Screening Serum Markers Of Primary Biliary Cirrhosis With High-throughput Protein Chip Encoded By The Human Gene

Screening serum markers of primary biliary cirrhosis with high-throughput protein chip encoded by the human geneBackgroundPrimary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology. It predominantly affects middle-aged women. Serum autoantibodies are crucial tools for diagnosis of PBC, but currently used serum markers are not perfect. it is very important to study the serum markers of PBC with newly research methods.MethodsHuman cDNA library was used as template to designed gene-specific primers. The PCR amplification of the target gene products were co-transfected into yeast (Y258) with the linearized yeast expression vector pEGH. Galactose was use to induced yeast containing the target gene to clone and express recombinant protein. All the target recombinant proteins were tagged with GST, and could be purified by glutathione-affinity chromatography material. High-throughput protein chip was fabricated and used to screen serum markers of primary biliary cirrhosis. ResultsThe fabricated high-throughput protein chip contains a total of 38 400 protein spots, includingl7 718 human genes encoding proteins. The detection rate of protein spots on the chip was 97.6%, and the signal intensity correlation coefficient of double protein spots was 0.98.18 serum markers were screened between PBC group and the control group with a statistically significant (P<0.01). Of the best of five markers, antibodies to PDHAl,DBT and DLAT were the component of AMA-M2 which has been used as the marker of PBC; antibodies to HK1 and ZNF364 were identified as new marker of PBC, whose positive rates were 52.38%(11/21) and 38.10%(8/21) respectively in PBC, and 0.00%(0/30) in the control group. There is no serum marker were screened between the AMA-M2 positive and negative PBC patients. Only antibody to CENPB was identified as marker between the ACA positive and negative PBC patients (P<0.01) ConclusionsHigh-throughput protein chip encoded by the human gene is a technology for screening of new markers of PBC quickly and comprehensively. Antibodies to HK1 and ZNF364 can be used as a new marker for PBC with highly sensitivity and specificity. No serum marker was found between the AMA-M2 positive and negative PBC patients whereas only antibody to CENPB was identified as marker between the ACA positive and negative PBC patients. ObjectiveTo explore the prevalence of the anti-Saccharomyces cerevisiae antibody (ASCA) in patients with primary biliary cirrhoses and evaluate it's clinical significance.MethodsThe subtypes of ASCA including IgA and IgG in specimens from 198 patients with PBC, 44 patients with AIH,41 patients with other non-autoimmune liver diseases controls (LDC), 169 patients with inflammatory bowel disease (IBD) and 167 other diseases controls were measured by ELISA.ResultsThe positive rate of ASCA-IgA in PBC is 24.2%, which is higher than in ulcerative colitis (UC) group (11.3%, x2=6.8 P<0.01) and other diseases controls (12.6%, x2=8.0, P<0.01). Compared with the AIH group (20.5%) or LDC group (14.6%) or Crohn's disease (CD) (33.9%), there is no difference statistically significant (P>0.05). The prevalence of ASCA-IgG in PBC is 13.6%, lower than the CD group (27.4%,x2=6.4, P<0.05), and other diseases controls (24.6%, x2=7.1, P<0.01). There is no statistically significant difference (P>0.05) between PBC and the AIH group (15.9%) or LDC group (7.3%) or UC group (7.2%). The prevalence of ASCA-IgA or ASCA-IgG in PBC is 29.8%, which is statistically lower than that of CD group (45.2%, x2=5.0, P<0.05), but higher than that of UC group (14.4%,x2=8.3,P<0.01) and other non-autoimmune liver diseases controls (9.8%,x2=7.0, P<0.01). ASCA was detected more frequently in PBC patients with positive anti-GP210 antibody than in anti-gp210 antibody negative PBC patients (39.7%&24.6%, x2=4.9, P<0.05). The positive rate of ASCA between AMA positive and negative patients with PBC or anti-SP100 antibodies positive and negative patients with PBC was not significant different. PBC patients with positive ASCA-IgA have higher level of TBIL, DBIL, ALP, AST, TBA, LD, IgA, IgM, ES Rand lower level of ALB, CHE than patients with negative ASCA-IgA. There is no statistically significant difference of liver injury indicators and immune function parameters between patients with positive ASCA-IgG and negative ASCA-IgG. ConclusionsASCA is not an IBD-specific antibody. There is a high prevalence of ASCA in patients with PBC, especially the subtype of ASCA-IgA. ASCA-IgA was found to be associated with the severity of liver damage and immune activity whereas ASCA-IgG was not found to be associated with.