Frequently Asked Questions
Fc Effector Function
The complete protocol for each Fc effector assay is available on the respective product page. For a complete list of assay product pages, see the Fc Effector Activity Bioassays.
Measuring Fc Effector Functions:
- What are Fc effector functions and why do they matter in antibody development?
- Fc effector functions are immune system activity mediated by an antibody’s Fc region, through engagement of Fc receptors and C1q, that trigger ADCC, ADCP, CDC, and regulate half-life via FcRn. They help shape mechanism of action, efficacy, safety (off-target killing/cytokine release), PK/PD, dosing, and drive design choices such as afucosylation, Fc silencing, half-life extension.
- How do I measure effector activity of the antibody Fc region?
- Measure Fc effector activity with a tiered approach: begin with biochemical immunoassays to confirm engagement of FcγR, C1q, and, when relevant, FcRn. Next, run cell-based functional assays for ADCC, ADCP, and CDC to determine relative potency. Finally, confirm orthogonally with a primary cell assay as a bridging method to support your data.
- How do I minimize immobilization artifacts seen in ELISAs?
- Use a no-wash, homogenous immunoassay such as Lumit® FcγR Binding Immunoassays that avoid surface immobilization, delivering fast, reliable quantitation for ranking before functional studies.
- How do I reduce variability from primary cell assays?
- Use primary effector cells, such as ADCC-qualified PBMCs and ADCP-qualified macrophage, that have been prescreened for activity in combination with the HiBiT® Target CelL Killing system. This reduces donor-to-donor variability while keeping a physiologically relevant assay design.
Measuring Antibody-Dependent Cellular Cytotoxicity (ADCC):
- What is ADCC?
- ADCC (antibody-dependent cellular cytotoxicity) is an Fc-mediated mechanism where antibodies bound to a target cell engage FcγRIIIa (CD16a) on NK cells triggering degranulation and perforin/granzyme-mediated lysis of the target. It’s a key effector function, especially for IgG1 antibodies.
- How do I measure ADCC activity?
- Measure ADCC by co-culturing antigen-positive target cells and effector cells expressing FcγRIIIa (V158/F158). Use either (a) reporter bioassays that measure FcγRIIIa signaling or (b) primary-cell killing assays that quantify target lysis (e.g., HiBiT Target Cell Killing Bioassays).
- Which FcγRIIIa allele (V158 vs. F158) should I use, and what’s the impact?
- Use FcγRIIIa V158 when you want a higher-affinity interaction and strong ADCC signal, often used for screening and routine potency work. Use FcγRIIIa F158 for a lower-affinity, more stringent readout that can better reveal differences from Fc engineering or glycan changes like afucosylation. For clinical relevance and comparability, many programs profile both alleles and clearly report which allele was used, keeping it consistent across studies.
Measuring Antibody-Dependent Cellular Phagocytosis (ADCP):
- What is ADCP?
- ADCP (antibody-dependent cellular phagocytosis) is an Fc-mediated mechanism where opsonized targets are engulfed and cleared by phagocytes (macrophages/monocytes), primarily via FcγRIIa (CD32a) with contribution from high-affinity FcγRI (CD64).
- How do I measure ADCP activity?
- Measure ADCP by co-culturing antigen-positive target cells and effector cells expressing FcγRIIa alleles (H131/R131). Use either (a) reporter bioassays that measure FcγRIIa signaling or (b) primary-cell killing assays that quantify target lysis via phagocytosis (e.g., HiBiT Target Cell Killing Bioassays).
- Which FcγRIIa alleles (H131/R131) and FcγRI should I use and what’s the impact?
- Use FcγRIIa-H131 for higher-affinity interactions and certain IgG subclasses, like IgG2 for a stronger ADCP signal. R131 provides a more stringent, discriminating readout. FcγRI (CD64) models high-affinity uptake on macrophages but can mask subtle variant differences observed with FcγRIIa.
Measuring Complement-Dependent Cytotoxicity (CDC):
- What is CDC?
- Complement-dependent cytotoxicity (CDC) is an immune mechanism triggered by antibody binding and a biochemical cascade to activate the complement system, beginning with C1q. This process culminates in the formation of the membrane attack complex (MAC) that punctures the target cell membrane, resulting in cell death.
- How do I measure CDC activity?
- Use the Lumit® C1q Binding Assay as a surrogate measure CDC and detect complement engagement across antibody isotypes and engineered variants; it’s useful for biosimilar comparability and Fc engineering programs.
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