Guohua Wang

Enhanced serodiagnostic utility of novel Mycobacterium tuberculosis polyproteins

OBJECTIVESnThe detection of Mycobacterium tuberculosis specific antibodies in human sera has been a rapid and important diagnostic aid for tuberculosis (TB) control and prevention. However, any single antigen is not enough to be used to cover the antibody profiles of all TB patients.nnnMETHODSnSeven single antigens (38xa0kDa, ESAT-6, CFP10, Mtb8.4, MPT64, TB16.3 and Mtb8) were evaluated serodiagnostically. Two novel M. tuberculosis polyproteins, 38kD-ESAT6-CFP10 (38F) and Mtb8.4-MPT64-TB16.3-Mtb8 (64F), were expressed and the novel 38F-64F indirect ELISA assay used to analyze antibody responses to polyproteins in serum samples.nnnRESULTSnThe sensitivity of the novel 38F-64F indirect ELISA alone was much higher than that of the sputum culture test (86.91% vs. 50.62%) and that of the sputum smear test (78.64% vs. 47.57%). The novel 38F-64F indirect ELISA had a sensitivity of 74.16% with sera from extrapulmonary TB patients and a sensitivity of 37.14% with sera from LTBI. The specificity of the novel 38F-64F indirect ELISA was 90.36% with the sera from healthy blood donors and 94.15% with the sera from non-TB patients.nnnCONCLUSIONSnThe novel 38F-64F indirect ELISA assay had effective diagnostic performance and would make meaningful contribution to the diagnosis of TB disease in developing countries.

IgG, IgM and IgA antibodies against the novel polyprotein in active tuberculosis

BackgroundThe present study was aimed to evaluate whether IgG, IgM and IgA antibodies levels detected against a novel Mycobacterium tuberculosis polyprotein 38xa0F-64xa0F (with 38xa0F being the abbreviation for 38kD-ESAT6-CFP10 and 64xa0F for Mtb8.4-MPT64-TB16.3-Mtb8) are suitable for diagnosing active tuberculosis, and for monitoring the efficacy of chemotherapy on TB patients.MethodsIn this study, a total of 371 active TB patients without treatment were selected and categorized into S+/C+u2009group (nu2009=u2009143), S-/C+u2009group (nu2009=u2009106) or S-/C- group (nu2009=u2009122). A series of serum samples were collected from 82 active TB patients who had undergone anti-TB chemotherapy for 0–6 months at one month interval. Humoral responses (IgG, IgM and IgA) were determined for the novel Mycobacterium tuberculosis polyprotein using indirect ELISA methods in all of serum samples.ResultsFor S+/C+, S-/C+u2009and S-/C- active tuberculosis patients before anti-TB chemotherapy, the sensitivities of tests based on IgG were 65.7%, 46.2% and 52.5% respectively; the sensitivities based on IgM were 21.7%, 24.5% and 18.9%; and the sensitivities based on IgA were 25.2%, 17.9% and 23.8%. By combination of three isotypes, for all active tuberculosis patients, the test sensitivity increased to 70.4% with the specificity being 91.5%. After anti-TB chemotherapy, there were no significant differences between groups with different courses of anti-TB chemotherapy.ConclusionsThe novel Mycobacterium tuberculosis polyprotein 38xa0F-64xa0F represents potential antigen suitable for measuring IgG, IgM and IgA antibodies. However, the serodiagnostic test based on the 38xa0F-64xa0F polyprotein appears unsuitable for monitoring the efficacy of chemotherapy.

Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies.

Cancerogenic effect of different fragments of the hepatitis C virus core protein.

The hepatitis C virus core protein plays an extremely important role in the hepatocarcinogenesis of hepatitis C virus. Little, however, is known about the oncogenic potency of fragments. Thus, the purpose of the present study is to investigate the cancerogenic effects of the different core protein fragments. Two series of recombinant plasmids containing hepatitis C virus core gene fragments encoding the different-length core protein were constructed using plasmid enhanced green fluorescent protein (pEGFP)-C1 and pcDNA3.1(+), respectively. Human hepatocyte L02 cells transiently transfected with pEGFP-C1-based plasmids were subjected to confocal laser scanning microscopy analysis to determine the localization of the different core protein fragments. The stably transfected L02 cells with the pcDNA3.1(+)-based core protein plasmids were used to investigate the ultrastructural effects of the core protein and the tumorigenicity of L02 cells expressing core protein fragments in athymic nude mice. The full-length core protein and Core130−191 were completely localized in the cytoplasm, while Core1−59 existed exclusively in the nucleus. On the other hand, Core50−140 and Core1−140 were observed in both the nucleus and the cytoplasm. Ultrastructural changes of L02 cells expressing the full-length core protein were comprehensive and included, for example, irregular nuclear, increased nuclear/cytoplasmic ratio and mitochondria swelling. The slight changes were observed in the cells expressing Core50−140 and Core130−191, whereas the ultrastructure of the cells expressing Core1−59 remained normal. All the L02 cells stably expressing different fragments of the core protein, with the exception of the C-terminal truncated fragment Core1−59, could induce the occurrence of tumor in the nude mice. The N-terminal fragment of the core protein, Core1−59, was not oncogenic, while the intermediate and posterior segments of the hepatitis C virus core protein had the cancerogenic potency. In view of the existence of many important immunogenic epitopes in it, the core protein anterior segment might be a safer candidate for the development of hepatitis C virus vaccine.

Construction, Expression, Purification and Biotin Labeling of a Single Recombinant Multi-Epitope Antigen for Double-Antigen Sandwich ELISA to Detect Hepatitis C Virus Antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.