RESUMEN
An immunoassay for IVA phospholipase A2 in human red blood cells is described. The assay is a non-competitive sandwich assay in which increasing amounts of the measured protein produce increased luminescence. The antibodies used in the assay are directed against two unique epitopes of the molecule, which sequentially trap and detect the protein. The standard curve covers the range 0.7ng to 23ng/mL (0.07 to 2.3ng/well). The intra-assay and inter-assay coefficients of variation were 9% and 12%, respectively. Evidence is presented that the assay is specific for the alpha paralog of IV PLA2. The assay allows simple and rapid quantification of IVAPLA2 in red blood cell lysates and other biological fluids.
Asunto(s)
Epítopos/metabolismo , Eritrocitos/enzimología , Fosfolipasas A2 Grupo IV/química , Fosfolipasas A2 Grupo IV/metabolismo , Adulto , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Sf9 , Spodoptera , Adulto JovenRESUMEN
Two studies of the behaviour of cytosolic phospholipase A(2) (cPLA(2)) in the red blood cell (RBC), as measured by ELISA, are described. In the first study we show a significant increase in cPLA(2) in patients with schizophrenia compared to controls and suggest that this measure, if corroborated, could be used as a diagnostic marker. In a second study we found that washing the RBC introduced an unknown confounding variable which led us to reject this study. A subsequent investigation of washing red cells showed that the washing effect may be due to a plasma factor likely to be more than 5kDa MW which can be removed from red cells by washing with buffers. When the cells are washed, the concentration of cPLA(2) in the red cell, as measured by ELISA, significantly increases. We advise against washing the red cell in any study that involves measuring cPLA(2) by ELISA.
Asunto(s)
Citosol/enzimología , Fosfolipasas A/metabolismo , Esquizofrenia/enzimología , Artefactos , Tampones (Química) , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Eritrocitos , Humanos , Fosfolipasas A/sangre , Fosfolipasas A2 , Esquizofrenia/sangreRESUMEN
Phospholipase A(2) type IVA (IVAPLA(2)) is a cytosolic enzyme that on activation selectively releases arachidonic acid (AA) from cell membrane phospholipids. Both AA and lysophospholipid, products of the enzymic reaction, can function as signal transducers in cellular interactions. The enzyme is present in most cells, including polymorphs, eosinophils, and platelets. This study used affinity purification to extract IVAPLA(2) from red cell lysate prepared from leukocyte- and platelet-depleted human blood to overcome the masking effect of hemoglobin on Western blot detection. We show that IVAPLA(2) is present in red cells as a 90-kDa protein.