Rapid protocol of dot-immunnoassay for orthopoxviruses detection

The aim of the work was to create a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the "point of care" format. This work presents the results of the comparative evaluation of a single-stage (rapid version) and two-stage protocol of dot-immunoassay based on plane protein array for detection of vaccinia virus (VACV), cowpoxvirus (CPXV) and ectromelia virus (ECTV) in viral culture materials with different degrees of purification. It has been established that rabbit polyclonal VACV-antibodies can be used in a one-stage dot-analysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the sensitivity of detection of OPXV is inversely related to the degree of purification of viruses. The one-stage variant of the dot-immunoassay allows reducing the analysis time to 39 min and increasing the detection sensitivity of all the studied orthopoxviruses in crude viral samples to a range of 10⁴-10³ PFU/mL. The increase in sensitivity in the rapid version of the analysis, presumably, occurs due to binding of capture antibodies to subviral structures that form large aggregates of gold particles. Ultrasonic treatment of culture virus reduces the detection sensitivity, presumably due to both the destruction of conformational epitopes located on the surface of subvirus structures, as well as the increase in the dispersion of cell debris, which limits diffusion and contacts of viral antigens with capture antibodies on the substrate. Both versions of the analysis are specific and do not detect interactions both with preparations of non-infected cell culture and with heterogeneous controls of the causative agents of erythematous infections. The rapid protocol of dot-immunnoassay described above can be used to detect, or help to exclude, the presence of threat viruses in samples and could be useful in a variety of biodefense applications. Ready-to-use setup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.