Characterisation of Liver membrane autoantibodies determined by indirect immunofluorescence (original) (raw)
Related papers
Clinical and Experimental Immunology, 1984
The reactivity of sera in liver diseases was investigated by an immunoradiometric assay for antibody to liver membrane antigens (LMAg). Serum reactivity was similar using glutaraldehyde treated hepatocytes from man and several animals, but the reactivity with human hepatocellular carcinoma cell lines was weak and irregular. F(ab)2 fragments from reactive sera were used in competitive inhibition experiments to ascertain whether anti-LMAg from different liver diseases reacted with the same or different antigenic determinants of the liver membrane. Sera from different patients with autoimmune CAH reacted predominantly with the same membrane components, as did sera from hepatitis B virus associated CAH (CAH-B); however sera from acute viral hepatitis A did not and, since such sera were reactive to LMAg in the assay, other membrane antigens are presumably involved. Enzymatic treatment of viable hepatocytes was performed to determine the nature of the reactive antigen(s): proteases or periodate resulted in reduced binding ofreactive sera, but neuraminidase did not, suggesting that the LMAg recognized by CAH sera comprises species non-specific membrane glycoproteins. We conclude, on the basis ofquantitative data from a immunoradiometric assay, that antibody to LMAg is more probably reactive than pathogenic, and that assessment of the antigenicity of specific membrane components is necessary.
Journal of Histochemistry & Cytochemistry, 1983
Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients wi...
Gut, 1973
Serum immunoglobulin G, A, and M and serum antinuclear, mitochondrial, and smooth muscle antibody have been measured in 223 patients with hepatitis-associated antigen (HAA)positive and -negative acute and chronic liver disease. In patients with acute hepatitis, chronic persistent hepatitis, and primary liver cell carcinoma, these indices failed to show any significant differences. However, in the group with chronic aggressive hepatitis the patients who were HAA negative had significantly higher levels of serum IgG, much higher titres of smooth muscle antibody, and often antinuclear and mitochondrial antibodies which were not found in the HAA-positive patients from this group. This suggests that different pathogenic mechanisms may be operative in HAA-positive and -negative chronic aggressive hepatitis.
Journal of Clinical Laboratory Analysis, 1987
Liver-kidney-microsomal (LKM) autoantibodies are diagnostic markers for a subgroup of HBsAg-negative chronic active hepatitis, presumably owing to autoimmunity. They were originally detected by indirect immunofluorescence and can now be evaluated by radioimmunoassay, enzyme-linked immunosorbent assay, and immunoblotting. In immunoblotting LKMpositive sera react strongly with a 50-kilodalton (KD) polypeptide band of microsomes. In immunoelectron microscopy, LKM-positive sera show a binding with membranes of the endoplasmic reticulum. The LKM antigen was further identified on various isoenzymes of cytochrome P-450. lmmunofluorescence is still the method of choice for screening sera routinely. However, cytoplasmic antigen-antibody systems often can hardly be distinguished by this method. A specific radioimmunoassay and an enzyme-linked immunosorbent assay can differentiate the various autoantibodies against cytoplasmic antigens. Immunoblotting and immunoelectron microscopy are specific tools for the characterization of the target antigens on an ultrastructural or molecular level. So far they have no use in routine testing of sera. However, since LKM antigen was localized on isozymes of cytochrome P-450, this subgroup of CAH might turn out to be a drug-induced autoimmune liver disease. Clinically, these patients are characterized by chronic active hepatitis or cirrhosis in liver histology, a slight predominance of females, low IgA immunoglobulin levels, and an often rapidly progressing disease. Patients can be treated with immunosuppressive drugs. However, controlled therapeutical trials are missing. Furthermore, an immunogenetic background still has to be proven for this autoimmune liver disease.
Journal of Clinical Laboratory Analysis, 2002
In the diagnosis of autoimmune hepatitis type I (AIH-I), the routine assay of indirect immunofluorescence (IFL), used for the detection of anti-smooth muscle antibodies (ASMAs), has a low predictive value. On the other hand, the enzyme-linked immunosorbent assay (ELISA), which detects anti-cytoskeleton antibodies (ACTAs), presents contradictory results concerning their specific antigenic target. In this study, we first looked for the immunological properties (isotypes and antigenic targets) of autoantibodies in AIH-I and two other control liver diseases: primary biliary cirrhosis (PBC) and viral hepatitis (VH), using ELISA based on cytoskeleton proteins: F-actin, Gactin, myosin, tropomyosin, troponin, desmin, vimentin, keratin, and an extract of HEp-2 carcinoma cells. We also compared the diagnostic value of IFL and ELISA. In contrast to previous studies, we found that actin was not specific for AIH-I. No autoantigen and no antibody class or subclass discriminated AIH-I from the control diseases. IFL is more suitable for AIH-I diagnosis, as 97% of AIH-I sera but only 22% of PBC sera were ASMA-positive. Additionally, 96% of ASMA-positive, and all ASMA-negative sera from all three liver diseases were ACTA-positive. ASMA were mainly IgG, while 450% of ACTA also contained IgA and IgM. These data suggest that ACTAs recognize additional epitopes as compared to ASMAs, and they frequently occur in all liver diseases.
The Lancet, 1987
Autoantibodies against a soluble liver antigen (SLA) were detected in 23 patients with HBsAg-negative chronic active hepatitis (CAH) but not in 502 patients with various other hepatic and nonhepatic disorders or 165 healthy blood donors. Anti-SLApositive serum samples were negative for antinuclear and liver-kidney-microsomal antibodies, markers of two subgroups of autoimmune-type CAH. 6 anti-SLA-positive patients were negative for all autoantibodies sought. Most of the anti-SLA-positive patients were young women (2 men, 21 women, mean age 37 years) with hypergammaglobulinaemia (mean 3·2 g/l, range 1·8-5·3 g/l); 18 of the 23 patients had received immunosuppressive treatment and all responded well. Anti-SLA titres declined during therapy, corresponding to disease activity. Anti-SLA cannot be detected by immunofluorescence. SLA is not organ-specific or species-specific, but the highest concentrations were found in liver and kidney. Anti-SLA autoantibodies characterise a third subgroup of autoimmune-type CAH and will allow a better differentiation of HBsAg-negative CAH which has therapeutic consequences.
Hepatology, 2008
Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n ؍ 65) and from 90 controls, that is, healthy blood donors (n ؍ 40) and patients with systemic diseases (n ؍ 20) or other liver diseases (n ؍ 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. Conclusion: Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1. (HEPATOLOGY 2008;47:937-948.) See Editorial on Page 786 Abbreviations: AIH, autoimmune hepatitis; ASGPR, asialoglycoprotein receptor; CK, cytokeratin; HSP, heat shock protein; LKM1, liver-kidney microsome type 1; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; VCP, valosin-containing protein.