Objective  To prepare nanobodies against N protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and apply them to the detection of N protein antigen of SARS-COV-2.

  Methods  The alpaca was immunized with N protein to construct a gene library of nanobody to N protein. The nanobody with affinity to N protein was screened with phage display technology; nanobody genes were cloned to yeast plasmids; positive clone strains were picked to induce expression; the secreted proteins were purified with affinity chromatography, desalted with dialysis and concentrated, then screened with enzyme-linked immunosorbent assay (ELISA) for the collection of high affinity nanobodies to N protein. A pair of nanobodies was screened with cross pairing experiment for the preparation of colloidal gold immunochromatography detection cards, and N-protein solution was dropwise added to the sample wells to observe the detection line and quality control line.

  Results  Totally 46 nanobodies with different sequences were screened out and 6 of them were selected to express nanobodies with the concentration > 1.0 mg/ml and high affinity to N protein after purification, desalting and enrichment. Finally, the selected pair of antibodies was assembled into colloidal gold N protein immunodetection card to detect N protein with sandwich method. Obvious detection line and quality control line were observed in the test of SARS-CoV-2 N protein antigen using the detection card and the detection limit was ≥ 10 μg/mL for the test.

  Conclusion  The prepared nanobody can be used in the colloidal gold nanobody immunochromatography detection card to detect SARS-CoV-2 N protein.