Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to His-tagged proteins

The Luminex system is a flow cytometry based tool that permits the simultaneous measurement of many analytes from just a single serum sample. The technology uses microspheres, which are available in different colors and can be coated with different kinds of biomolecules. For the immobilisation of His-tagged proteins, two types of beads can be used: chemically activated carboxylated beads or Penta-His beads, which have antibodies against His-tags on their surface. In this study, we compared carboxylated and Penta-His beads. For carboxylated as compared to Penta-His beads, the non-specific background is lower (Median Fluorescence Intensity; MFI > 250, 0% versus 15%), the specific signal intensity is higher (mean MFI 2860 versus 722) and not dependent on the configuration of the protein. Above all, the protein coupled carboxylated beads are useful over longer periods of time. Therefore, we conclude that for developing a multiplex assay for semi-quantitative measurement of antibody responses against His-tagged proteins the best microspheres to use are the carboxylated ones.