Anti-Human IgG Fc Specific Antibodies

Bio-Rad offers four antibody clones specific to human IgG Fc that can be used to detect human or humanized therapeutic antibodies in non-human primate samples.

Clone AbD27686 (HCA285) is a recombinant monovalent Fab antibody, specific to human IgG1 Fc, with an affinity of 76 nM. It has been shown to have a greatly reduced affinity (about 90-fold) for IgG from cynomolgus macaque compared to human IgG1. It also has strongly reduced binding to rhesus macaque IgG. It does not recognize other human immunoglobulin (Ig) isotypes or mouse IgG2a (Figure 1).

This antibody has been generated by Bio-Rad using the HuCAL^® antibody library and CysDisplay^® technology. It is sold for research use, with implicit permission for use for the detection of therapeutic antibodies from samples obtained from experimental animals. When tested by Bio-Rad in an antigen capture pharmacokinetic (PK) assay format, this antibody performs similarly to Mouse Anti-Human IgG (CH2 Domain) Antibody, clone R10Z8E9, in 10% cynomolgus macaque serum (Figure 2). It recognizes a different epitope on human IgG1 Fc than clone R10Z8E9 (Figure 3).

Clone R10Z8E9 (MCA5748G) is a mouse monoclonal antibody specific to human IgG CH2 domain. It has been demonstrated by ELISA not to recognize IgG from the non-human primate species baboon, marmoset, rhesus and cynomolgus macaques; nor does it recognize mouse or rat immunoglobulin.

Important note: Clone R10Z8E9 may not be used for the detection of therapeutic antibodies from samples obtained from experimental animals without prior consent by the owner of EP 1 853 921 (F. Hoffmann-La Roche AG, Basel, Switzerland) and /or US 7,955,806 (Hoffmann-La Roche, Inc., Nutley, NJ).

Clone AbD13299 (TZA098) is a recombinant Fab antibody, specific to the CH2 domain of human IgG, with an affinity of 4 nM. Clone AbD13299 recognizes the same epitope as Mouse Anti-Human IgG (CH2 Domain) Antibody, clone R10Z8E9 and shows reduced reactivity to immunoglobulins from rat, mouse, or non-human primate species (rhesus and cynomolgus macaques).

Clone AbD13299 is available as a monovalent Fab antibody incorporating a SpyTag at the C-terminus end of the antibody heavy chain for conversion by the end user into alternative formats using any of the SpyCatchers available in our catalog under product code TZA098. It is also available as a bivalent Fab antibody already conjugated to HRP (TZA098P) via coupling to the HRP-conjugated BiSpyCatcher2 (TZC002P) or conjugated to biotin (TZA098B) via coupling to the biotin-conjugated BiSpyCatcher2 (TZC002B).

This antibody has been generated by Bio-Rad using the HuCAL antibody library and is sold for research use only, with implicit permission for the detection of therapeutic antibodies from samples obtained from experimental animals.

Note: TZA098 is not available for sale in North America.

Clone AbD27686 (TZA043) is a recombinant monovalent Fab antibody that specifically recognizes the human IgG1 Fc region, with an affinity of 76 nM. It does not recognize other human immunoglobulin isotypes (IgG2, IgG3, IgG4, IgA, IgE, IgM) or mouse IgG2a and it has been shown to have a greatly reduced affinity for cynomologue macaque IgG (89-fold) and rhesus macaque compared to human IgG1.

This antibody has been generated by Bio-Rad using the HuCAL antibody library and CysDisplay technology. It is sold for research use, with implicit permission for use for the detection of therapeutic antibodies from samples obtained from experimental animals. When tested by Bio-Rad in an antigen capture pharmacokinetic assay format, this antibody performs similarly to Mouse Anti-Human IgG (CH2 Domain) Antibody clone R10Z8E9 in 10% cynomolgus macaque serum. It recognizes a different epitope on human IgG1 Fc than clone R10Z8E9.

Antibodies Specific to Human IgG Fc
 

¹ F=DYKDDDDK-tag H=His-6-tag
² 89-fold lower affinity to cynomolgus macaque IgG than to human IgG1
³ Consent required by the owner of EP 1 853 921 (F. Hoffmann-La Roche AG, Basel, Switzerland) and /or US 7,955,806 (HOFFMANN-LA ROCHE, INC., Nutley, NJ)
 ⁴ Fab-F-Spy2-H = Monovalent Fab antibody with (F) DYKDDDDK, (Spy2) SpyTag version 2, and (H) His-6-tags
⁵ Fab2-FH-X22-HRP= Bivalent Fab antibody with (F) DYKDDDDK, (H) His-6-tags, and Fab-SpyTag coupled to HRP-conjugated BiCatcher
⁶ Fab2-FH-X22-bio= Bivalent Fab antibody with (F) DYKDDDDK, (H) His-6-tags, and Fab-SpyTag coupled to biotin-conjugated BiCatcher

Specificity of Antibody Clone AbD27686 (HCA285)

Fig. 1. Demonstration of specificity to human IgG1 and strongly reduced binding to rhesus and cynomolgus macaque IgG.

