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Surface plasmon resonance

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Surface plasmon resonance is a technique used to study the interactions between molecules in real-time. It measures changes in the refractive index near a metal surface, which can be used to determine the affinity and kinetics of molecular binding events.

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Lab products found in correlation

Biacore 3000, by Cytiva (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) C digit blot scanner, by LI COR (1 mentions) Goat anti human fc specific antibody, by Merck Group (1 mentions) Human male ab plasma, by Merck Group (1 mentions) Superdex 200 5 150 gl column, by GE Healthcare (1 mentions) Protein g sepharose, by GE Healthcare (1 mentions) Phosphate buffered saline (pbs), by Lonza (1 mentions) Br 1008 39, by GE Healthcare (1 mentions)

10 protocols using surface plasmon resonance

1

Determining Antibody-Antigen Affinity by SPR

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Example 10

Affinity of antibodies and antigen-binding fragments thereof described herein for to target protein can be assessed using conventional techniques such as, for example, surface plasmon resonance (SPR; Biacore).

Affinity constants for the binding of the various selected antibodies and antigen-binding fragments to target protein are determined by SPR using, for example, a BIAcore™ 3000 analytical system equipped with a CM5 sensor chip (BIAcore AB). The selected antibodies or antigen-binding fragments are covalently coupled to the CM5 sensor chip up to 1500 resonance units (using a concentration of 10 μg/mL in 10 mM acetate buffer and pH appropriate for the specific selected antibody or antigen-binding fragment tested). Target protein is injected (40 μL) at concentrations between 5 and 250 nM at a flow rate of 30 μL/min. Ten microliters of a 10 mM HCl solution is used to regenerate the chip after each cycle. Association and dissociation rate constants are calculated with the software of the BIAcore™ 3000 (Langmuir binding model).

US10982258B2. Methods of sequencing, determining, pairing, and validating therapeutic agents and disease specific antigens (2021-04-20). AbVitro LLC [US]. Inventors: Francois Vigneault [US], Adrian Wrangham Briggs [US], Christopher Ryan Clouser [US], Stephen Jacob Goldfless [US], Sonia Timberlake [US].
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2

Benchmarking Automated Holdup Assay

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Automated holdup assay and the protocol of binding intensity calculations have been benchmarked on a previously published data set. This data set consists of 210 interactions and non-interactions between 5 His-MBP-PDZ domain constructs and 42 biotinylated C-terminal peptides for which binding intensities had been obtained in a previous study using Surface Plasmon Resonance (BIAcore) 12 (link). Like in that study, normalized Response Units (RUs) obtained for the interaction experiments (see Supplementary Table 1) were used instead of dissociation constants to interpret binding intensities of measured interactions.
Uncorrected binding intensities obtained for crude protein samples using the holdup assay correlate well with the reference binding intensities determined using BIAcore (r=0.69). Better correlations can even be obtained when comparing input-corrected holdup assay binding data with the BIAcore data (r=0.76). This analysis indicates that input correction using standard peaks results in more reliable binding intensities (see Supplementary Data 1 and 2 (folder (“benchmarkHU”) as well as Supplementary Table 1 for exported caliper data, parameters and binding intensities).
Vincentelli R., Luck K., Poirson J., Polanowska J., Abdat J., Blémont M., Turchetto J., Iv F., Ricquier K., Straub M.L., Forster A., Cassonnet P., Borg J.P., Jacob Y., Masson M., Nominé Y., Reboul J., Wolff N., Charbonnier S, & Travé G. (2015). Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. Nature methods, 12(8), 787-793.
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3

Antibody Binding Affinity to FcRs

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The ability of antibodies to bind to FcRs was determined using surface plasmon resonance (Biacore). The CM5 sensor chip was activated by injecting 25 µL of a mixture of an equal mixture of NHS (0.1 M in water) and EDC (0.1 M in water) at 7 µL/min. Next, 4.5 µg/mL of FcR diluted in 10 mM acetate buffer (pH 4.5) was injected over the chip surface at 7 µL/min for 10 min to coat. Unreacted sites were subsequently deactivated by injecting 25 µL of 1 M ethanolamine (pH 8.5) at 7 µL/min. 125 ug/mL of antibody were then run over the chip at a flow rate of 25 µL/min. The affinity (RU), on-rate (Ka), off-rate (Kf) and overall binding affinity (Kd) was compared among the different groups.
Dugast A.S., Chan Y., Hoffner M., Licht A., Nkolola J., Li H., Streeck H., Suscovich T.J., Ghebremichael M., Ackerman M.E., Barouch D.H, & Alter G. (2014). Lack of Protection following Passive Transfer of Polyclonal Highly Functional Low-Dose Non-Neutralizing Antibodies. PLoS ONE, 9(5), e97229.
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4

