Proteon surface plasmon resonance biosensor

Steady-state equilibrium binding of post-vaccination sera was monitored at 25 °C using a ProteOn surface plasmon resonance biosensor (BioRad). The recombinant RSV G proteins from E. coli (REG) or from mammalian 293 T cells (RMG; Recombinant Mammalian cell derived G) were coupled to a GLC sensor chip via amine coupling with 500 resonance units (RU) in the test flow channels. For polyclonal post-vaccination serum antibody kinetics against individual RSV-G peptides, the SPR analysis was performed using peptides biotinylated at C-terminus captured on a NLC chip surface. Samples of 100 μl freshly prepared sera dilutions were injected at a flow rate of 50 μl/min (120 sec contact duration) for association, and disassociation was performed over a 600 second interval. Responses from the protein surface were corrected for the response from a mock surface and for responses from a buffer only injection. Pre-vaccination cotton rat sera and a IgG depleted sera were used as a negative control. Total antibody binding and data analysis results were calculated with BioRad ProteOn manager software (version 3.1).

Steady-state equilibrium binding of post-H5N1-vaccinated human sera was monitored at 25 °C using a ProteOn surface plasmon resonance biosensor (Bio-Rad).^{22 (link)} The recombinant HA globular domain (rHA1-His₆) or HA stalk domain (rHA2-His₆) for the A/Indonesia/05/2005 (clade 2.1) or from H5N1- A/Vietnam/1203/2004 influenza virus strain was coupled to a GLC sensor chip with amine coupling with 100 and 500 resonance units (RU) in the test flow cells. Samples of 60 µL sera at 10-fold and 100-fold dilutions were injected at a flow rate of 50 µL/min (120-s contact time) for association, and disassociation was performed over a 600-s interval (at a flow rate of 50 µL/min). Responses from the protein surface were corrected for the response from a mock (no coating) surface and for responses from a separate, buffer only injection. Binding kinetics for the human vaccine sera and the data analysis were calculated with Bio-Rad ProteOn manager software (version 3.0.1). Antibody off-rate constants, which describe the fraction of antigen−antibody complexes that decay per second, were determined directly from the serum/plasma sample interaction with rHA1 or rHA2 protein using SPR in the dissociation phase and calculated using the Bio-Rad ProteOn manager software for the heterogeneous sample model as described before.^{22 (link)} Off-rate constants were determined from two independent SPR runs.

Steady-state equilibrium binding of polyclonal antibodies in human sera was monitored at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad). The purified recombinant F or G proteins were coupled to a GLC sensor chip via amine coupling with 200 resonance units (RU) in the test flow channels and spatial density of the proteins were optimized such that there is single monovalent interaction for each antibody molecule irrespective of their isotype. For peptide analysis in SPR, chemically synthesized biotinylated RSV-F or RSV-G peptides were captured on NLC chips. Samples of 300 μl freshly prepared sera at 10-fold dilution or MAbs (starting at 1 μg/ml) were injected at a flow rate of 50 μl/min (120 sec contact duration) for association, and disassociation was performed over a 600-second interval. Responses from the protein surface were corrected for the response from a mock surface and for responses from a buffer-only injection. Total antibody binding and data analysis results were calculated with BioRad ProteOn manager software (version 2.0.1).