Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques

doi:10.1016/0022-1759(95)00192-X

Journal of Immunological Methods

Volume 187, Issue 2, 1 December 1995, Pages 259-264

https://doi.org/10.1016/0022-1759(95)00192-X Get rights and content

Abstract

Liver cytosol specific antibody type 1 (anti-LC1) was first described in a proportion of patients with liver/kidney microsomal antibody type 1 (anti-LKM1)-positive autoimmune hepatitis (AIH) and is routinely evaluated by immunodiffusion (ID). Using human liver cytosol as the source of antigen, we have used ID, counterimmunoelectrophoresis (CIE) and immunoblotting (IB), to test sera from 167 patients with documented chronic liver diseases of different etiology. 15 patients had antinuclear antibody (ANA) and/or smooth muscle antibody (SMA)-positive AIH, 13 had anti-LKM1-positive AIH, four had ANA/SMA/anti-LKM1-negative AIH, 76 had anti-LKM1-positive hepatitis C (recently renamed unclassified chronic hepatitis-UCH), 40 had chronic hepatitis C, 15 had chronic hepatitis B, and 4 had chronic hepatitis D. A precipitin line of identity with an anti-LC1 reference serum was detected both by ID and CIE in 16 patients: six with anti-LKM1-positive ‘definite’ AIH, four with ANA/SMA/anti-LKM1-negative ‘definite’ AIH, and six with anti-LKM1-positive UCH. By IB, 14 out of the 16 anti-LC1-positive sera (87.5%) reacted with a 58 kDa human liver cytosolic polypeptide, whereas three out of 16 (19%) recognised an additional 60 kDa band. Compared to ID, CIE is more economical in terms of both time and reagents and provides more clear-cut results. The 58 kDa reactivity by IB was detectable in nearly all CIE/ID anti-LC1-positive patients, was not found among CIE/ID anti-LC1-negative patients. In conclusion, CIE is the ideal screening test for the detection of anti-LC1, an autoantibody that can be regarded as an additional serological marker of AIH and is especially useful in ANA/SMA/anti-LKM1 negative cases.

