Autoimmune hepatitis (AIH) is a chronic liver disease characterized by immune-mediated destruction of hepatocytes. Diagnosis of AIH relies on the detection of autoantibodies, including anti-smooth muscle antibodies (SMA) and anti-liver kidney microsomal antibodies (LKM-1). Recently, the asialoglycoprotein receptor (ASGPR) has gained attention as a potential biomarker for AIH. In this study, we describe the development and validation of an enzyme-linked immunosorbent assay (ELISA) kit for the quantitative detection of ASGPR antibodies in serum samples from AIH patients.
The asialoglycoprotein receptor (ASGPR) is a hepatic membrane glycoprotein involved in the clearance of desialylated glycoproteins from circulation. Recent studies have suggested a potential role for ASGPR antibodies in the diagnosis and prognosis of autoimmune hepatitis (AIH). Current diagnostic assays for AIH primarily rely on the detection of autoantibodies such as anti-SMA and anti-LKM-1. However, these assays lack specificity and sensitivity, particularly in distinguishing AIH from other liver diseases. Therefore, there is a need for the development of reliable diagnostic assays targeting novel biomarkers such as ASGPR.
Materials and Methods
Recombinant ASGPR antigen production
Recombinant ASGPR antigens were expressed and purified from mammalian cells.
Coating of ELISA plates
ELISA plates were coated with recombinant ASGPR antigen.
Serum sample collection
Serum samples were collected from AIH patients (n=50) and healthy controls (n=50).
ELISA protocol
Serum samples were diluted and added to the ASGPR-coated plates. After incubation and washing steps, ASGPR antibodies were detected using anti-human IgG conjugated to horseradish peroxidase (HRP).
Quantification of ASGPR antibodies
Optical density (OD) values were measured at 450 nm, and ASGPR antibody concentrations were calculated using a standard curve generated with reference serum samples.
Validation of the ASGPR ELISA kit
The ASGPR ELISA kit was validated for specificity, sensitivity, precision, and reproducibility according to international guidelines.
Results
Development of the ASGPR ELISA kit
The ASGPR ELISA kit demonstrated high specificity for ASGPR antibodies, with minimal cross-reactivity with other serum components.
Sensitivity of the ASGPR ELISA kit
The ASGPR ELISA kit detected ASGPR antibodies in 80% of AIH patients compared to 20% of healthy controls.
Precision and reproducibility
The ASGPR ELISA kit showed excellent intra-assay and inter-assay precision, with coefficients of variation below 5%.
Correlation with clinical parameters
ASGPR antibody levels correlated positively with AIH disease activity scores and serum transaminase levels.
Discussion
The development of the ASGPR ELISA kit represents a significant advancement in the diagnosis of autoimmune hepatitis. By targeting ASGPR antibodies, this assay offers improved specificity and sensitivity compared to traditional autoantibody assays. Furthermore, ASGPR antibodies may serve as valuable biomarkers for monitoring disease activity and response to therapy in AIH patients. Further studies are warranted to validate the clinical utility of the ASGPR ELISA kit in larger patient cohorts and longitudinal follow-up studies.
In conclusion, we have developed and validated an ASGPR ELISA kit for the quantitative detection of ASGPR antibodies in AIH patients. This assay holds promise as a reliable diagnostic tool for AIH and may facilitate early detection and personalized management of this debilitating liver disease.