[The rapid ELISA method for detection of orthopoxviruses].

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.

[Rapid immunochemical method for the detection of orthopoxviruses (Orthopoxvirus, Chordopoxvirinae, Poxviridae).]

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: Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care¼ 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.

[The selection of system of detection for multiplex dot-immune analysis of antibodies].

The comparative evaluation was carried out concerning systems of detection using conjugates on the basis of peroxidase, alkaline phosphatase, colloid gold and system of amplification "Super-CARD" for multiplex dot-immune assay of antibodies. It is established that sensitivity of system of detection with colloid gold is approximately 8 times higher than with amplification "SuperCARD"; 30 times higher than with conjugate of alkaline phosphatase and 250 times higher than with peroxidase conjugate. The immunesols of gold maintain limit of detection of human IgG 10 pg with dynamic range of signal alteration from 5 ng to10 pg overlapping the range of concentrations of specific IgG in case of typical natural immune response. The most specific results provides conjugate of colloid gold with monoclonal antibodies to human IgG.

[The multiplex method of estimation of humoral immunity to vaccine regulated childhood infections].

The goal of this work was to present the results of the laboratory tests of the multiplex dot immunoassay method using protein microarray for complex estimation of humoral immunity to measles, mumps, and rubella viruses. It was shown that the obtained results were in a good agreement with data of commercial monospecific ELISA kits. The developed method is fast, requires fewer resources, and may be used in the field.

[The application of cobaltferous markers in serodiagnostic assay of infectious diseases].

The study was targeted to the investigation of possibilities of using in immunological analysis the sols on basis of bivalent cobalt compounds as a catalytic marker to ensure the trusted visual registration of analysis results in immunology reactions plats. The results of comparative evaluation of immune-enzyme and immune catalytic tests on the basis of several commercial diagnostic test systems of various national manufacturers are presented. The conclusion is derived that by the sensitivity and specificity the cobaltferous immunosols are not inferior to immuneperoxidase conjugated metabolites and even have an advantage due to the relative simplicity of immunosol preparation and the possibility of trusted non-instrument registration of results. The application of cobaltferous immunosols permits to develop the diagnostic test systems with trusted visual registration of results following the "yes-no" principle to ensure the immunodiagnostics in poorly equipped laboratories.

[Development of a method for rapid detection of Ebola virus antibodies and antigen].

Despite the wide spectrum of reliable methods for identifying Ebola virus, their performance requires highly-skilled personnel, specialized laboratories, complicated equipment, and much time. Therefore, there is a need for a method that allows a physician or a medical attendant to identify the causative agent in field or bedside tests without special equipment as soon as possible. The immunoassay involving nitrocellulose membrane immuno-filtration, by using a fixed antigen (antibodies) or their immunosols, is a tried-and-true method. The time of the analysis is 7-15 min.

[Multiprofile immunochemical indication of the causative agents in infectious diseases].

A variant of multiprofile immunochemical indication of the causative agents of infectious diseases is described on the basis of a protein microarray with a low-density antibody population. The method is a multiplex variant of dot ELISA whose sensitivity increased due to the use of a colloid gold-based conjugate and an efficient system of its development. The method shows both its multiprofile character with its easiness-to-prepare and to use protein microchips. Having the similar sensitivity, it has obvious advantages over the routine ELISA in the rate of obtaining results, the consumption of reagents, labor intensiveness, and specific cost.

[Dynamics of humoral immune response to Treponema pallidum proteins p17 and p41 at early stages of syphilis]. / Dinamika gumoral'nogo immunnogo otveta na belki P17 i P41 Treponema pallidum na rannikh stadiiakh sifilisa.

In 60 blood sera from syphilis patients the titers of IgG to T. pallidum antigens p17 and p41 were detected with the use of the test system based on the recombinant analogues of T. pallidum proteins. The study revealed that primary syphilis was characterized by considerable prevalence of IgG to protein p41 with the total antibody level being low, while early latent syphilis was characterized mainly by considerable prevalence of IgG to protein p17 in the presence of high titers of antibodies. In secondary syphilis the sera contained a high total antibody level and a wide range of IgG ratios to individual antigens. On the basis of the data obtained the dynamics of immune response to antigens p17 and p41 at the early stages of the disease was hypothetically plotted. The curves of antibody levels had a wave-like character with the phase shifts of peaks for individual proteins and very low antibody titers (less than 1:100) in the negative peak areas. Conclusions were made that it was necessary to use the mixture of antigens in the production of the test systems and, when designing reference panels of sera, to include sera with extremely low titers of antibodies to individual proteins.

[Use of silver sols as markers for immunoassay on microtiter plates]. / Ispol'zovanie zolei serebra kak merkerov immunoanaliza na mikrotitroval'nykh planshetakh.

Some basic characteristics of silver sols, facilitating their use as possible markers for the enzyme immunoassay on microtitration plates, were studied. Dispersion characteristics of silver sols obtained from AgNO3 solutions with a concentration of 0.005-0.5%, as well as the efficiency of their detection with physical developers on the basis of methol, paraphenylene diamine and amidol, were determined. The aggregative stability of the sol and the protective effect of a number of nonionic detergents, synthetic and biopolymeric, were evaluated. Similar sensitivity at the level of 1 ng/ml was attained in the comparative detection of lgG adsorbed on microtitration plates with the use of immunoperoxidase conjugate and silver immunosols. The possibility of using silver sols in the enzyme immunoassay as an alternative to enzymatic markers was shown.

[Use of silver immunosalts for detecting HIV antibodies in microtiter plates]. / Primenenie serebrianykh immunozolei dlia vyiavleniia VICh-antitel v mikrotiroval'nykh planshetakh.

The authors examined the possibility of replacing immunoperoxidase conjugates with silver sol (mean diameter of particles 9 nm) adsorption-bound to antispecies IgG in solid-phase enzyme immunoassay in microtitration plates. Experiments with commercial test systems Antigen and Recombitest-antiHIV-1,2 manufactured by the Vektor State Research Center of Virology and Biotechnology, Ministry of Health and Medical Industry of the Russian Federation, showed that the sensitivity of detecting anti-HIV with silver immunosols and boosting of the signal with physical development was no less sensitive than standard EIA with immunoperoxidase conjugate. Preparation of silver immunosols is simple, easily reproducible, and is based on sparing and economic use of immunoreagents.

[Use of catalytic properties of iron-containing salts in immunoanalysis]. / Ispol'zovanie katalitcheskikh svoistv zhelezosoderzhashchikh zolei v immunoanalize.

The possibility of using inorganic catalysts as markers for solid-phase immunoassay on the plates is shown with iron-containing hydrosols: ferrum hydroxide, ammonium ferriglycerate, and ferrum hydroxide modified with potassium ferrocyanide. Diagnostic agents based on iron-containing sols with dispersion of 0.5 to 0.8 mm bound to immunoglobulins or staphylococcal protein A are no less sensitive than the immunoperoxidase agents, provided the same developing system is used (o-phenylenediamine + hydrogen peroxide). The sensitivity of solid-phase immunocatalytical assay of purified vaccinia and Venezuelan equine encephalomyelitis virus antigens was about 2 and 10 ng/ml, respectively. Hence, inorganic catalysts are prospective markers for immunoassay.