Surface Plasmon Resonance and ITC of PI(4,5)P2 Liposomes

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Surface plasmon resonance (Biacore) and ITC experiments were performed using PI(4,5)P2 liposomes as described4 (link).
Chopard C., Tong P.B., Tóth P., Schatz M., Yezid H., Debaisieux S., Mettling C., Gross A., Pugnière M., Tu A., Strub J.M., Mesnard J.M., Vitale N, & Beaumelle B. (2018). Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells. Nature Communications, 9, 2251.
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5

Measuring IGF-1 Receptor Binding Kinetics

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Example 3

High affinity binding of hIGF-1 and IGF-1 variants to rhIGF1R was measured using surface plasmon resonance (Biacore). A direct binding assay was performed. Human IGF-1-Ea_Fc_Mut_13/2_A and hIGF-1 were immobilized on a chip and the IGF-1 receptor served as analyte in solution. The resulting sensorgrams were fitted to a 1:1 interaction model to calculate the equilibrium dissociation constants (KD). The results show that binding of hIGF-1-Ea_Fc_Mut_13/2_A to IGF-1R is comparable to hIGF-1 (FIG. 1).

US10167329B2. Stabilized insulin-like growth factor polypeptides (2019-01-01). None [CH], None [CH], None [CH]. Inventors: Mara Fornaro [CH], Thomas Huber [CH], Mauro Zurini [CH].
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6

Surface Plasmon Resonance Kinetic Analysis

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Surface plasmon resonance (Biacore) assays were performed on CM-5 dextran chips (GE) covalently coupled to the ligand via amine coupling. The running and injection buffers were matched and consisted of 150 mM NaCl, 0.01% Tween-20, 0.1 mg/ml BSA, and 10 mM HEPES at pH 7.5. Response unit (RU) values were measured as a function of analyte concentration at 298 K. Kinetic analysis was performed using the global fitting feature of Scrubber 2 (BioLogic Software) assuming a 1:1 binding model. For experiments using hAPN as a ligand, between 300 and 400 RU were coupled to the CM-5 dextran chips. For experiments using 9.8E12, 1900 RU was immobilized.
Wong A.H., Tomlinson A.C., Zhou D., Satkunarajah M., Chen K., Sharon C., Desforges M., Talbot P.J, & Rini J.M. (2017). Receptor-binding loops in alphacoronavirus adaptation and evolution. Nature Communications, 8, 1735.
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7

PD-1 Binding and Blockade Assays

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In vitro binding of spartalizumab to PD-1 was assessed using surface plasmon resonance (Biacore). PD-1 immunoglobulin was covalently bound as ligand to a CM-5 chip, and spartalizumab was passed over in serial dilutions at a rate of 50 µL/min.
Spartalizumab was tested for its ability to block the binding of PD-L1 and PD-L2 to PD-1 in a competitive flow cytometry binding assay. Murine 300.19 cells expressing PD-1 were incubated with solutions that contained a constant concentration of PE-labeled PD-L1-Fc or PD-L2-Fc and serial dilutions of spartalizumab at 4°C for 4 hours. Bound labeled PD-L1-Fc or PD-L2-Fc were then quantified using fluorescence-activated cell sorting (FACS), and half maximal inhibitory concentration (IC50) values were derived from best-fit competition curves generated with Prism GraphPad software.
Naing A., Gainor J.F., Gelderblom H., Forde P.M., Butler M.O., Lin C.C., Sharma S., Ochoa de Olza M., Varga A., Taylor M., Schellens J.H., Wu H., Sun H., Silva A.P., Faris J., Mataraza J., Cameron S, & Bauer T.M. (2020). A first-in-human phase 1 dose escalation study of spartalizumab (PDR001), an anti–PD-1 antibody, in patients with advanced solid tumors. Journal for Immunotherapy of Cancer, 8(1), e000530.
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8

