Determination of misfolded albumin in serum

The experiments were designed to test the hypothesis that presence of misfolded albumin in serum can be determined by the albumin-specific flu-PC liposome assay. The assay was similar to that described above with minor modifications. In these experiments, FAF-albumin or albumin in serum was partially denatured under heating conditions or treated with β-mercaptoethanol (BME). In the albumin heat-treatment experiment, an aliquot of 20μl of FAF-albumin solution (0.325 mg in Tris-HCl buffer, 0.01 M, pH 7.4) in a vial was heated to 40C, 60C, or 80C as specified in a thermal controller for 10 min followed by cooling on ice. The 20μl of heat-treated albumin was then added to a glass tube containing 669μl 0.01 M Tris-HCl (pH 7.4, 10 mM CaCl₂) and 9μl of flu-PC UL. Then, an aliquot of 2μl sPLA2 working solution was added to the mixture with agitation. An aliquot of 0.3 ml of the final 0.7 ml assay mixture was transferred to a well of a 96-well microplate in duplicates for assay as described above. Similarly, an aliquot of 10μl serum was mixed with 30μl water in a vial and heated to a desired temperature for 10 min followed by cooling on ice. An aliquot of 9μl of heat-treated serum was applied to a glass tube containing the assay buffer and flu-PC UL prior to the addition of 2μl sPLA2 as described above. An aliquot of 0.3 ml of the final 0.7 ml assay mixture was used for assay in duplicates. In the BME-treatment experiment, an aliquot of 20μl FAF-albumin solution (0.65 mg in Tris-HCl buffer) or 20μl serum was mixed with 1μl of BME (Fisher Scientific) on ice for 1 h. An aliquot of 5 or 10μl of BME-treated albumin or 2.3μl BME-treated serum was applied to the flu-PC liposome assay mixture followed by addition of sPLA2 solution to a final volume of 0.7 ml for duplicate assays, similar to that described above for heat treatment experiments. Untreated albumin or serum was used as control and assayed in parallel.