OPTIMIZATION OF THE CONDITIONS FOR THE DEVELOPMENT OF THE RESULTS OF THE DOT-IMMUNOASSAY IN AN AUTONOMOUS KIT FOR THE DETECTION OF ORTHOPOXVIRUSES

Abstract

The aim of the work is to create a sensitive, fast and easy-to-use immunochemical assay for the detection of orthopoxviruses in the “point of care” format. The objectives of the study included: the fine-tuning of technology of preparation and use of the developer for colloidal gold conjugates in autonomous diagnostic systems based on flat protein matrices, as well as testing of this technology for detecting vaccinia virus. The quality of available reagents (metol, citric acid and silver nitrate) for the manufacture of the developing system was assessed: the most suitable reagents were selected. A technology has been fine-tuned for the preparation of a developer. The developer includes the dry component in the form of tablets (3.5-4 mg) containing metol and citric acid in a ratio of 2:3, and the liquid component - 0.4% silver nitrate solution. To increase the contrast and stability of optical signals, it was proposed to use the processing of the developed protein matrix with an alkaline solution of thiourea. The use of a two-component developer allows the detection of vaccinia virus in non-laboratory conditions with a sensitivity of 5.4 × 104 PFU/ml.

Keywords

orthopoxviruses / diagnostics / dot-immunoassay / colloidal gold / optical signal develop and amplification

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