Fc Receptors: Key Reagents for Antibody Drug Half-Life Assessment and In Vitro Functional Validation

The purity of Canine FCGRT&B2M Heterodimer Protein, His Tag&Tag Free (Cat. No. FCM-C52W8) is more than 90% and the molecular weight of this protein is around 43-60 kDa verified by SEC-MALS.

Immobilized Biotinylated Human FCGRT&B2M Heterodimer Protein, His,Avitag (Cat. No. FCM-H82W7) on SA Chip can bind Herceptin® with an affinity constant of 0.267 μM as determined in a SPR assay (Biacore 8K) (QC tested).

Loaded Cynomolgus / Rhesus macaque FCGRT&B2M Heterodimer Protein, His Tag&Strep II Tag (Cat. No. FCM-C5284 ) on SA Biosensor via Biotin his antibody, can bind Herceptin with an affinity constant of 0.13 μM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).

The kit has been used to detect different subclasses of Human IgG, which exhibit different IC50 results as expected.

The kit has been used to detect different subclasses of Human IgG Fc proteins, which exhibit different IC50 results as expected.

Inhibition Assay of interaction of Human FcRn and Human IgG1 antibody by Human IgG standard in a homogeneous (no wash) TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) competition assay, with a typical IC50 of 20.14 μg/mL (QC tested).

The kit has been used to detect the binding activity between Human FcRn and Human IgG standard under different pH. The IC50 shows a significant increase with increasing pH, which corelates with a decreased binding between Human FcRn and Human IgG standard.

The half-lives of these 3 monoclonal antibodies currently in clinical use generally correlate with the binding affinity to FcRn. The kit has been used to detect 3 FDA approved antibody drugs of different binding affinity to FcRn, and the IC50 trends are consistent with affinity constant from SPR as well as the actual in vivo half-life published

The kit is suitable for the detection of FcRn inhibitors. It shows that both Efgartigimod and its biosimilar, Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F) His Tag (Cat. No. IG1-H52H8) exhibit good inhibitory activity in this TR-FRET competition assay.

FACS assay shows that FITC-Labeled Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F) Protein, His Tag (Cat. No. IG1-HF2H3) can bind to HEK293/Human FcRn (FCGRT & B2M) Stable Cell Line. HEK293/Human FcRn (FCGRT & B2M) Stable Cell Line (Cat. No. CHEK-ATP079) was red line, negative control HEK293 cells was grey line(QC tested).

FACS analysis of FcRn (FCGRT & B2M) on HEK293/FcRn (FCGRT & B2M), GFP Tag Stable Cell Line.HEK293/FcRn (FCGRT & B2M), GFP Tag Stable Cell Line (Cat. No. CHEK-ATP132) was red line, negative control HEK293 cells was grey line(QC tested).

The purity of Human CD32a (R167), His Tag (Cat. No. CDA-H5221) is more than 90% and the molecular weight of this protein is around 25-35 kDa verified by SEC-MALS.

Human CD64, His Tag (Cat. No. FCA-H52H1) captured on CM5 chip via anti-His antibody can bind Herceptin® with an affinity constant of 4.92 nM as determined in a SPR assay (Biacore 8K) (QC tested).

Loaded Human CD16a (V176), His Tag (Cat. No. CD8-H52H4) on HIS1K Biosensor, can bind Herceptin with an affinity constant of 0.447 μM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).

Batch consistency of Human Fc gamma RIIB / CD32b (Cat. No. CDB-H5228). The similarity of Human Fc gamma RIIB / CD32b (Cat. No. CDB-H5228) for different batches is more than 90%.

Inhibition Assay of interaction of Europium-chelate labeled human Fc gamma RIIIA / CD16a (F176) and FA labeled human lgG by Human IgG standard in a homogeneous (no wash) TR-FRET (Cat.No.FRT-06) competition assay, with a typical IC50 of 1.246 μM (QC tested).

The kit (Cat.No.FRT-06) has been used to detect different subclasses of Human IgG (Human IgG1, Human IgG2, Human IgG3 and Human IgG4), which exhibit different IC50 results as expected. As shown in the figure, human CD16a (F176) binds to human IgG1, IgG2, IgG3 and IgG4 with low affinity, and IgG1 and IgG3 show the higher affinity than IgG2 and IgG4.

The kit (Cat.No.FRT-06) has been used to detect different subclasses of mouse IgG (mouse IgG1, mouse IgG2a and mouse IgG2b), which exhibit different IC50 results as expected. As shown in the figure, human CD16a (F176) almost has no binding to mouse IgG1, mouse IgG2a and mouse IgG2b.

Verify potential matrix effects by adding different levels of DEME, RPMI1640, FBS and HSA to the Sample Diluted buffer (Cat.No.FRT-06).

Cell surface staining using PE-labeled anti-human CD16a antibody was performed on CHO/Human CD16a (158V) Stable Cell Line (Cat.No. SCCHO-ATP059H) with different expression levels: CHO/Human CD16a (158V) Stable Cell Line (Low Expression); CHO/Human CD16a (158V) Stable Cell Line (Medium Expression); CHO/Human CD16a (158V) Stable Cell Line (High Expression).

Cell surface staining using PE-labeled anti-human CD32a antibody was performed on CHO/Human CD32a Stable Cell Line (Cat.No.SCCHO-ATP061H) with different expression levels: CHO/Human CD32a Stable Cell Line (Low Expression); CHO/Human CD32a Stable Cell Line (Medium Expression); CHO/Human CD32a Stable Cell Line (High Expression).