A multiplexed and miniaturized serological tuberculosis assay identifies antigens that discriminate maximally between TB and non-TB sera.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Use of horseradish peroxidase- and fluorescein-modified cisplatin derivatives for simultaneous labeling of nucleic acids and proteins.

BACKGROUND: Microarray platforms will change immunochemical and nucleic acid-based analysis of cell homogenates and body fluids compared with classic analyses. Microarrays use labeled target and immobilized probes, rather than fixed targets and labeled probes. We describe a method for simultaneous labeling of nucleic acids and proteins. METHODS: Horseradish peroxidase- and fluorescein-modified cisplatin derivatives were used for labeling of nucleic acids and proteins. These reagents, called the Universal Linkage System (ULS), bind to sulfur- and nitrogen-donor ligands present in amino acids and nucleotides. For automated screening of proteins and nucleic acids on microarrays, it is advantageous to label these biomolecules without pre- or postpurification procedures. The labeling of antibodies and nucleic acids in whole serum was therefore pursued. RESULTS: Immunoglobulins in nonpurified serum were labeled efficiently enough to be used for immunochemistry. To investigate whether protein-adapted labeling allowed nucleic acid labeling as well, 1 microg of plasmid DNA was added to 1 microL of serum. DNA and serum proteins were simultaneously labeled, and this labeled DNA could be used as a probe for direct fluorescence in situ hybridization. CONCLUSION: ULS provides a direct labeling tool for the (simultaneous) modification of proteins and nucleic acids even in unpurified samples.