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The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (K d ), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the K d  of antigen-antibody interactions in solution. The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the K d  values of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent K d  values. This HiBiT-qIP assay showed a considerable variation in the K d  values among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.

HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions