title: Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay.
abstract: The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, p<0.05) and BmNPV (R(2)=0.9834, p<0.05). In conclusion, a novel, rapid and semi-automated procedure for titrating baculovirus was developed based on the specific immunostaining of infected cells followed by automated spot counting.
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361005, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Tong
authors.initials: T
authors.lastname: Cheng
authors.forename: Ke
authors.initials: K
authors.lastname: Ma
authors.forename: Dezhen
authors.initials: D
authors.lastname: Xia
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Hailian
authors.initials: H
authors.lastname: Du
authors.forename: James Wai Kuo
authors.initials: JW
authors.lastname: Shih
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Qinjian
authors.initials: Q
authors.lastname: Zhao
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D057205:Automation, Laboratory / Q000379:methods; D016367:Baculoviridae / Q000302:isolation & purification*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D061987:Sf9 Cells; D018411:Spodoptera; D019562:Viral Load / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral