Table 1 Biophysical characterization that aids in iterative immunogen design.

X-ray Crystallography

Determine the exact positions and orientations of amino acids in the immunogen vs WT antigen when bound to the target Ab, especially at the paratope-epitope interface¹⁵.

Biolayer Interferometry

Quantify differences in binding affinity to WT antigen vs. the immunogen against the target antibody, as well as the off-target antibodies to verify immunofocusing¹³⁵.

Protein melts

Measure melting temperatures of an immunogen compared to the native antigen to detect differences in thermostability that may be indicative of premature unfolding¹⁴².

Cryo-EM

Capture the epitope specificity of polyclonal sera elicited by an engineered immunogen in a high-throughput manner, specifically via methods such as Cryo-EMPEM¹⁴³.

Competition ELISA

Precisely map preferential targeting of a given epitope by polyclonal sera by measuring immunogen binding in the absence or presence of competing Ab interactions¹⁴⁴.

Isothermal calorimetry

Calculate differences in the theoretical and measured heat capacity of binding to determine if immunogen-Ab binding recapitulates WT epitope-Ab binding¹⁰¹.