We used the recombinant expression plasmid pCBD2-2J-2-9-1 [33] to generate VLP mutants. Various VLP mutants were generated by site-directed mutagenesis derived from pCBD2-2J-2-9-1 as a template. PCR was performed using pfu ultra DNA polymerase (MERCK) and all mutant constructs were confirmed by sequencing. BHK-21 cells at 80%-90% confluency in 48-well plates were transfected with plasmids of various VLPs. After two days transfection, the cells were washed with PBS supplemented with 1% FBS, fixed with 3.7% formaldehyde at 4uC for 10 min, and then permeabilized in PBS supplemented with 1% FBS, 0.1% saponin (Sigma-Aldrich) at 4uC for 10 min. For staining, cells were incubated with mAbs at 4uC for 30 min, DB32-6, DB25-2, 3H5 and mix mAbs (4G2, DB2-3, DB13-19, DB21-6 and DB42-3) at a concentration of 0.1, 1, 1 and 1 mg/ml, respectively. After being washed twice, R-Phycoerythrin (PE)-conjugated AffiniPure F(ab9) 2 fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories) diluted to 1:250 was then added at 4uC for 30 min and analyzed by flow cytometry. Relative recognition was performed according to previously described procedures and calculated as [intensity of mutant VLP/intensity of WT VLP] (recognized by a mAb)6[intensity of WT VLP/intensity of mutant VLP] (recognized by mixed mAbs) [34] .