Anti human igg fc capture biosensors

Manufactured by Molecular Devices

The Anti-human IgG Fc capture biosensors are a type of lab equipment designed to detect and measure the presence and concentration of human immunoglobulin G (IgG) Fc fragments in a sample. These biosensors utilize a capture surface that binds specifically to the Fc region of human IgG antibodies, allowing for the quantification of IgG levels in the tested sample.

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15 protocols using anti human igg fc capture biosensors

Kinetic Analysis of Anti-β2GPI Antibody

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Label-free kinetic assays were determined by bio-layer interferometry on a BLItz instrument (ForteBio). Manufacturer’s instructions were followed unless otherwise noted. Sensors were soaked in and reagents diluted in kinetics buffer (ForteBio). Measurements consisted of 5 steps: Initial baseline 30 s, loading 180 s, baseline 120 s, association 300 s, dissociation 300 s. Purified anti-β₂GPI mAb P1-117 was immobilized onto anti-human IgG Fc capture biosensors at a concentration of 10 μg/ml (ForteBio). Sensorgrams were fit globally to a 1:1 binding model by BLItz Pro v1.1.0.28. The equilibrium dissociation constant (K_D) and association (k_a) and dissociation (k_d) rate constants were calculated from a minimum of 4 molar concentrations ranging from 200 nM to 25 nM with a 0 nM control.

Ruff W.E., Dehner C., Kim W.J., Pagovich O., Aguiar C.L., Yu A.T., Roth A.S., Vieira S.M., Kriegel C., Adeniyi O., Mulla M.J., Abrahams V.M., Kwok W.W., Nussinov R., Erkan D., Goodman A.L, & Kriegel M.A. (2019). Pathogenic Autoreactive T and B Cells Cross-React with Mimotopes Expressed by a Common Human Gut Commensal to Trigger Autoimmunity. Cell host & microbe, 26(1), 100-113.e8.

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Binding Kinetics of CHIKV E1 Mabs

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BLI experiments were performed using an Octet Red96 (ForteBio) at 25°C in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant, and 1% BSA (w/v). MAbs were immobilized onto anti-human IgG Fc capture biosensors (ForteBio) and dipped into wells containing 1 μM CHIKV E1 (Native Antigen) to assess E1 reactivity of mAbs.

Raju S., Adams L.J., Earnest J.T., Warfield K., Vang L., Crowe JE J.r., Fremont D.H, & Diamond M.S. (2023). A chikungunya virus-like particle vaccine induces broadly neutralizing and protective antibodies against alphaviruses in humans. Science translational medicine, 15(696), eade8273.

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Binding Kinetics of HCT-mono-mIL12 and HER2

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Binding kinetics and affinity for the interactions of HCT-mono-mIL12 with HER2 antigen (Sino Biological Inc.) were measured using an Octet QKe instrument (ForteBio), as described previously (28 (link)). All kinetic experiments were conducted at 25°C with orbital shaking at 1000 rpm in a volume of 200 μL in 96-well black flat-bottom plates (VWR International, 82050-784). Each purified HCT and HCT-mono-mIL12 variant was diluted to 5 μg/mL in kinetics buffer [phosphate-buffered saline (PBS), pH 7.4, containing 0.02% (v/v) Tween 20] and directly immobilized onto anti-human IgG Fc capture biosensors (ForteBio) with an approximate 1.0 nm response. After an equilibration step of 180 s, the binding isotherms were monitored by exposing separate sensors simultaneously to different concentrations of HER2 antigen. The association of the antigen was measured for 100 s, followed by a dissociation step for 300 s. The association (k_on) and dissociation rate (k_off) constants as well as the equilibrium dissociation constant (K_D) were determined by fitting to sensorgrams via the 1:1 binding model with a correlation coefficient (R²) value equal to or greater than 0.99 in Octet Data Analysis software version 11.0 (ForteBio).

Jung K., Yoo S., Kim J.E., Kim W, & Kim Y.S. (2022). Improved intratumoral penetration of IL12 immunocytokine enhances the antitumor efficacy. Frontiers in Immunology, 13, 1034774.