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Citation Excerpt :
As a result, in cases of anti-LC1 coexistence with anti-LKM1 antibodies, IIF fails to detect them because anti-LKM1 stains the cytoplasm of hepatocytes in the entire liver lobule (Fig. 5B). Therefore, additional methods like immunodiffusion, ELISA or immunoblot are required [54–56]. The formiminotransferase cyclodeaminase, a liver enzyme involved in folate metabolism, has already been documented as the molecular target of anti-LC1 [57].
Diagnosis of autoimmune hepatitis (AIH) is in most cases challenging for clinicians as there is not a single specific laboratory or histological marker to diagnose or exclude the presence of the disease. The clinical spectrum of AIH varies from completely asymptomatic to acute-severe or even rarely fulminant hepatic failure, while everybody can be affected irrespective of age, gender, and ethnicity. The old revised and the newer simplified diagnostic scores have been established by the International Autoimmune Hepatitis Group (IAIHG) in 1999 and 2008, respectively, which are based on several clinical, laboratory and histological parameters. Additionally, a thorough differential diagnosis from other diseases mimicking AIH is absolutely indicated. In this context, autoantibodies detection in patients with suspected AIH is mandatory -even though not pathognomonic- not only for AIH diagnosis but furthermore, for AIH classification (AIH-type 1 and AIH-type 2). Although autoimmune serology can be supportive of AIH diagnosis in ≥95% of cases if testing has been performed according to the IAIHG guidelines, this is not the case under real-life circumstances in routine clinical laboratories. Clinicians should be careful both for the importance of the required testing and how to interpret the results and therefore, they should communicate and discuss with the laboratory personnel to achieve the maximum benefit for the patient. Herein, a detailed and updated review of the diagnostic work-up for AIH diagnosis under real-life conditions is given to minimize the underestimation and misdiagnosis of AIH which can result in progression of the disease and unfavourable outcomes.
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This chapter describes perturbations in the signaling, apoptotic, and regulatory pathways within the innate and adaptive immune systems that favor the development of autoimmune hepatitis. Molecular mimicry, neoantigens, epitope spread, and promiscuous targeting by activated lymphocytes are discussed, and the genetic predispositions implicated in the occurrence and severity of autoimmune hepatitis are presented. Cytokine pathways that support a proinflammatory response and the chemokines that influence the trafficking of immune cells are reviewed. Receptor-mediated and mitochondrial-induced apoptosis are identified as principal mechanisms of hepatocyte loss, and apoptotic bodies that are incorporated into self-amplification loops strengthen the adaptive immune response, activate hepatic stellate cells, and increase the production of reactive oxygen species. Dendritic cells, natural killer cells, natural killer T lymphocytes, gamma delta lymphocytes, and regulatory T lymphocytes are identified as key cell mediators, and the pathogenic mechanisms are interwoven into a counter-regulatory network that affords opportunities for site-specific therapeutic interventions.
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Patients with autoimmune hepatitis (AIH) can present with symptoms ranging from those that are insidious and nonspecific to acute hepatitis with jaundice. However, some patients have no symptoms at diagnosis and are identified incidentally. We investigated disease progression and outcomes of these 2 groups of patients.
We performed a retrospective study to compare clinical, immunologic, and histologic features and outcomes of patients with asymptomatic vs symptomatic AIH. We analyzed data collected from 305 patients (90 asymptomatic and 215 with symptoms), diagnosed with AIH from 1994 and 2013, at the Center for the Study and Treatment of the Autoimmune Diseases of the Liver and Biliary System in Bologna, Italy.
At diagnosis, patients with asymptomatic AIH had significantly lower mean levels of alanine aminotransferase (7.0- ± 8.0-fold the upper limit of normal) than patients with symptomatic disease (23.0- ± 18.0-fold the upper limit of normal; P < .001), and lower mean levels of bilirubin (1.4 ± 1.4 mg/dL vs 8.6 ± 10.4 mg/dL; P < .001). Asymptomatic patients also had significantly lower histologic grades (7.0 ± 2.5) than symptomatic patients (9.0 ± 2.9; P < .001). However, larger proportions of asymptomatic patients had anti–liver/kidney microsomal antibodies type 1 (26.8% vs 13.1%; P < .006), and associated autoimmune thyroid (26.7% vs 12.6%; P = .003) or skin (8.9% vs 2.3%; P = .010) disorders. Age at onset, sex, response to therapy, disease progression, genetic factors, and other autoantibody markers did not differ between patients with asymptomatic vs symptomatic disease.
Patients with asymptomatic vs symptomatic AIH have similar courses of disease progression and responses to immunosuppressive agents, and therefore should receive the same treatment. Patients affected by thyroid or dermatologic autoimmune disorders are at increased risk of developing subclinical liver disease, and should be assessed routinely for AIH.
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Citation Excerpt :
It stains the cytoplasm of liver cells with relative sparing of the centrilobular area (Fig. 5), but is usually obscured by the concurrent presence of anti-LKM1 [2]. In the presence of anti-LKM1, anti-LC1 can be detected by the use of liver cytosol in double-dimension immunodiffusion, or counterimmunoelectrophoresis, and a positive reference serum [54]. In Western blot, anti-LC1 reacts with a 58–60 kDa protein when human liver cytosolic fraction is used as a substrate.
The accurate diagnosis and classification of autoimmune hepatitis (AIH) rely upon the detection of characteristic autoantibodies. Positivity for anti-nuclear (ANA) and/or anti-smooth muscle (SMA) autoantibodies defines AIH type 1 (AIH-1), whereas anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) define AIH type 2 (AIH-2). ANA and SMA, and less commonly anti-LKM1, have also been detected in de-novo autoimmune hepatitis developing after liver transplantation, a condition that may affect patients transplanted for non-autoimmune liver disease. The diagnostic autoantibodies associated with AIH-1 are also detected in the paediatric AIH/sclerosing cholangitis overlap syndrome, referred to as autoimmune sclerosing cholangitis (ASC). ASC, like adult primary sclerosing cholangitis, is often associated with atypical perinuclear anti-neutrophil cytoplasmic autoantibodies (p-ANCA), although p-ANCA are also detected in other autoimmune liver diseases. These associations highlight the necessity for simple and prompt diagnostic autoantibody testing, and the requirement for the accurate interpretation of the results of the tests in the clinical context. Fine-mapping of antigenic autoantibody targets has facilitated the development of rapid molecular assays that have the potential to revolutionise the field if properly standardised and when used in combination with classical immunofluorescence. Despite their diagnostic significance, the pathogenic role of the various autoantibodies and the mechanisms by which they can potentially inflict damage onto the liver cell remain a topic for further research.
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Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with varying major involvement of diverse organs and tissues. This chapter considers the issues raised by the nosologic problems of autoimmune hepatitis with features of SLE and liver lesions that occur in classical SLE. It opens with a brief discussion of the problems of disease nomenclature, particularly with those diseases in which autoimmunity is prominent among multifactorial components. Serologically, there are commonalities in SLE and autoimmune hepatitis (AIH), namely the presence of antibodies to chromatin/histones as the major antinuclear antibody (ANA) reactant. However, there are also unique differences, including a strong expression of anti-dsDNA in SLE but only weak or no expression in AIH and the absence in SLE of reactivities that are characteristic of one or another type of AIH. Another commonality is a degree of sharing of immunogenetic determinants including HLA DR3. For AIH and SLE, the differences seem to outweigh the similarities. The study concludes by stating that when there does appear to be a convergence of SLE and AIH, this is usually attributable to multisystemic features that accumulate during the course of an initially occurring AIH, like that seen with other SLE overlaps rather than the reverse wherein a case presenting initially as SLE traverses to AIH.

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Research reportDetection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques

Abstract

Hepatology

J. Immunol. Methods

Lancet

J. Hepatol.

Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis

Hepatology

Anti-liver cytosol antibodies in hepatology: autoimmune hepatitis, viral hepatitis C and graft-versus-host disease

Hepatitis C viraemia in adults with type 2 autoimmune hepatitis

J. Med. Virol.

Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of ‘autoimmune’ hepatitis

Hepatology

Research report
Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques