A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis

Abstract

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Keywords: Aquafex aeolicus; Fluorescent chromatography; Lumazine synthase; Mpox; Time-resolved fluorescence immunoassay.