Aims: To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo-blood group antigens (HBGA)-conjugated magnetic beads.
Methods and results: HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM-MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15min of incubation with PGM-MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real-time RT-PCR analyses suggest that NoV could be concentrated as much as 1000-fold at neutral pH, up to 3·4-fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0-4·2. Variation between GI and GII viral binding to the PGM-MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM-MB, ranging from 28-fold (GI.4) to 1502-fold (GI.1). Eleven of 13 GII strains were concentrated from 30-fold (GII.5) to 1014-fold (GII.4, lab strain) by PGM-MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery.
Conclusions: All GI and 85% of GII NoVs tested could be captured and concentrated by PGM-MB method. The binding occurred rapidly and was enhanced at low pH.
Significance and impact of the study: These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.
Journal of Applied Microbiology © 2010 The Society for Applied Microbiology. NO claim to US Government works.