Luminescence-guided engineering of anti-idiotype nanobody-NanoLuc fusion reagents for sensitive monitoring of free therapeutic antibodies.

Therapeutic antibody monitoring requires bioanalytical platforms combining high sensitivity, specificity, and robustness in complex biological matrices. Here, we report the development of a sensitive bioluminescent bridging immunoassay for detecting free, paratope-accessible therapeutic antibodies using anti-idiotype nanobody-NanoLuc fusion reagents. To streamline reagent generation, we implemented a luminescence-guided workflow integrating camelid immunization with a therapeutic monoclonal antibody cocktail, competitive phage display enrichment for paratope-directed binders against adalimumab, and direct screening of enriched pools at the nanobody-NanoLuc fusion level in Escherichia coli. This approach enabled early identification of highly active chimeras while preserving functional specificity, overcoming common nanobody-dependent expression bottlenecks during assay development. Selected binders were incorporated into a bridging assay format using a biotinylated nanobody as capture reagent and a nanobody-NanoLuc chimera for signal generation. The resulting assay showed a linear response over the 0-50 ng/mL range (R2 = 0.998), with a limit of detection of 0.22 ng/mL. Recovery studies performed in adalimumab-spiked human serum within the clinically relevant therapeutic range demonstrated high accuracy and precision. Application to a limited panel of clinically stratified patient samples yielded measurements consistent with the expected treatment response status. These results support a modular bioanalytical workflow for free therapeutic antibody monitoring and the broader selection of functional Nb-NanoLuc reagents for immunoassay applications.