title: Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay.
abstract: The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 μl of a 0.1-pg/ml dilution of the probe (2.6 × 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 μl 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).
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, M5B 2K3, Canada.
authors.forename: Angelique
authors.initials: A
authors.lastname: Florentinus-Mefailoski
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, M5B 2K3, Canada. Electronic address: 4marshal@ryerson.ca.
authors.forename: John G
authors.initials: JG
authors.lastname: Marshall
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008297:Male; D015335:Molecular Probes / Q000032:analysis* / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019809:Streptavidin / Q000032:analysis / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D019809:Streptavidin; D000469:Alkaline Phosphatase; D017430:Prostate-Specific Antigen
keywords: AMP; AP–SA; Antibody; ELIMSA; ELISA; LC–MS; PSA