Binding kinetics and affinity of 17T2 mAb for RBD were evaluated by surface plasmon resonance on a BIAcore T100 instrument (Cytiva) with a running buffer composed of 10 mM HEPES at pH 7.2, 150 mM NaCl and 0.05% Tween 20. The assay format involved antibody capture on a Series S CM5 chip. Briefly, amine coupling was used to create a human IgG capture surface (anti-human Fc mAb) following instructions provided with the Cytiva human IgG capture kit. 17T2 mAb was captured on flow cell 2, leaving flow cell 1 as a subtractive reference. Capture levels of IgG were targeted between 100 and 200 resonance units, after which serial dilution of RBD was flowed over immobilized IgG (50 μL/min for 2 min) and allowed to dissociate up to 30 min. The capture surface was regenerated with a 60-s injection of 3 M MgCl2 (50 μL/min for 1 min). A 2-fold concentration series of each RBD variants ranging from 2 to 0.25 nM was used to analyze binding to 17T2. All sensorgrams were analyzed using a 1:1 Langmuir binding model with software supplied by the manufacturer to calculate the kinetics and binding constants. Where no decay in the binding signal was observed during the time allowed for dissociation, KD based on the kd limit was determined by the “5% rule54 (link).
Human igg capture kit
Manufactured by Cytiva
The Human IgG Capture Kit is a laboratory product designed for the purification of human immunoglobulin G (IgG) from various sample types. It provides a simple and efficient method for the isolation and concentration of IgG molecules.
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2 protocols using human igg capture kit
1
Measuring RBD-17T2 mAb Kinetics
2
SPR Binding Kinetics of Antibodies
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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.
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.
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