Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6–200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%–102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications. •A double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on Nanobody.•The assay exhibited good analytical performance, including a broad linear range, low detection limit.•High sensitivity, as well as outstanding reproducibility.•Laying the foundation for the development of a CRP diagnostic kit.