RESUMO
Determination of creatine kinase isoenzymes by inhibition assay is a useful tool for the diagnosis and monitoring of myocardial infarction. We have established several mouse hybridoma lines secreting monoclonal antibodies with creatine kinase M-subunit inhibitory capacity. One of the monoclonal antibodies (MAK33) inhibits creatine kinase-MM by 80% without influencing the activity of creatine kinase-MB. A combination of two monoclonal antibodies increased the inhibition of creatine kinase MM up to 99.4%. Poly(A) + RNA of hybridoma cells secreting MAK33 was isolated and used for cloning cDNA of both heavy and light chains of this antibody. Full-length cDNA clones were obtained by hybridization with gamma 1 and kappa constant region cDNA probes. The complete nucleotide sequences from the variable regions including signal peptide and part of the 5'-untranslated regions have been determined.
Assuntos
Anticorpos Monoclonais/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Isoenzimas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sequência de Bases , Creatina Quinase/imunologia , DNA/genética , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genéticaRESUMO
Elevated phospholipase A2 activities in serum were measured in patients suffering from acute pancreatitis or various inflammatory diseases. The photometric phospholipase A assay of Hoffmann & Neumann (Klin. Wochenschr. 67 (1989) 106-109) was combined with immunoabsorption by different monoclonal antibodies directed against pancreatic phospholipase A2. Pancreatic phospholipase A2 was purified from human duodenal juice. Monoclonal antibodies were prepared by fusion of spleen cells from immunized mice with P3X63-Ag8-653 myeloma cells. Samples with phospholipase A2 activity were incubated in monoclonal antibody-coated microtitre plates. Phospholipase A2 activities were determined in the monoclonal antibody-treated samples as well as in control samples. The method allows the determination of the fraction of human phospholipase A2 isoenzymes in various biological materials. For pancreatic phospholipase A2 the specific binding capacity was about 60-80%, the unspecific binding was 5-30%. Practically no cross-reactivity was seen with partially purified serum phospholipase A2, with recombinant platelet phospholipase A2, or with the sera of patients with non-pancreatic diseases. In conclusion, the present study confirmed the presence of pancreatic phospholipase A2 in human duodenal juice and in the ascites of necrotizing pancreatitis. However, pancreatic isoenzyme was absent in non-pancreatic inflammatory diseases. Therefore, elevated phospholipase activities in non-pancreatic inflammatory diseases cannot be attributed to the pancreas.