrast to the recombinant proteins) were found to be mostly acetylated with no other posttranslational modifications. Human S100A8 and S100A9 were purchased from Bachem AG. The protocol of the supplier was followed except that the Sangtec 100 IRMA calibrators (containing lyophilized partly purified bovine S100 peptides) were replaced by the same concentration (0.5–60 g/L) of human recombinant S100 proteins. The reagent blank (negative control) contained no protein calibrators. As illustrated in Fig. 1, the assay recognized the human S100B protein equally well and in the same concentration range as the bovine S100B calibrator. The Sangtec IRMA was linear over a range of 0–20 g/L; the same linearity was obtained with identical concentrations of the recombinant human S100B. To test the specificity of this assay, we replaced the bovine S100B calibrator with the various human recombinant S100 proteins in concentrations of 10–30 g/L. There was no interference/cross-reactivity by the other S100 proteins tested, including S100A6 and S100A4, which are highly expressed in brain tissue. According to these results this diagnostic assay is specific and reliable for measurement of S100B in human body fluids.