This study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system “Super-CARD” for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification “Super-CARD”, 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 μg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads. The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.

The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver developer. Rabbit polyclonal antibodies against the vaccinia virus were used as capture and detection reagents. The sensitivity of detection of OPV and the specificity of the analysis were assessed using culture crude preparations (monkeypox virus, vaccinia virus, rabbitpox virus, cowpox virus, and ectromelia virus), a suspension from a crust from a human vaccination site as well as blood and tissue suspensions of infected rabbits. It has been shown that the assay using the kit makes it possible to detect OPV within 36 min at a temperature of 18–40 °C in unpurified culture samples of the virus and clinical samples in the range of 10³–10⁴ PFU/mL. Tests of the kit did not reveal cross-reactivity with uninfected cell cultures and viral pathogens of exanthematous infections (measles, rubella and chicken pox). The kit can be used to detect or exclude the presence of a virus threat in samples and can be useful in various aspects of biosecurity. The simplicity of analysis, the possibility of visual accounting the and interpretation of the results make it possible to use the test in laboratories with a high level of biological protection and in out-of-laboratory conditions.

Introduction. Following the successful eradication of smallpox, mass vaccination against this disease was discontinued in 1980. The unvaccinated population continues to be at risk of infection due to military use of variola virus or exposure to monkeypox virus in Africa and non-endemic areas. In cases of these diseases, rapid diagnosis is of great importance, since the promptness and effectiveness of therapeutic and quarantine measures depend on it. The aim of work is to develop a kit of reagents for enzyme-linked immunosorbent assay (ELISA) for fast and highly sensitive detection of orthopoxviruses (OPV) in clinical samples. Materials and methods. The efficiency of virus detection was evaluated by single-stage ELISA in the cryolisate of CV-1 cell culture samples infected with vaccinia, cowpox, rabbitpox, and ectromelia viruses, as well as in clinical samples of infected rabbits and mice. Results. The method of rapid ELISA was shown to allow the detection of OPV in crude viral samples in the range of 5.0 1025.0 103 PFU/ml, and in clinical samples with a viral load exceeding 5 103 PFU/ml. Conclusions. The assay involves a minimum number of operations and can be performed within 45 minutes, which makes it possible to use it in conditions of a high level of biosecurity. Rapid ELISA method was developed using polyclonal antibodies, which significantly simplifies and reduces the cost of manufacturing a diagnostic system.

Introduction. The abolition of smallpox vaccination has led to the disappearance of population immunity to pox viruses. However, the threat of infection by pathogenic orthopoxviruses persists and determines the need to develop sensitive and operational methods for indicating pathogens.Objectives and purposes. Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care» format.Material and methods. We used preparations of cultural vaccinia virus (VV) with varying degrees of purification, polyclonal antibodies from hyperimmune rabbit serum, and equipment from a previously developed autonomous kit for dot-immunoassay on flat protein arrays.Results and discussion. It has been established that rabbit polyclonal antibodies can be used in a single-stage dotanalysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the effectiveness of the detection of VV is inversely related to the degree of purification of viruses from sub-viral structures. The sensitivity of the rapid detection of viruses in a crude preparation was about 30 times higher than in pure viral material. The increase in sensitivity, presumably, occurs due to binding to the capture antibodies of subviral structures, which form large aggregates of sensitized gold particles. The test does not detect cross-reactions with heterogeneous viruses (measles, rubella and chickenpox) that cause exantematous diseases.Conclusion. The one-stage variant of the dot-immunoassay reduces the analysis time to 40 minutes and improves the detection sensitivity of orthopoxviruses in crude viral preparations to the range of 105-104 PFU / ml. Full makeup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.