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New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10(-6)). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

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Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum.

We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.

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