Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay

The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (similar to 267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 mu l of a 0.1-pg/ml dilution of the probe (2.6 x 10(-22) mol). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m/z (M + H) with liquid chromatography, electrospray ionization, and quadrupole mass spectrometry (LC-ESI-MS). The safe limit of detection and quantification of the AP-SA probe was approximately 0.5 pg/well or 5 pg/ml. Enzyme-linked immuno mass spectrometric assay (ELIMSA) using the AP-SA probe provided a linear signal response for prostate-specific antigen (PSA) against external standards from 1 to 500 pg/ml. The ELIMSA showed a safe limit of detection and quantification at 5 pg PSA/well or 50 pg/ml (false positive detection rate P <= 0.01). Female samples of 100 mu l plasma/well were read against standards and blanks made in normal female plasma, and the lowest sample quantified was approximately 9.8 pg/well or 98 pg/ml. Here ELIMSA was applied to measure PSA in plasma from female, normal male, prostatectomy patient, and cancer patient samples that showed significant differences by analysis of variance (ANOVA). (C) 2016 Published by Elsevier Inc.