Cm5 sensor

Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 with a CM5 sensor and human IgG capture kit (Cytiva) at 25°C using HEPES buffered saline (HBS) with 0.005% P20 as the running buffer. Anti-human IgG was captured using the protocol recommended by Cytiva over all four flow paths. Between 535 and 580 RU of each Ab was captured using a contact time of 60 s and a 10 μL/min flow rate in an HBS running buffer containing 3 mM EDTA and 0.005% P20. MN.B gp120 (AIDS Reagent Repository) was used as the analyte diluted between 1.6 and 200 nM. gp120 MN.B was injected over flow paths 1, 2, 3, and 4 with a contact time of 120 s, flow rate of 30 μL/min, and a long dissociation of 1,200 s.
Flow path 1 was used as reference, while antibodies were bound in flow paths 2, 3, or 4. 3 M magnesium chloride was injected over all four flow paths for regeneration using a contact time of 30 s and flow rate of 30 μL/min. 1:1 Langmuir model was used to determine the kinetics of each Ab. Because of the low level of binding of gp120 (or the saturation at low gp120 MN.B concentrations), steadystate kinetics were attempted for both pre-immune 1098B8 and germline 1098B8 by reducing the captured Ab to 120 RU and increasing the contact time of analyte to 300 s. The pre-immune 1098B8 mAb did not fit well to 1:1 Langmuir kinetics and the lower binding could only be estimated.