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Objective To diagnose the process of hepatic injury,a method of quantitating the concentration of sH2a subunit from asialoglycoprotein receptor (ASGPR) in serum by enzyme-linked immunosorbent assay was established and clinical evaluated.Methods 210 serum samples were collected in Suzhou Kowloon Hospital.Among them,70 subjects with cirrhosis,viral hepatitis and fatty liver disease were as hepatic injury group and 140 subjects of healthy and high fat,hemolysis,jaundice and with autoimmune disease were as control group.The serum sH2a of two group were measured by ELISA kit.The results of sH2a ELISA were analyzed by four table chi-square test and SPSS20.0 software.Results sH2a protein level in liver injury group and control group were 105.92+ 53.41 ng/ml and 69.25+27.45 ng/ml,respectively.The difference between the control group and the liver injury group was statistically significant (F=14.375,t=5.397,P=0.000).The sensitivity and specificity of the sH2a ELISA kit were 68.57％ (95％CI:56.37～79.15％) and 82.86％ (95％CI:75.58％～88.70％),and the total compliance rate was 78.10％ (95％CI:71.88％～83.49％) with KAPPA coefficient:0.510 6 (95％ CI:0.3877～0.6336).Conclusion SH2a serum ELISA kit with positive and negative coincidence rate between SH2a serum level and meet the clinical requirement,which could be used as new marker for diagnosis of heptieal injury related diseases.
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Objective To detect the serum anti‐sialic acid glycoprotein receptor antibody (anti‐ASGPR) levels in the patients with chronic hepatitis B(CHB) ,chronic hepatitis C (CHC)and healthy people ,and to observe the relationship between anti‐ASGPR and the disease development in the patients with hepatitis .Methods Totally 60 patients with hepatitis B virus (HBV) infection(30 cases of CHB and 30 cases of CHB cirrhosis) and 60 patients with hepatitis C virus (HCV) infection(30 cases of CHC and 30 cases of CHC cirrhosis) were selected with 60 persons undergoing the physical examination as the control group .The anti‐ASGPR and ALT levels in all research subjects were detected with ELISA .Results (1)The anti‐ASGPR level in the HBV and HCV infection groups was significantly higher compared with the control group ,and the difference was statistically significant(P<0 .01) .The an‐ti‐ASGPR level in the CHB cirrhosis group was significantly higher than that in the CHB group ,and the difference was statistically significant(P<0 .05) .The anti‐ASGPR level in the CHC cirrhosis group was significantly higher than that in the CHC group ,and the difference was statistically significant(P<0 .05) .No correlation between anti‐ASGPR and ALT was found .(2) The anti‐ASG‐PR level in the CHC group was significantly higher than that in the CHB group ,and the difference was statistically significant(P<0 .01) .Conclusion The detection of anti‐ASGPR is helpful for clinical differential diagnosis and has an important significance for the treatment and prognosis .
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Objective To verify the interaction between asialoglycoprotein receptor (ASGPR)and hepatitis B virus (HBV)preS1 protein in vivo and in vitro ,and identify ASGPR as a cell-surface receptor for HBV,which could elucidate the molecular mechanism of HBV infection.Methods The preS1-ASGPR interaction was examined in mammalian two-hybrid and coimmunoprecipitation system by strictly following the manufacturer’s instructions.Results ASGPR interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro .Conclusion ASGPR may be a candidate receptor for HBV that mediates further step of HBV entry.
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To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR Hlb) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH 1 a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector plRES2EGFP,pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRHla and H2c in 4-1-6 were confirmed by RT-PCR,Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H lb/pCDNA3.1 (neo)was transfected into cell line 4-1-6, Hlb did not down-regulate the ligand binding ability of ASGPR.The eukaryotic expression plasmid Hlb/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single Hlb nor Hlb and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both Hla and H2c stably was established. The new split variant Hlb has no effect on ASGPR binding to ASOR. ASGPRHlb alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.
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AIM: To prepare and identify mouse polyclonal antibody against protein Hlb, which is the variant of major subunit of human ASGPR. METHODS: Hlb specific peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH) for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. ELISA assay was used to detect the titer of the antibody. Antibody was also identified by Western blot and immunohistochemistry assays. RESULTS: Mouse antibody against Hlb was prepared after injection of H1bKLH conjugation. The titer of H1b antibody was about 1:10~5.Western blot confirmed its high specificity. This antibody could also be used for immunohistochemistry analysis. CONCLUSION: The successful preparation of the polyclonal antibody against protein H1b, which can discriminate the two variants of the major subunit of ASGPR with high specificity, will provide an efficient reagent for further study of the physiologic functions of H1b and its role in the pathogenesis of human disease.