Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2.
The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology*; D010740:Phospholipases / Q000276:immunology*; D010741:Phospholipases A / Q000276:immunology*; D054467:Phospholipases A2; D051381:Rats
D000911:Antibodies, Monoclonal; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2