title: Evaluation of enzyme-linked immunosorbent assays for quantitation of eicosanoid mediators of sepsis syndrome.
abstract: Thromboxane, prostacyclin, and the leukotrienes are eicosanoids that have been implicated as mediators of the host inflammatory response to infection and injury. Commercial enzyme-linked immunosorbent assays (ELISA) are being increasingly utilized to quantitate plasma eicosanoid concentrations in both clinical and experimental systemic inflammatory conditions. The objectives of this study were to 1) critically examine the performance characteristics of commercial ELISA kits for thromboxane B2, 6 keto-prostaglandin F1 alpha, leukotriene B4, and leukotrienes C4, D4, and E4; 2) apply the four ELISA kits in obtaining actual eicosanoid plasma values under both baseline and septic conditions; and 3) recommend quality control measures for general use. Although averages of multiple standard curves from individual assays were variable, there was a strong linear regression relationship between the backfit dose and nominal dose for each level of standard. Precision profiles constructed for each assay type defined a working range where the intra-assay coefficient of variation is less than 10%. Backfit doses deviated most from nominal doses at both extremes of the standard curves and this variation is reflected in the higher percentage errors in these regions. Recovery of each eicosanoid analyte was 96-103%. Calculated sensitivities were somewhat higher than the manufacturer's specifications. When applied to actual measurements in human and porcine plasma, the assays yielded values that fell within the working ranges of the standard curves with the exception of leukotriene B4. Thus, the ELISAs examined were reproducible, precise, and accurate within a specific working range for each assay type. However, internal controls were lacking in the commercial kits examined, which made assessment of intra- and inter-assay variation difficult.
authors.affiliation: Department of Surgery, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School at Camden, USA.
authors.forename: J V
authors.initials: JV
authors.lastname: Quinn
authors.forename: S
authors.initials: S
authors.lastname: Bilgrami
authors.forename: G J
authors.initials: GJ
authors.lastname: Seidel
authors.forename: G J
authors.initials: GJ
authors.lastname: Slotman
mesh_terms: D015121:6-Ketoprostaglandin F1 alpha / Q000097:blood; D000328:Adult; D000818:Animals; D015777:Eicosanoids / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D015289:Leukotrienes / Q000097:blood; D008297:Male; D008875:Middle Aged; D011785:Quality Assurance, Health Care; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D018746:Systemic Inflammatory Response Syndrome / Q000097:blood*; D013929:Thromboxane B2 / Q000097:blood
chemical_list: D015777:Eicosanoids; D015289:Leukotrienes; D011933:Reagent Kits, Diagnostic; D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha