Two sensitive time-resolved fluoroimmunoassays for cellular fibronectin.
Two sensitive sandwich-type immunoassays for determination of cellular fibronectin (cFN) in cell culture supernatants and in human plasma were developed. Both assays used a monoclonal antibody with specificity against the EDA sequence, which is characteristic for the cellular form of human and rat FN. Assay 1 involves binding of FN on gelatin-coated microwells followed by reaction with the anti-cFN antibody, whereas in assay 2 the anti-cFN antibody was immobilized first and detection was with an anti-FN antiserum. Time-resolved fluorescence spectrophotometry with measurement of an Eu3+ chelator after dissociation of the solid-phase complexes with urea/sodium dodecyl sulfate in the presence of excess Eu3+ was the detection method. The detection limit of the new assays was between 2.6 and 4.0 micrograms/L cFN. In serial dilution of human plasma samples, parallelism with the calibration curve was obtained over the whole measuring range (12-1000 micrograms/L) with assay 2, whereas assay 1 had deviations of the dilution curves at concentrations > or = 100 micrograms/L. The between-run CVs for assays 1 and 2 were 11.4% and 7.2%, respectively, at a concentration of 200 micrograms/L (median value of 18 experiments). Respective within-series CVs of 4.3% and 4.7% were obtained at the same concentration. The recovery of added cFN from human plasma was between 90% and 96%.
Kropf
J
J
Abt. Klinische Chemie und Zentrallaboratorium, Philipps-Universität, Marburg, Germany.
Gressner
A M
AM
D000818:Animals; D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D005063:Europium; D005353:Fibronectins / Q000032:analysis*; D015200:Fluoroimmunoassay / Q000706:statistics & numerical data; D006801:Humans; D051381:Rats; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D005353:Fibronectins; D005063:Europium