ABSTRACT
<p><b>OBJECTIVE</b>To establish a high-throughput chemiluminescence assay of serotype 5 specific neutralizing antibody and understand the epidemiology of this antibody in the healthy adults and children in Guangzhou.</p><p><b>METHODS</b>Using rAd5 carrying the reporter gene of secreted alkaline phosphatases (SEAP), serum samples from 116 healthy adults and 94 healthy children were examined with chemiluminescence assay to detect the presence of Ad5 neutralizing antibody. The reliability of this assay was tested against conventional cytopathic effect observation.</p><p><b>RESULTS</b>The chemiluminescence assay using secreted alkaline phosphatases (SEAP) as the reporter allowed rapid, sensitive, specific and reproducible detection of serotype 5 specific neutralizing antibody for epidemiological study of Ad5, which was positive in 26.72% of the adults and 17.02% of the children in this study.</p><p><b>CONCLUSION</b>Ad5 neutralizing antibody is prevalent in the population of Guangzhou, suggesting the necessity of developing other serotype adenovectors for better vaccination and therapeutic effects.</p>
Subject(s)
Adult , Animals , Female , Humans , Male , Middle Aged , Young Adult , Adenoviridae , Classification , Genetics , Allergy and Immunology , Alkaline Phosphatase , Genetics , Antibodies, Neutralizing , Allergy and Immunology , Artifacts , Blotting, Western , China , DNA, Recombinant , Genetics , Genes, Reporter , Genetics , High-Throughput Screening Assays , Luminescent Measurements , Methods , Reproducibility of Results , Serotyping , Time FactorsABSTRACT
<p><b>OBJECTIVE</b>To clone, express and characterize the capsid protein of human Norwalk virus Guangzhou strain NVgz01.</p><p><b>METHODS</b>On the basis of successful construction of full-genome clones and sequence analysis of human norovirus Guangzhou strain NVgz01, the full capsid gene was ligated into pET28a (+) for expression. After IPTG induction, the recombinant protein was purified through metal (Ni(2+)) chelating affinity chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to determine the antigenicity of the recombinant protein.</p><p><b>RESULTS</b>The recombinant capsid gene was overexpressed in E.coli, yielding the recombinant protein with relative molecular mass of 62x10(3) that was highly purified through metal (Ni(2+)) chelating affinity chromatography. IDEIA Norovirus Kit and immunoassay showed that the recombinant protein had good antigenicity.</p><p><b>CONCLUSION</b>The capsid gene of norovirus Guangzhou strain has been cloned and expressed, which can be useful for developing diagnostic reagents or vaccine of norovirus.</p>