RESUMO
In this study we have optimised the enzyme immunoassay (ELISA) to quantify CD59 antigen in human serum or plasma. The glycosyl-phosphatidylinositol (GPI)-linked form of CD59 is known to complex with serum high-density lipoprotein. For ELISA optimisation, therefore, we investigated the effect of detergents, added to the sample diluent, on the determined values of CD59. Values obtained in the presence of octyl-glucoside (OG) for 20 adults aged 18-35 years and 17 children 1-5 years old were, respectively, 33-119 ng/ml (mean +/- S.D.: 66+/-22 ng/ml) and 37-143 ng/ml (76+/-33 ng/ml). These results were higher than those measured without OG and were in contrast with published results showing absence, or eight to nine times lower levels, of the protein in serum. A known range for serum concentrations of CD59 in healthy individuals will establish an important reference point for clinical work and for the investigation of diseases involving the complement membrane attack complex (MAC) and its regulation.
Assuntos
Antígenos CD59/sangue , Antígenos CD59/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Glucosídeos/química , Glicosilfosfatidilinositóis/metabolismo , Adolescente , Adulto , Antígenos CD59/química , Antígenos CD59/imunologia , Pré-Escolar , Glicosilfosfatidilinositóis/química , Humanos , Lactente , Octoxinol , Polietilenoglicóis/químicaRESUMO
The number of CR1 on podocytes is reduced in nephropathies with severe glomerular damage, especially in the diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE). Reduction of CR1 number on erythrocytes is due to proteolysis of CR1 by macrophage proteases activated by the reaction of their complement receptors, which leaves a 'CR1 stump peptide' on the erythrocyte. In the present study, we demonstrated the presence of the terminal complement complex (TCC) and the CR1 stump in histological sections of biopsies from patients with SLE by the indirect immunoperoxidase technique. Less severe glomerular lesions presented TCC deposits mainly in the mesangium (mesangial pattern). In lupus nephritis, with more severe glomerular damage, TCC deposits were detected both in the mesangium and in the capillary loops with podocyte involvement (mixed pattern). Patients with highly active DPGN presented a marked reduction of intact podocyte CR1 receptors in association with increased reactivity to the anti-CR1 stump antibody and with glomerular TCC deposits of mixed histological pattern. These results suggest that the decrease in the number of podocyte CR1 receptors in severe glomerular lesions of SLE may be due to a local proteolytic activity associated with activation and deposition of TCC.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/análise , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Peptídeos/análise , Receptores de Complemento 3b/análise , Autoantígenos/análise , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Humanos , Coloração e RotulagemRESUMO
Previous studies of erythrocyte CR1 levels in systemic lupus erythematosus (SLE) and other diseases with in vivo complement activation have led to the conclusion that CR1 levels fall because of loss of CR1 from erythrocytes by proteolysis--predominantly in the liver. In order to measure the existence of proteolysed CR1 remnants on erythrocytes an antibody was raised to a peptide corresponding to the CR1 sequence between the proximal standard consensus repeat (SCR) and the transmembrane segment. This antipeptide antibody recognizes a neo-antigen found on trypsinized erythrocytes which has been demonstrated to represent the 'CR1-stump'. The anti-'CR1-stump' antiserum detects proteolysed CR1 on the ex vivo erythrocytes of a patient with cold haemolytic antibody disease (CHAD). However, higher affinity antibodies will be needed to make anti-CR1-stump a satisfactory diagnostic reagent.
Assuntos
Anticorpos/imunologia , Eritrócitos/imunologia , Peptídeos/imunologia , Receptores de Complemento/imunologia , Sequência de Aminoácidos , Animais , Epitopos/análise , Testes de Hemaglutinação , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Coelhos , Receptores de Complemento/análise , Receptores de Complemento 3b , Tripsina/farmacologiaRESUMO
Factors B and D as well as the total activity of the alternative pathway of complement activation were measured using a functional assay in sera from 29 patients with sickle cell anaemia and 18 normal controls. Total alternative pathway activity was reduced in the patients compared with controls. In patients with abnormally low total alternative pathway activity factor D levels were normal, whereas factor B levels were significantly depresed to a mean level of about half of normal. Regression analysis in patients also showed a singnificant relation between total alternative pathway activity and factor B levels. A deficiency of factor B is the likely cause of the defect in the complement system in patients with sickle cell anaemia. Such a defect may contribute to the excessive proneness of such patients to severe infection.(AU)