Nanobody based immunoassay for alpha fetal protein detection using streptavidin-conjugated polymerized horseradish...

The enzyme-linked immunosorbent assay (ELISA) offers several advantages, including simple operation, high throughput, and low cost, making it an ideal immunoassay method for efficient screening of disease-related biomarkers in clinical samples. However, the traditional colorimetric ELISA has relatively low sensitivity, which promotes the continuous emergence of various novel signal amplification technologies. In this work, we fused the AFP-specific nanobody (A1) with the streptavidin-binding peptide (SBP) to develop a fusion protein (A1-SBP) as biorecognition element in a colorimetric ELISA for detecting AFP. Besides, to further improve the sensitivity of the traditional colorimetric ELISA, the streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) were selected as a detection probe for signal amplification. The proposed signal enhancement strategy demonstrated a limit of detection (LOD) of 0.597 ng/mL for the SA-polyHRP-based ELISA, which is 7.67-fold lower than that of the traditional SA-HRP-based ELISA without additional steps. Furthermore, the proposed SA-polyHRP-based ELISA showed a good correlation with the detection of clinical samples using the Roche E601 chemiluminescence immunoassay analyzer. Therefore, the proposed signal enhancement strategy is an attractive approach for improving the sensitivity of immunoassay without requiring additional steps.