Nanobody multimerization strategy to enhance the sensitivity of competitive ELISA for detection of ochratoxin A in coffee samples

Enzyme-linked immunosorbent assay (ELISA) is an ideal immunoassay technique for large scale screening detection of various chemical contaminants in food due to its high throughput, low cost and ease of automation. However, the widespread use of traditional colorimetric ELISA has been hindered by the relatively low sensitivity. In this paper, the OTA-specific nanobody (Nb28) was fused with the C4-binding protein (C4bp) C-terminal fragment to form a self-assembled heptamer fusion protein (Nb28-C4bp alpha). In addition, the heptamer with higher affinity was employed to develop an ultrasensitive competitive ELISA for the detection of OTA in coffee samples. The effect of this sensitivity enhancement strategy was demonstrated with the IC50 of 0.13 ng/mL and LOD of 0.009 ng/mL for the heptamer-based ELISA, which is 26.54-fold and 175.56-fold lower than that of the monomer-based ELISA, respectively. The proposed nanobody multimerization can be a particularly appealing strategy to enhance the sensitivity or analytical signals in various immunoassays.

Automated summary

In this study, a heptamer fusion protein was developed by fusing the OTA-specific nanobody with the C4-binding protein, which was utilized to enhance the sensitivity of detecting OTA in coffee samples using a competitive ELISA. The sensitivity enhancement strategy resulted in a significantly lower IC50 and LOD compared to traditional monomer-based ELISA, demonstrating the potential of nanobody multimerization in enhancing analytical signals in immunoassays.