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Kinetic Analysis of Antibody-Virus Interactions

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We used BLI using an Octet Red 96 instrument to estimate the relative k_obs, which is the observed rate constant. Human monoclonal antibodies (clones S9-1-10/5-1, CR9114, 4-6-19/6, or 1429C6/3-3) at 2 μg/ml in binding buffer (PBS pH 7.4 including 0.1% BSA) were loaded onto anti-human IgG Fc capture biosensors (ForteBio) and incubated with 3 concentrations of the purified CA04/PB2-KO virus, which is a replication incompetent PB2-knockout PR8 virus possessing HA and NA segments derived from CA04 (Ozawa et al., 2011 (link)). All binding data were collected at 30 °C. The experiments comprised 3 steps: (1) antibody loading onto the biosensor (300 s); (2) baseline acquisition (60 s); and (3) association of CA04/PB2-KO virus for the measurement of k_obs (600–3600 s).

Yamayoshi S., Uraki R., Ito M., Kiso M., Nakatsu S., Yasuhara A., Oishi K., Sasaki T., Ikuta K, & Kawaoka Y. (2017). A Broadly Reactive Human Anti-hemagglutinin Stem Monoclonal Antibody That Inhibits Influenza A Virus Particle Release. EBioMedicine, 17, 182-191.

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Biolayer Interferometry Kinetics of HIV-1 Env Binding

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BLI using the Octet.Red instrument (Forte Bio) were performed at room temperature with shaking at 1,000 RPM following the manufacturer’s instructions. Briefly, 100 μg/mL of cloned antibodies were loaded on anti-human IgG Fc capture biosensors (Forte Bio) for 240 seconds followed by a wash step in kinetics buffer (1X DPBS, 0.01% BSA, 0.02% Tween 20, and 0.005% NaN_3, pH 7.4) for 60 seconds. After washing, 1 μM HIV-1 Env was associated for 300 seconds followed by dissociation into kinetics buffer for an additional 300 seconds. The HIV-1 envelope-specific antibody VRC01 was used as a positive control in all experiments, and the RSV-specific antibody palivizumab was used as a negative control.

Steach H.R., DeBuysscher B.L., Schwartz A., Boonyaratanakornkit J., Baker M.L., Tooley M.R., Pease N.A, & Taylor J.J. (2019). Cross-reactivity with self-antigen tunes the functional potential of naïve B cells specific for foreign antigens. Journal of immunology (Baltimore, Md. : 1950), 204(3), 498-509.

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Kinetics of Antibody-Antigen Binding

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The kinetics of NHP and human MAb binding to a panel of Env variants were assessed with an Octet RED96 system (ForteBio) with Bio-Layer Interferometry (BLI) in a 96-well format following the manufacturer's instructions as described previously [30] . Abs at 10 µg/ml in binding buffer (PBS/0.2% Tween 20) were captured on the surface of the anti-human IgG Fc capture biosensors (ForteBio) for 1 minute, followed by 1 minute wash in binding buffer to establish a new baseline signal, and the biosensor is then immersed in wells containing the Env variants which are 2-fold serially diluted in binding buffer at an initial starting concentration of 250 nM. Ab-Env association rate (on-rate) and the dissociation rate (off-rate) were measured over a 2-min interval, respectively. K_D values (in nanomolar) were calculated as off-rate/on-rate.

Navis M., Tran K., Bale S., Phad G.E., Guenaga J., Wilson R., Soldemo M., McKee K., Sundling C., Mascola J., Li Y., Wyatt R.T, & Karlsson Hedestam G.B. (2014). HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization. PLoS Pathogens, 10(8), e1004337.

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MACV GP1-Fc Competition Assay

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All assays were performed using an Octet RED96 system (ForteBio, Fremont, CA, USA). For MACV GP1-Fc competition experiments, MACV GP1-Fc was loaded onto anti-human IgG Fc capture biosensors (ForteBio) at 17 nM for 100 s in kinetic buffer PBS containing 0.02% (v/v) Tween and 0.1% (w/v) bovine serum albumin. After equilibration in the buffer for 120 s, sTfR1 alone at 1.5 µM or in the presence of soluble ligands (ch128.1 Fab or Tf) at 2 µM were associated for 300 s. This was followed by a 300 s dissociation step.

Hickerson B.T., Daniels-Wells T.R., Payes C., Clark L.E., Candelaria P.V., Bailey K.W., Sefing E.J., Zink S., Ziegenbein J., Abraham J., Helguera G., Penichet M.L, & Gowen B.B. (2022). Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections. Nature Communications, 13, 558.

