Detection of sPLA₂ in human synovial fluid.

(A) Measurement of human synovial fluid sPLA₂ enzyme activity. Assays to measure human synovial fluid sPLA₂enzyme activity were performed as described in “Material and methods” using a 96-well microtiter plate. Enzyme activity was monitored for 15 minutes at 25°C by measuring absorbance at 414 nm. The background absorbance in the absence of added human synovial fluid was subtracted from each sample. Bee venom PLA₂ (0.01 μg) was included as a positive control. Data points represent the mean ± SE from triplicate determinations of a representative experiment. HSF indicates human synovial fluid. (B) Western blotting of human synovial fluid sPLA₂. Aliquots of human synovial fluid (15 μL/lane corresponding to 82.5 μg of total protein content) or purified human synovial sPLA₂ (1 μg/lane) were fractionated on a 16% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was probed with a monoclonal antisynovial sPLA₂ antibody (diluted 1:1000). Antibody reactions were detected using horseradish peroxidase–conjugated sheep antimouse Ig (diluted 1:2500) and the enhanced chemiluminescence detection system. Shown on the left are molecular weight markers expressed in kilodaltons. The arrow on the right indicates the 14-kd sPLA₂. Lane 1 is human synovial fluid. Lane 2 is human synovial sPLA₂ that was used as a positive control.