Development of an antibody-based assay for determination of baculovirus titers in 10 hours.
The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.
Kwon
M S
MS
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
Dojima
T
T
Toriyama
M
M
Park
Enoch Y
EY
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis* / Q000235:genetics; D016367:Baculoviridae / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D015203:Reproducibility of Results; D018411:Spodoptera
D000914:Antibodies, Viral; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins