Proteon manager software

Steady-state equilibrium binding of post-H5N1 human vaccine sera was monitored at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad)[7] . The rHA0-His₆, rHA1-His₆ or rHA2-His₆ protein sequence for the H5N1- A/Vietnam/1203/2004 influenza strain (were identical to the boosting vaccine strain; H5N1-A/Vietnam/1194/2004) was coupled to a GLC sensor chip with amine coupling with 500 resonance units (RU) in the test flow cells. Samples of 60 µl sera at 10-fold & 100-fold dilutions were injected at a flow rate of 30 µl/min (120-sec contact time) for association, and disassociation was performed over a 600 second interval (at a flow rate of 30 µl/min). Responses from the protein surface were corrected for the response from a mock surface and for responses from a separate, buffer only injection. Binding kinetics for the human vaccine sera and the data analysis were calculated with BioRad ProteOn manager software (version 2.0.1). 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 rHA0, rHA1 or rHA2 protein using SPR in the disassociation phase as described before[7] and calculated using the BioRad ProteOn manager software for the heterogeneous sample model. Off-rate constants were determined from two independent SPR runs.

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.

SPR was performed to measure and compare the affinity of ReBif (hIFN-β) to recombinant forms of hIFNAR1-ECD and hIFNAR1-ECD-Del. All experiments were carried out in the absence of IFNAR2. All SPR experiments were performed on a ProteOn XPR36 (Bio-rad Labs) using a HTG chip for His-tagged proteins and TBS as the running buffer. Both ligands (hIFNAR1-ECD and hIFNAR1-ECD-Del) were immobilized to the nickel activated chip via their C-terminal His-tags after dilution to 25 µg/ml in TBS. ReBif, (hIFN-β) previously dialyzed into TBS, was also diluted in TBS to various concentrations. All data were referenced according to the manufacturer’s instructions (Bio-rad) and analyzed using the Langmuir binding model. Data were considered for inclusion in the analysis only if the Chi₂ value (the measure of error between measured and fitted values) was less than 10% of the R_max as per the manufacturer’s instructions (Bio-rad). K_a (1/Ms), K_d (1/s), and K_D (nM) were calculated by the ProteOn Manager software (Bio-rad) and are represented as mean ± S.D. from at least three independent experiments.