Development of an Antibody‐Based Assay for Determination of Baculovirus Titers in 10 Hours

Abstract

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.

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

“…A commercially available immunocytochemical assay using a monoclonal antibody against the baculovirus gp64 protein provides virus titers within 48 h 18 . Two additional antibody-based assays have been developed that provide rapid virus titrations 19,20 . Quantification of baculovirus particles by flow cytometry 21 and real-time PCR 22 has been described.…”

Requirements for baculoviruses for clinical gene therapy applications

“…One option is a rapid immunological assay for detection of a viral protein (e.g. GP64) with monoclonal antibodies (Kitts and Green, 1999;Kwon et al, 2002;Mulvania et al, 2004). Other options are to detect infected cells by measuring increased cell-diameter following infection (Janakiraman et al, 2006) or analyzing cell viability by microculture tetrazolium (MTT) assays (Mena et al, 2003).…”

New baculovirus expression tools for recombinant protein complex production

“…the number of infectious viral particles (plaque forming units, pfu) present in a defined volume of viral supernatant. Also for this purpose, useful alternatives to the time intensive (5-7 days) plaque assay were developed based on an immunological assay or a PCR reaction, which can also be used on automated platforms (Bahia et al, 2005;Chambers et al, 2004;Kitts and Green, 1999;Kwon et al, 2002;Lo and Chao, 2004;Shen et al, 2002).…”