Target N297a Mutations with Precision—Now Available with R-PE Labeling for FACS Applications

Our monoclonal Mouse anti-Human IgG1(N297A) antibody is designed for precise detection of the N297A mutation in the Fc region of human IgG1, offering unmatched specificity with no cross-reactivity to the wild-type Fc region or other Fc mutations. This antibody is ideal for research and development of therapeutic antibodies incorporating the N297A mutation to avoid Fc receptor binding, commonly used in clinical applications to reduce immune activation. Available in both untagged and R-PE conjugated forms, it is optimized for a range of applications including flow cytometry (FACS), Western blotting, and ELISA, ensuring consistent, reliable results across diverse experimental platforms.

Product Summary:

Monoclonal Mouse anti-Human IgG1(N297A) (Clone: CB42) antibodies recognize mutated (N297A) Fc fragment of Human IgG1. Do not cross-react with wild type or other mutations in the Fc region. Can be used for a highly specific detection of therapeutic recombinant antibodies that carry N297A mutation.  N297A mutation prevents glycosylation of human IgG1 Fc region, and thus abrogates immunoglobulin interaction with the Fc-receptors. This mutation is commonly introduced to recombinant therapeutic antibodies to prevent Ab-mediated adverse reactions in patients. 

Available Products

Select from our available antibody formats, including unconjugated, biotinylated, and R-PE tagged options

Key Features and Benefits

1. Highly Specific Detection of N297A Mutation

• Selective Binding: This monoclonal antibody specifically recognizes the N297A mutation in the Fc region of human IgG1 without cross-reacting with the wild-type Fc region or other Fc mutations, enabling researchers to reliably distinguish N297A-modified antibodies from others in their assays.
• Targeted Use: Ideal for therapeutic antibody development, such as detecting recombinant antibodies carrying the N297A mutation, including drugs like TECENTRIQ® (Atezolizumab), where preventing Fc receptor binding is critical for therapeutic safety.

2. Available in Biotinylated, R-PE Conjugated, and Untagged Formats

• Flexible Conjugation Options: Offered in purified, unconjugated form, biotinylated, and as an R-PE conjugate, the antibody provides options for various applications in flow cytometry (FACS), Western blotting, ELISA, and other assay types where fluorescence detection or traditional labeling may be required.
• Bright and Stable Fluorescence: The R-PE conjugate enables highly sensitive detection, ensuring clear and specific signals, particularly in flow cytometry applications for cellular or protein analysis.
• Additional Options: Fill out the form and request a quote for custom labeling with the fluorophore of your choice.

3. Application Versatility

• Validated Across Multiple Techniques: This antibody has been internally validated for use in Western blotting, ELISA, and flow cytometry, making it a versatile choice for researchers needing consistency across diverse assay platforms.
• High Sensitivity in Detection Assays: Exhibits high sensitivity for detecting low levels of N297A-modified antibodies in complex samples, providing robust results in assays where precise quantification and sensitivity are essential.

4. Supporting Development of Safer Therapeutic Antibodies

• Designed for Immune Modulation Studies: The N297A mutation in the Fc region disrupts glycosylation, preventing immunoglobulin interaction with Fc receptors, thus reducing antibody-mediated immune responses. This makes the antibody a valuable tool in developing and validating therapeutic antibodies with reduced FcγR binding, lowering potential adverse reactions in patients.
• Optimized for Antibody Engineering: The specificity for the N297A mutation supports research on therapeutic antibodies engineered to avoid immune activation, helping to streamline therapeutic development pipelines and enhance drug safety profiles.

Applications and Protocols

Applications

• Therapeutic Antibody Detection: Detect and validate therapeutic antibodies containing the N297A mutation, such as Atezolizumab (TECENTRIQ®), by distinguishing them from wild-type IgG1 and other Fc variants.
• Flow Cytometry (FACS): Leverage the R-PE conjugated anti-N297a antibody to perform high-sensitivity staining and detection of N297A-mutated antibodies on cell surfaces. Useful for profiling immune cells or antibody screening in PBMC and similar samples.
• Western Blotting and ELISA: Use the unconjugated version to probe N297A mutations in recombinant antibodies, allowing clear detection and quantification of these specific mutations through traditional blotting and ELISA techniques.

Protocols

• Flow Cytometry Protocol:
  • Stain PHA-activated human PBMCs with anti-N297A-PE to detect N297A-modified antibodies, with guidance on cell staining concentrations, incubation times, and appropriate negative controls.
• ELISA Setup:
  • Bind target antibodies to ELISA plate wells and incubate with the anti-N297A antibody (unconjugated). Detect with a secondary anti-mouse HRP conjugate and TMB substrate for visualization.
• Western Blot Protocol:
  • Following PAGE and nitrocellulose transfer, incubate with anti-N297A (100 ng/ml). Use goat anti-mouse HRP and ECL substrate for clear visualization of N297A-labeled antibodies.

Scientific Data and Validation Studies

Flow Cytometry Data:

PHA-activated Human PBMC were stained with: (A) anti-PDL1 IgG1(wt); (B) anti-PDL1 IgG1(LALA-PG); and (C) anti-PDL1 IgG1(N297A). Then the bound Abs were detected with PE conjugated to mouse anti-human IgG1(N297A) (filled histograms). Cells stained only with anti-human IgG1(N297A)-PE conjugate (open histograms) were used as a negative control.

ELISA Sensitivity:

Detection of wild type (blue) or N297A human IgG1. Target antibodies (2.5 µg/ml) were directly bound to ELISA plate wells and then incubated with indicated amount of purified mouse anti-human IgG1(N297A) Mab. After 2h’ incubation and plate washing the bound murine Mab were detected with goat anti-mouse HRP conjugate (HRP-112) and stabilized TMB substrate.

Western Blot Validation:

The indicated recombinant human antibodies were resolved on PAGE (4-12% NuPage) transferred to nitrocellulose membrane and probed with mouse anti-human IgG1(N297A) Mab (100 ng/ml, 2h’ at room temperature). Then the bound MAb were detected with goat anti-mouse HRP conjugate (HRP-112) and stabilized ECL substrate.

Additional Resources and Support

Document Library: Access our document library for easy retrieval of COAs specific to each batch of antibody, ensuring confidence in product quality and consistency.

What Makes a Good Conjugate…Great?: Learn about the standards of high-quality conjugates and factors that impact conjugate stability and performance in assays.

Custom Conjugation Services: Learn more about our custom conjugation services, including fluorophore labeling options to support your specific research applications.

Contact Us: Please let us know if you need additional information or assistance. We look forward to your inquiry.