The ECL assays for FRA, MSLN, and MPF used in the present analyses have been described previously.43 (link),44 Samples (serum, urine) from healthy or diseased subjects and standards were added to wells of 96-well plates previously coated with marker specific capture monoclonal antibody (MAb) and incubated at room temperature for two hours. The ruthenium labeled detection MAbs were diluted in assay buffer, added to washed plates, and incubated for an additional two hours at room temperature. Plates were washed, read buffer added, and signals measured using an MSD DISCOVERY WORKBENCH® (Mesoscale Discovery, Gaithersburg, MD). Optimal sample dilutions were: FRA (80-fold dilution of urine and a 20-fold dilution of serum), MSLN (60-fold dilution of urine and an 80-fold dilution of serum), and MPF (4-fold dilution of urine and a 20-fold dilution of serum).
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ECL Assays for FRA, MSLN, and MPF
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Other organizations : Morphotek (United States)
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Variable analysis
independent variables
- None explicitly mentioned
- Levels of FRA, MSLN, and MPF
- Sample dilutions: FRA (80-fold dilution of urine and a 20-fold dilution of serum), MSLN (60-fold dilution of urine and an 80-fold dilution of serum), and MPF (4-fold dilution of urine and a 20-fold dilution of serum)
- Incubation time: 2 hours at room temperature for both capture and detection antibodies
- Plates: 96-well plates previously coated with marker-specific capture monoclonal antibody (MAb)
- Detection: Ruthenium-labeled detection MAbs diluted in assay buffer
- Positive control: Standards (not explicitly mentioned what these standards represent)
- Negative control: Samples from healthy subjects (in addition to diseased subjects)
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