Serum Stability Evaluation of Seldegs

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For serum stability assays, endogenous IgGs were depleted from human male AB plasma (Sigma) by passage through protein G-Sepharose (GE Healthcare). Seldegs were incubated in serum at a concentration of 100 nM at 37 °C for 3 or 5 days. Following incubation, Seldegs were immunoprecipitated using agarose beads coupled to goat anti-human Fc-specific antibody (Sigma). Immunoprecipitated Seldegs were run on 12% SDS–polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore) and membranes incubated with horseradish peroxidase-conjugated goat anti-human Fc-specific (H+L) antibody (Jackson ImmunoResearch). Bound secondary conjugate was detected using Westernsure substrate, followed by scanning with a C-DiGit blot scanner (LI-COR).
Seldegs were also incubated in PBS (Lonza) at 4 °C (30 days) or 37 °C for 5 days, followed by analyses using a Superdex 200 5/150 GL column (GE Healthcare), 12% SDS–polyacrylamide gel electrophoresis and surface plasmon resonance (BIAcore).
Devanaboyina S.C., Khare P., Challa D.K., Ober R.J, & Ward E.S. (2017). Engineered clearing agents for the selective depletion of antigen-specific antibodies. Nature Communications, 8, 15314.
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9

Antibody Binding Affinity Kinetics

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EXAMPLE 2

This example determines affinity kinetics for an exemplary antibody (F7) disclosed herein. Table 1 shows affinity kinetics for antibody F7.

TABLE 1
Binding characteristics of antibody F7
mAbka (1/Ms)kd (1/s)KD (M)Chi2
F72.94E52.17E−47.38E−100.805

This example illustrates binding affinities of exemplary anti-CTLA4 antibodies disclosed herein. Affinities were determined using surface plasmon resonance (Biacore). Briefly, Anti-human Fc antibody (GE, BR-1008-39) was immobilized on CM5 sensor chip to approximately 1000 RU using standard NHS/EDC coupling methodology. Antibodies (about 10 μg/ml) were captured for 60 s at a flow rate 10 μl/min Recombinant human CTLA4/His was serially diluted in running buffer (HBS-EP). All measurements were conducted with a flow rate of 30 μL/min Surfaces were regenerated with 3M MgCl2 for 60 s. A 1:1 (Langmuir) binding model was used to fit the data.

US10202453B2. Antibody therapeutics that bind CTLA4 (2019-02-12). Sorrento Therapeutics, Inc. [US]. Inventors: John Dixon Gray [US], Heyue Zhou [US].
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10

Benchmarking Automated Holdup Assay

Cited 1 time
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Automated holdup assay and the protocol of binding intensity calculations have been benchmarked on a previously published data set. This data set consists of 210 interactions and non-interactions between 5 His-MBP-PDZ domain constructs and 42 biotinylated C-terminal peptides for which binding intensities had been obtained in a previous study using Surface Plasmon Resonance (BIAcore) 12 (link). Like in that study, normalized Response Units (RUs) obtained for the interaction experiments (see Supplementary Table 1) were used instead of dissociation constants to interpret binding intensities of measured interactions.
Uncorrected binding intensities obtained for crude protein samples using the holdup assay correlate well with the reference binding intensities determined using BIAcore (r=0.69). Better correlations can even be obtained when comparing input-corrected holdup assay binding data with the BIAcore data (r=0.76). This analysis indicates that input correction using standard peaks results in more reliable binding intensities (see Supplementary Data 1 and 2 (folder (“benchmarkHU”) as well as Supplementary Table 1 for exported caliper data, parameters and binding intensities).
Vincentelli R., Luck K., Poirson J., Polanowska J., Abdat J., Blémont M., Turchetto J., Iv F., Ricquier K., Straub M.L., Forster A., Cassonnet P., Borg J.P., Jacob Y., Masson M., Nominé Y., Reboul J., Wolff N., Charbonnier S, & Travé G. (2015). Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. Nature methods, 12(8), 787-793.
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