A microtiter plate was coated overnight with various antibodies at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, increasing concentrations of the Human Anti-Human IgG1 Fc Antibody (clone AbD27686, HCA285) was added. Detection was performed using a Mouse Anti-Penta Histidine Tag:HRP (MCA5995P) in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.

Pharmacokinetic Assay Antigen Capture Format

Fig. 2. PK ELISA antigen capture format demonstrating detection of a humanized therapeutic antibody with anti-human IgG Fc-specific antibodies in cynomolgus serum.

A microtiter plate was coated overnight with VEGF at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, 10% cynomolgus macaque serum was added spiked with increasing concentrations of bevacizumab (hIgG1 kappa). Detection was performed using either HRP-conjugated Anti-Human IgG1 Fc Antibody (clone AbD27686, HCA285P), HRP-conjugated Mouse Anti-Human IgG (CH2 Domain) Antibody (clone R10Z8E9, MCA5748G) or, as a control for an antibody that is not human Fc specific, HRP-conjugated Mouse Anti-Human Fc CH2 Domain Antibody (clone MK 1 A6, MCA647P) at a concentration of 2 µg/ml in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Inhibition ELISA Demonstrating Antibodies Binding to Different Epitopes of Human IgG

Fig.3. Antibody clones AbD27686 and R10Z8E9 bind to different epitopes on human IgG.

A microtiter plate was coated overnight with Mouse Anti-Human IgG (CH2 Domain) Antibody clone R10Z8E9, at a concentration of 1 µg/ml. After washing and blocking with PBST + 5% BSA, a pre-incubated mixture of Recombinant Human IgG1 Kappa (HCA192) (0.5 µg/ml) plus increasing concentrations of either Human Anti-Human IgG1 Fc Antibody (clone AbD27686, HCA285) or Mouse Anti-Human IgG (CH2 Domain) Antibody (clone R10Z8E9, MCA5748G) was added. Free human IgG1 kappa antibody, still capable of binding to the R10Z8E9 coated plate, was detected using HRP-conjugated R10Z8E9 antibody at a concentration of 2 µg/ml in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Licensed use of clone AbD27686 (HCA285 and HCA285P): for in vitro research purposes and for commercial applications for the provision of in vitro testing services to support preclinical and clinical drug development. Any re-sale in any form or any other commercial application needs a written agreement with Bio-Rad.

Specificity of Antibody Clone AbD13299 (TZA098)

A microtiter plate was coated overnight with various antibodies at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, increasing concentrations of the Human Anti-Human IgG (CH2 Domain)  (clone AbD13299, TZA098) was added. Detection was performed using a Mouse Anti-Penta Histidine Tag:HRP (MCA5995P) in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.

Pharmacokinetic Assay Antigen Capture Format

Fig. 5. PK ELISA antigen capture format demonstrating detection of a humanized therapeutic antibody with anti-human IgG Fc-specific antibodies in cynomolgus serum.

A microtiter plate was coated overnight with human TNF alpha at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, 10% cynomolgus macaque serum was added spiked with increasing concentrations of infliximab (hIgG1 kappa). Detection was performed using either Human Anti-Human IgG1 (Fc):Biotin (clone AbD27686, TZA043B), or biotin-conjugated Mouse Anti-Human IgG (CH2 Domain) Antibody (clone R10Z8E9, MCA5748G). This was followed by HRP-conjugated in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Fig. 6. Antigen capture ELISA comparing the various antibodies available.

A microtiter plate was coated overnight with human TNFalpha at a concentration of 5 μg/mL. After washing and blocking with PBST + 5% BSA, infliximab titrated in PBST + 10% cynomolgus monkey serum was added. After washing, detection was performed using various anti-human IgG antibodies (clone AbD13299pap, coupled to BiCatcher-HRP; TZA098P, (0.2μg/mL); clone MCA5748 (2μg/mL), conjugated to HRP; clone AbD27686pab, coupled to BiCatcher-Biotin, TZA043B (0.2μg/mL); clone AbD13299pab, coupled to to BiCatcher-Biotin, TZA098B (0.2μg/mL), clone MCA5748 (2μg/mL), conjugated to biotin). Clones carrying a HRP label were detected directly with QuantaBlu fluorogenic peroxidase substrate. Biotinylated clones were detected by HRP labeled Neutravidin in HISPEC Diluent and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

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