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@#Autophagosomes derived from tumor cells have been proved to induce potent T cell response both in mouse and human. In human in vitro study, dendritic cells(DC)loaded with cytomegalovirus(CMV)pp65 antigen-containing DRibble were capable to efficiently re-stimulate pp65-specific T-cell recall responses from freshly isolated or frozen humanperipheral blood mononuclear cell(PBMC). This study developed more robust assays using in vitro expanded antigen-specific T cells that contained a much higher percentage of antigen-specific T cells. DC cross-presentation efficiency of OX40 and CD80 modified pp65-DRibble was detected by intracellular IFN-γ staining. Compared with Ctrl/pp65 DRibble, the percentage of IFN-γ+ in total CD8+ T cells andCD4+ T cells was improvedwith OX40/pp65 DRibbleand CD80/pp65 DRibble stimulation. In addition, vaccine induced IL-12indendritic cells, whichpolarizes Th cells toward the IFN-γ high Th1 phenotype, evaluated by ELISA inco-culture supernatantwas dramatically higher in OX40/pp65 DRibble and CD80/pp65 DRibblegroups than in Ctrl/pp65 DRibble group. These results have implications for the immuneactivity of OX40 and CD80 modified DRibble and choice for prospective clinical use ofDRibble-based cancer immunotherapy.
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Objective To establish an anti-hepatitis E virus (HEV) enzyme-linked immunosorbent assay (ELISA) one-step assay based on seven glutathione S-transferase (GST)-fusion recombinant proteins derived from different HEV genotypes and subtypes. Methods Concentration of the coating antigen was optimized by block titration. The cut-off values were determined for anti-HEV IgG and IgM, respectively. Assay sensitivity, specificity and reproducibility were investigated using samples with confirmed anti-HEV positive. Results An optimal concentration of mixture of recombinant proteins (Mix166) was 1.5 mg/L for antigen coating. Coefficient of variations (CV) of anti-HEV within-run and between-run were 8.67% and 10.85%, respectively. CV of anti-HEV IgM within-run and between-run were 4.56% and 5.99%, respectively. Positive rates of anti-HEV IgG and IgM were both 94% for 50 HEV-polymerase chain reaction (PCR) positive sera tested with the one step assay. Using one-step assay to detect 674 serum samples from healthy people, 52 samples were found anti-HEV IgG positive and 3 samples were anti-HEV lgM positive. A series of serum specimens collected at different time points until 76 weeks from a chimpanzee challenged with HEV Mexican strain were anti-HEV IgM positive during week 1--6 and anti-HEV IgG positive during week 2--76 determined by the one step ELISA. However, import ELISA kits were lack of both the IgM and lgG reactivity to all of the serial chimpanzee sera. Conclusions The sensitivity and specificity of anti-HEV ELISA one-step assay based on the Mix166 antigen are high and could be used for the diagnosis of HEV infection.
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Objectives To evaluate accurately hepatocyte injury degree of severe hepatitis patients by quantifying plasma DNA of severe hepatitis patients and study its clinical application in diagnosis of severe hepatitis comparing with ALT.Methods Six milliliters of peripheral blood samples were collected from 185 patients with hepatitis B which are divided into four groups, severe hepatitis with 30 cases, acute hepatitis with 20 cases, chronic B hepatitis with 90 cases, and liver cirrhosis with 45 cases. 100 healthy controls were enrolled. Circulating DNA was extracted from plasma by the BILATEST virus DNA/RNA extraction kit and quantified with a novel duplex real-time PCR assay, respectively.Results Plasma DNA levels of hepatitis B patients were significantly higher than those of healthy controls (104.2 ng/ml vs. 23.4 ng/ml (median),P=0.0000).A significant difference of plasma DNA concentration was found between severe hepatitis and acute hepatitis (P=0.0018), and chronic B hepatitis (P=0.0000), and liver cirrhosis (P=0.0000).The median value of serum ALT of hepatitis B patients was 107.5 U/L, much higher than that of the healthy controls (24.1 U/L,P=0.0000).The levels of serum ALT were significantly different between severe hepatitis and acute hepatitis (P=0.0024), while there was no remarkable difference between severe hepatitis and chronic B hepatitis (P=0.0600), liver cirrhosis (P=1.0000). Moreover, for distinguishing severe hepatitis from liver cirrhosis and chronic B hepatitis, the plasma DNA assay was notably superior to ALT by the analysis of receive operating characteristic (ROC) curves (AUC, 0.95 vs. 0.51,P=0.0000; 0.86 vs. 0.34,P=0.0000).Conclusion The results by measuring plasma DNA of hepatitis B patients with the novel duplex real-time quantitative PCR showed that plasma DNA may be considered as a robust predictive marker for accurately evaluating hepatocyte injury degree of severe hepatitis patients.
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Objective To establish a double-antigen sandwich ELISA(S-ELISA) for detection of total antibodies against hepatitis C virus(HCV).Methods Recombinant HCV proteins fusion-expressed with His-tag and GST-tag were separately used as coating and HRP-labeling antigen of the S-ELISA.Serum samples were tested with both the S-ELISA and another commercial indirect ELISA(I-ELISA) kit(Beijing Wantai Biological Pharmacy Enterprise Co.Ltd.).HCV RNA in some of the samples were tested by RT-nested PCR.Results Among 1 968 tested samples,190(9.7%) were total anti-HCV-positive while 1 761(89.5%) were negative by both of the S-ELISA and I-ELISA,with a resultant concordance rate of 99.1% of the two ELISA assays.However,the results of 17(0.9%) were not consistent in the two assays,in which 14 were S-ELISA negative but I-ELISA positive and 3 were S-ELISA positive but I-ELISA negative.One of the 14 samples(0.7%) with S-ELISA negative was detected as HCV RNA-positive,while 2 of the 3(66.7%) samples with S-ELISA positive were detected as HCV RNA-positive.In addition,HCV RNA was detectable in 23 of 31(74.19%) samples which were total anti-HCV positive in both S-ELISA and I-ELISA.Taking the PCR data together into account,the sensitivity of the S-ELISA and I-ELISA were 99.48% and 98.96% and specificity were 99.94% and 99.27%,respectively.Conclusions The established S-ELISA in this study may provide a novel means for detection of HCV antibody with high sensitivity and specificity.