At4g01130 Antibody

How to validate antibody specificity for novel targets like At4g01130?

• Methodology:

  • Perform flow cytometry or surface plasmon resonance to measure binding affinity (e.g., KD ≈ 178 pM in IL-4Rα studies ).

  • Use alanine scanning mutagenesis to map epitopes and confirm target specificity (as in IL-4Rα antibody engineering ).

  • Validate with negative controls (e.g., HEK293T cells lacking the target antigen ).

What experimental designs minimize off-target effects in antibody development?

• Strategies:

  • Employ bispecific formats (e.g., PD-L1×4-1BB antibodies) to localize activity to disease microenvironments .

  • Integrate transgenic mouse models expressing humanized targets to assess in vivo toxicity and efficacy .

How to resolve discrepancies in antibody-antigen binding data?

• Troubleshooting framework:

How to engineer antibodies for resistant targets?

• Approach:

  • Use computational design tools (e.g., RosettaAntibodyDesign) to model CDR loops and optimize interface energy .

  • Validate with flexible-backbone protocols to account for conformational changes .

  • Test bispecific formats to overcome resistance mechanisms (e.g., ATG-101 for PD-L1/4-1BB co-targeting ).

What metrics define antibody developability?

• Key parameters:

  • Risk ratio (DRR): Native CDR recovery frequency ÷ sampling frequency (benchmarked at DRR >1.0 in RosettaAntibodyDesign ).

  • Thermodynamic stability: Assessed via differential scanning calorimetry (DSC) or accelerated aggregation assays .

How to prioritize antibodies for translational studies?

• Criteria:

  • In vitro potency: ≥50% inhibition in reporter cell systems (e.g., IL-4/IL-13 signaling blockade ).

  • Safety profile: Normal liver function in primate models (e.g., ABL503 bispecific antibody ).