The IgG and IgM antibodies against each phospholipid were measured by an enzyme-linked immunosorbent assay method. Microtiter plates were coated with each type of phospholipid and diluted with methanol to 50 Mg/ml. After drying at 50°C for 30 min and washing 3 times in 0.01 mol/L phosphate-buffered saline (PBS), 50 Mg/ml of 10 percent bovine serum was added to each well and kept at room temperature for 1 h in order to eliminate adhesion nonspecific IgG and IgM. Then 50 ц\ of sera diluted to a 1:100 solution with PBS was placed in each well and incubated at room temperature for 1 h and washed 3 times in PBS. Next, 50 д1 each of peroxidase-labeled antihuman IgG antibodies and IgM antibodies was diluted to a 1:100 solution with PBS. Fifty microliters of Нг02-0-рЬепу1епе diamine was added as a substrate to the wells and incubated at room temperature for 15 min, followed by the addition of 50 д1 of IN H2SO4 to each well to stop the reaction.
The optical absorbance was read at 492 nm by a microplate reader. As a control, the serum obtained from the pooled blood of ten healthy adults was used. Canadian neighbor pharmacy read more To determine the cutoff index, the optical absorbance of individual sera from 70 healthy volunteers who were free from all diseases and age- and sex-matched with patients with sarcoidosis was measured. Each cutoff index of the 70 sera was derived by dividing the optical absorbance of the individual sera by that of the control serum. From these values, the mean±3 SD values were considered as the upper limits of the normal values. Using this method, the cutoff indices exceeding the upper limit of the normal values were considered positive. A class of antibodies was considered IgG antibody-positive or IgM antibody-positive when either was positive to one of the five types of phospholipids. For statistical analysis, x2 tests were used for independency.