Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay

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 48 h 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 (R2 = 0.9980, p < 0.05) and BmNPV (R2 = 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.

► Sensitive and specific mAbs against both AcMNPV and BmNPV VP39 capsid protein. ► Specific immunostaining of infected cells as blue spots in the ELISPOT assay. ► Automated spot counting using ELISPOT hardware and software. ► Development of a 96-well microtiter plate ELISPOT assay for baculovirus titration. ► The ELISPOT assay functioned well for both AcMNPV and BmNPV quantitation.