Engineered Nanobodies-Based Sandwich-Competitive Immunoassay Broadens the Detection Range for Aflatoxin B1

Competitive immunoassays for small-molecule haptens such as aflatoxin B₁ (AFB₁) are often limited by insufficient sensitivity and narrow dynamic ranges. This study seeks to address these issues through the genetic engineering of nanobodies and the development of novel immunoassay formats using AFB₁ as a model. An innovative elution-free biopanning strategy was developed for the <i>in vitro</i> evolution of an AFB₁-specific nanobody, which proved highly suitable for screening high-performance anti-hapten nanobodies. Using this strategy, several mutants with improved analytical performance were obtained, including one that showed a 3.5-fold reduction in the half-maximal inhibitory concentration (IC₅₀) compared with the parent nanobody. Affinity kinetics of five representative mutants demonstrated a positive correlation between the IC₅₀ values and their affinity for the artificial antigen. By leveraging these mutants, a novel sandwich-competitive immunoassay (SCI) format was constructed using two nanobodies along with a special artificial antigen. The SCI demonstrated a markedly broadened detection range (IC₁₀-IC₉₀) with excellent linearity (<i>R</i>² > 0.98), outperforming conventional competitive assays, which showed compromised linearity outside the IC₂₀-IC₈₀ range (<i>R</i>² < 0.92). The broader detection range of SCI can be attributed to the two-stage competitive binding process of the analyte to the sandwich immunocomplex. Spike-recovery experiments confirmed the accuracy and applicability of the SCI in actual sample detection. This study offers a strategy that revitalizes the <i>in vitro</i> evolution of nanobodies targeting haptens and advances immunoassay development for such molecules.