Preparation of antibodies to phospholipases A2

Publisher Summary Application of antibodies provides useful information to gain an understanding of the physiological functions, distribution, dynamics, and structural characteristics of phospholipases A 2 . Such antibodies are also useful for identification and purification of the enzymes. This chapter describes the preparation and possible application of antibodies to mammalian group II phospholipases A 2 obtained from both rat platelets and human rheumatoid synovial fluid. Phospholipase A 2 in the fluid is purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW gel filtration [high-performance liquid chromatography (HPLC)]. Phospholipase A 2 is purified to near homogeneity from the synovial fluid of patients with rheumatoid arthritis by the sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl (Tosoh), and reversed-phase HPLC. Immunoaffinity-purified phospholipase A 2 is used as an immunogen for raising antibodies in Japanese albino rabbits. Intact or denatured (treated with 2-ME for 10 min at 100°) phospholipase A 2 is injected subcutaneously into the footpads. Animals are bled 1 week after each booster injection and the titer of antiplatelet phospholipase A 2 antiserum is examined by enzyme-linked immunosorbent assay (ELISA).