B6VitaStat represents a microarray assay for the rapid detection of the vitamin B6 status from blood. In comparison to established methods, B6VitaStat directly monitors PLP bound to proteins in erythrocytes. Advantages comprise the speed, ease and reliability of this technology which is less prone to fluctuations e.g. due to nutrition uptake. The methodological workflow starts with the isolation of erythrocytes from blood, which are lysed and incubated with a chemical probe mimicking PLP. The probe binds to unoccupied PLP binding sites facilitating a fluorescence readout which indirectly reports the PLP status. The inverse correlation of PLP saturation and probe signal was shown via mass spectrometry analysis of intact proteins using recombinantly expressed PLP proteins. These results were successfully transferred to a microarray format. The development of the array included the determination of a suitable PLP probe and appropriate PLP dependent enzymes in erythrocytes, the selection of suitable antibodies for immobilization on glass slides and the optimization of signal-to-noise ratios. Further the sample preparation workflow was carefully optimized, especially with regard to erythrocyte isolation and depletion of the high abundant protein hemoglobin in erythrocytes to avoid signal suppression of the probe. Fine-tuning of all parameters contributed to the successful development of an assay format reporting the PLP status based on the three PLP dependent enzymes PLPBP, SHMT1 and OAT. The extention and implementation of the invented assay in a point of care device has great potential to facilitate an easy monitoring, including third world countries and a correlation of the received results with severe global issues of diseases and malnutrition.