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Kinetic Analysis of Antibody-Antigen Binding

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The antibody and gp120 variants binding kinetics analyses were performed as previously described (16 ). Briefly, an Octet RED96 system (ForteBio) was used to assess the kinetics of rabbit MAb and human MAb binding to gp120 core or RSC3 with Bio-Layer Interferometry (BLI). Prior to the assay, both gp120 core and RSC3 were subjected to size exclusion chromatography to remove undesired oligomeric forms when applicable. Human MAbs at 10 μg/ml in PBS/0.2% TWEEN 20 were captured on the surface of the anti-human IgG Fc capture biosensors (ForteBio) for 1 min. SA-sensor (ForteBio) was used to initially capture biotin-labeled mouse anti-rabbit IgG Fc (Sigma-Aldrich) MAb at 10 μg/ml, followed by loading the rabbit MAbs. The biosensor tip was then immersed in wells containing gp120 core or RSC3 in PBS/0.2% TWEEN 20. The samples were two-fold serially diluted from an initial starting concentration of 250 nM. K_D, the affinity constant value in nanomolars was calculated as off-rate/on-rate. The sensograms were corrected with the blank reference and fit with the software ForteBio Data Analysis 6.4 using a 1:1 binding model with the global fitting function (grouped by color, Rmax).

Chen Y., Wilson R., O’Dell S., Guenaga J., Feng Y., Tran K., Chiang C.I., Arendt H.E., DeStefano J., Mascola J.R., Wyatt R.T, & Li Y. (2016). An HIV-1 Env-antibody Complex Focuses Antibody Responses to Conserved Neutralizing Epitopes. Journal of immunology (Baltimore, Md. : 1950), 197(10), 3982-3998.

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Quantifying TRAIL-R2 Binding Affinity

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Binding affinity measurement was performed using an Octet RED96 System (ForteBio, Menlo Park, CA, USA). Anti-Human IgG Fc Capture biosensors (ForteBio) were used to immobilized rhTRAIL-R2/Fc and rmTRAIL-R2/Fc chimera proteins (R&D systems). Biosensors were hydrated in the kinetic buffer for 10 min prior to protein immobilization. rhTRAIL-R2/Fc and rmTRAIL-R2/Fc chimera proteins were immobilized for 50 s. Then, the sensors were washed with kinetic buffer for 60 s and reacted with various concentrations (from 6.25 to 100 nM) of TLY012 for 200 s association steps. After association, the sensors were moved to the kinetic buffer for dissociation steps. The association and dissociation data were corrected by the baseline response in the kinetic buffer and the equilibrium dissociation constant (K_D) was calculated using Octet Data Analysis software (ForteBio).

Park J.S., Oh Y., Park Y.J., Park O., Yang H., Slania S., Hummers L.K., Shah A.A., An H.T., Jang J., Horton M.R., Shin J., Dietz H.C., Song E., Na D.H., Park E.J., Kim K., Lee K.C., Roschke V.V., Hanes J., Pomper M.G, & Lee S. (2019). Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma. Nature Communications, 10, 1128.

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Biolayer Interferometry Binding Assay

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Biolayer interferometry (BLI) assays were performed on the Octet Red instrument (ForteBio) as previously described (Lin et al., 2020 (link); Parks et al., 2019 (link)). Briefly, anti-human IgG FC capture biosensors (ForteBio) were used to immobilize mAbs (20 μg/μL in PBS) for 5 min, followed by baseline interference reading for 60 s in kinetics buffer (PBS, 0.01% BSA, 0.02% Tween-20, 0.005% NaN₃). Sensors were then immersed into wells containing Env Core monomers (2 μM) diluted in kinetics buffer for 300 s (association phase) and another 300 s (dissociation phase). mVRC01 and glVRC01 mAbs were used as internal controls for comparison. All measurements were corrected by subtracting the signal obtained from simultaneous tracing of the corresponding Env using an irrelevant IgG Abs in place of the mAbs tested. Curve fitting was performed using the Data analysis software (ForteBio).

Knudsen M.L., Agrawal P., MacCamy A., Parks K.R., Gray M.D., Takushi B.N., Khechaduri A., Salladay K.R., Coler R.N., LaBranche C.C., Montefiori D, & Stamatatos L. (2022). Adjuvants influence the maturation of VRC01-like antibodies during immunization. iScience, 25(11), 105473.

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