ABSTRACT
Objective To establish a flow cytometry-based assay for the detection of monocyte-me-diated antibody-dependent cell-mediated cytotoxicity ( ADCC ) .Methods P815 cells double stained with PKH26 and carboxyfluorescein succinimidyl ester ( CFSE ) were used as target cells and coated with P 815 specific antibodies to form antigen-antibody complexes .The peripheral blood mononuclear cells were isolated as effector cells and co-cultured with the antigen-antibody complexes .The CD3-CD14+PKH26+CFSE-cell population were gated by flow cytometry .Optimized effector/target cell ratio and incubation time for killing assay were identified .Monocyte-mediated ADCC in 23 patients with chronic HCV infection and 22 healthy subjects were analyzed .Results The monocyte-mediated ADCC could be evaluated through analyzing the CD3-CD14+PKH26+CFSE-cells with flow cytometry .The optimized effector/target cell ratio was 10 ∶1 and the optimized time for incubation was 4 h.Monocyte-mediated ADCC was inhibited in patients with chronic HCV infection as compared with healthy subjects (P=0.009).Conclusion A flow cytometry-based assay for the detection of monocyte-mediated ADCC was established , which could be used as a fast , sensitive and safety method for the evaluation of monocyte-mediated ADCC during viral infections and the research and de-velopment of drugs .
ABSTRACT
Objective To evaluate the analytical performance of a novel HBV DNA assay based on automated DNA extraction and real-time fluorescence quantitative PCR .Methods Analytic verification studies.Accuracy and lower limit of detection were assessed by determining a panel of HBV standard plasma of WHO.HBV standard plasma (genotype A, B, C and D) at 6 different concentrations were measured 18 times to evaluate precision and reproducibility .Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range .One hundred and forty-four clinical specimens were quantified for HBV DNA so as to evaluate the correlation between the new test and COBAS ? system. Results Quantification of HBV standard plasma showed acceptable accuracy , with each deviation between observed and expected values within ±0.35 lg IU/ml (-0.17-0.32 lg IU/ml).Intra-assay coefficients of variation ( CV) for genotype A , B, C and D were 3.87% -6.32%, 0.45% -14.68%, 0.16% -8.36% and 0.64%-13.01%respectively, and the inter-assay CV were 5.67%-9.69%, 1.28%-15.68%, 0.36%-9.05%and 1.69%-13.65%, separately.Linearity assessment exhibited an excellent dynamic range of linear quantification from 20 to 1.0 ×1010 IU/ml ( r =0.998, P <0.001 ) .And the satisfactory results obtained at 3 levels of HBV DNA concentration (10, 20, 50 IU/ml, respectively) confirmed the claimed lower limit of detection with 5/5 detectable rate at 20 IU/ml.Furthermore, good correspondence was observed between the new HBV DNA assay and the COBAS ? system with 100% ( 144/144 ) qualitative coincidence and significant correlation based on 104 positive data ( r=0.984, P<0.000 1).Conclusions The novel fully-automated real-time PCR assay displayed good analytical and clinical performance for highly sensitive detection of HBV DNA.It was well suited for monitoring antiviral responses as well as drug resistance according to current clinical practice guidelines for the management of chronic HBV infection .