Highly Sensitive and Novel Point-of-Care System, aQcare Chlamydia TRF Kit for Detecting Chlamydia trachomatis by Using Europium (Eu) (III) Chelated Nanoparticles

BACKGROUND: The bacterium Chlamydia trachomatis is one of the leading causes of sexually transmitted diseases worldwide. Since no simple and effective tool exists to diagnose C. trachomatis infections, we evaluated a novel point-of-care (POC) test, aQcare Chlamydia TRF kit, which uses europium-chelated nanoparticles and a time-resolved fluorescence reader. METHODS: The test performance was evaluated by comparing the results obtained using the novel POC testing kit with those obtained using a nucleic acid amplification test (NAAT), using 114 NAAT-positive and 327 NAAT-negative samples. RESULTS: The cut-off value of the novel test was 20.8 with a detection limit of 0.27 ng/mL. No interference or cross-reactivity was observed. Diagnostic accuracy showed an overall sensitivity of 93.0% (106/114), specificity of 96.3% (315/327), positive predictive value (PPV) of 89.8% (106/118), and negative predictive value (NPV) of 97.5% (315/323). The sensitivity of the novel test was much higher than that of currently available POC tests. Furthermore, the relative ease and short turnaround time (30 min) of this assay enables C. trachomatis-infected individuals to be treated without a diagnostic delay. CONCLUSIONS: This simple and novel test is a potential tool to screen a larger population, especially those in areas with limited resources.

Confirmation of HCV Positivity for Indeterminate Donors in Anti-HCV Antibody Immunoblot Assay among Blood Donors / 대한수혈학회지

BACKGROUND: In Korean Red Cross, recombinant immunoblot assay (RIBA) has been used for the confirmatory test of HCV positive units since 1995. To certify the HCV infection in blood donors who showed the 'indeterminate result on the RIBA test, this study was performed. METHODS: Three enzyme immunoassay (EIA) kits (LG HCD 3.0, DONG-A HCV 3.0, and ORTHO HCV 3.0)and RNA detection method were employed to evaluate infection state of 135 samples of the 'indeterminate in the RIBA test. RESULTS: The 52.6% of the samples showed the same test results with three EIA kits. Fifteen samples (11.1%) were HCV RNA positive with RT-PCR-hybridization technique. Among 15 samples of HCV RNA positive, 13 (86.7%), 13 (86.7%), and 14(93.3%) of samples were positive in LG HCD 3.0, Ortho HCV 3.0 and Dong-A HCV 3.0 EIA, respectively. In the analysis of RIBA band reaction, HCV RNA positivity were correlated with core14, core518, and 897 antigen. However, among 64 samples which react with core antigen only, five samples (7.8%) were HCV RNA positive. CONCLUSION: Based on the results of the present study, it is recommend that the HCV RNA test be used as a method of confirmatory test in order to notify exact HCV positivity status to blood donor who showed indeterminate RIBA result.

Performance of HCV and HIV-1 Nucleic Acid Amplification Test(NAT) in Korean Blood Donors / 대한수혈학회지

BACKGROUND: There is still risk of acquiring HCV and HIV by transfusion due to window phase. Screening for HCV and HIV-1 by nucleic acid amplification testing (NAT) may improve blood safety allowing detection during the preseroconversion window in donors. METHODS: We investigated NAT usefulness using COBAS AMPLICOR analyzer (Roche). The following sample population were tested:1) 15,552 HCV/HIV-1 seronegative random blood donor samples for HCV and HIV-1 NAT;2) 696 high ALT and 271 HCV EIA positive samples for HCV NAT;3) 1,152 HIV-1 EIA reactive samples for HIV-1 NAT. NAT was performed on pools of 24 donations according to the assay protocol. RESLUTS: Six pools showed initial reactive reactions in HCV NAT and one pool showed initial reactive reaction in HIV-1 NAT. But no donor sample was found repeatedly reactive by this assay. CONCLUSIONS: Although there were false positive reactions, specificity of the NAT assay was high enough for the assay to be applied as a blood screening test and implementation of this assay is expected to improve blood safety and be useful for blood products use.

cDNA Cloning and Expression of Angiostatin, an Angiogenesis Inhibitor , from Human Liver Tissue mRNA / 대한암학회지

PURPOSE: Angiostatin, a 38 kDa internal fragment of plasminogen, is a potent inhibitor of angiogenesis. It blocks neovascularization and growth of primary and metastatic tumors in mice. To produce recombinant angiostatin protem comprising kringle 1-4 of plasminogen, we cloned the angiostatin cDNA from human liver tissue mRNA and expressed it in E. coli. MATERIALS AND METHODS: We cloned angiostatin cDNA from human liver tissue mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Cloned cDNA was ligated to pET22b (+) expression vector, transformed into E. coli stram BL21 (DE3) and expressed by IPTG induction. Recombinant human angiostatin protein was purified from the inclusion bodies of lysated bacterial pellet with 8 M urea solubilization, refolding, single step Lysine-Sepharose 4B affinity chromatography and 0.2 M E-aminocarproic acid elution. The anti-angiogenic activity of purified recombinant angiostatin was assayed with endothelial cell proliferation assay and chorioallantoic membrane assay (CAM). RESULTS: The identification of cloned angiostatin cDNA was confirmed by Southern hybridization and Pst I restriction enzyme digestion pattern. Angiostatin cDNA was expressed in E. coli, refolded in vitro and purified by Lysine Sepharose 4B affinity chromatography. The molecular weight of purified recombinant angiostatin was about 55 kDa on the SDS-PAGE. It inhibited the proliferation of bovine capillary endothelial (BCE) cells in vitro with a half-maximal inhibition concentration (ED50) of approximately 500 ng/mL. It also suppressed neovasculrization on the CAM assay. CONCLUSION: These results demonstrated that recombinant human angiostatin has similar function and biological activity compared with human angiostatin which is purified from porcine elastase digested human plasminogen fragment.