Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals.

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.

Measurement of antibodies to varicella-zoster virus using a virus-free fluorescent-antibody-to-membrane-antigen (FAMA) test.

The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Evaluation of a commercial glycoprotein enzyme-linked immunosorbent assay for measuring vaccine immunity to varicella.

PURPOSE: To evaluate a recently marketed commercial glycoprotein enzyme-linked immunosorbent assay (gpEIA) kit, the VaccZyme™ VZV gpEIA, for measuring the immunity of varicella-vaccinated children. MATERIALS AND METHODS: We investigated the accuracy and reproducibility of the VaccZyme™ VZV gpEIA kit for the detection of antibodies to VZV. We also examined the sensitivity, specificity, and correlation between antibody titers calculated with gpEIA versus fluorescent antibody to membrane antigen (FAMA) by using sera of 349 children, ranging from 1 to 6 years old. RESULTS: VaccZyme™ VZV gpEIA gave precise and reproducible intra- and inter-assay results. FAMA and gpEIA titers showed a linear correlation (Pearson correlation coefficient=0.987). The sensitivity and specificity of the VaccZyme™ gpEIA was 31.4% and 100%, respectively, when the guidelines of the gpEIA (<100 mIU/mL) and FAMA 1:4 were adopted as cutoff values. However, the maximum sensitivity and specificity were 88.9% and 95.1%, respectively, with the highest correlation (κ=0.840), if the cutoff values were set with gpEIA at 49.7 mIU/mL and FAMA 1:16. CONCLUSION: These results demonstrate that the VaccZyme™ VZV gpEIA kit gave precise and reproducible data for measuring antibody titer after varicella vaccination. The results also showed that the antibody titer calculated with the VaccZyme™ gpEIA kit strongly correlated with the FAMA titer. However, cutoff values should be re-optimized for the evaluation of vaccine immunity.

Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection.

The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.

Enhanced production of recombinant B-domain deleted factor VIII from Chinese hamster ovary cells by propionic and butyric acids.

Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities.

Selection and characterization of human antibodies against hepatitis B virus surface antigen (HBsAg) by phage-display.

Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma. Human plasma-derived antibody to HBV is being used as a prophylactic for postexposure to HBV and liver transplantation currently. However, it is required to replace the plasma-derived anti-HBs antibody (Ab) to a recombinant antibody because of limited availability of human plasma with high anti-HBs Ab titer and possible contamination of human pathogens. We constructed an anti-HBs Ab-enriched phage-display library from peripheral blood B cells of vaccinated volunteers and the size of library was approximately 1.0 x 10(7). The library was panned against hepatitis B surface antigen (HBsAg) and five different clones were isolated. All five clones exhibited the same heavy chain sequence; in contrast, light-chain exhibited one lambda and four different kappa sequences. The Fabs were expressed soluble in E. coli and exhibited affinities of 2.1 x 10(8) approximately 7.7 x 10(8) M(-1).