Detection of reticuloendotheliosis antibody by enzyme-linked immunosorbent assay with baculovirus expressed envelope protein
Date Issued
2014
Date
2014
Author(s)
Hsiao, Ting-Hsiang
Abstract
Reticuloendotheliosis virus (REV) infects variety animals including chickens, ducks, geese, pheasants, peafowl and other birds. REV is ignored in Taiwan because the infected poultry without obvious clinical signs. REV causes severe economic losses in commercial poultry, such as runting disease syndrome and immunosuppression. Currently, the disease lacks effective prevention and control in the field. The purpose of this study is to develop a rapid, reliable, convenient and economic method for detecting REV antibody. We use baculovirus/insect cells expression system to express REV envelope protein, and to evaluate the potential of the recombinant protein to be used as the antigen in enzyme linked-immunosorbent assay (ELISA). To use the laboratory isolated Taiwan strain goose/3410/06 as the cloning template. The full-length of REV envelope was cloned into bacmid vector and then the constructed recombinant baculoviruse-env was infected the Sf9 insect cells. The infected insect cells showed expected recombinant envelope protein in western blotting which size was about 62 kDa. The expressed recombinant envelope protein was purified by Ni-NTA (nitrilotriacetic acid) column. The purified recombinant envelope protein used as the coated antigen. Then, we test optimization condition of indirect ELISA by checkerboard method. Using 182 field samples (138 broilers and 44 Taiwan Country Chickens) to compare virus neutralization tested as the gold standard with the indirect ELISA. The result showed the sensitivity and the specificity of the indirect ELISA were 88.5% and 97.7%, respectively. Both values were good enough to screen detection in the field. The indirect ELISA of recombinant envelope protein possesses potential of development. Additional, we used blocking ELISA which envelope protein was expressed by E. coli to compare with the result of indirect ELISA. The result showed the sensitivity and the specificity were 84.6% and 95.3%, respectively. The two kind of methods displayed similar result, but the indirect ELISA possess advantages of lower usage amount of samples, easier obtainable secondary antibody and cheaper.
Subjects
家禽網狀內皮增生症病毒
桿狀病毒/昆蟲細胞表現
重組封套蛋白質
間接型酵素連結免疫吸附法 (ELISA)
Type
thesis
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