Application of Latex-Agglutination for Rapid Detection of Pathogenic Burkholderia

Abstract

The aim . Development of a drug for the identification of Burkholderia pseudomallei and Burkholderia mallei grown in solid nutrient medium at the stage of rapid diagnosis of pathogenic bulkholderia. Materials and methods . Latex agglutination method, in which suspensions of polymers are used as a carrier of agglutinins. The agent recognizing the antigen of the target cells is a monoclonal antibody directed to the epitopes of the glycoprotein capsule of melioidosis causative agent. A liquid latex diagnosticum was prepared from a suspension of polymer polystyrene with a microsphere diameter of 0.8-1.1 pm, the surface of which was loaded with a pre-selected concentration of antibodies. We used typical strains of melioidosis and glanders agents with a full antigenic structure, as well as closely related and heterologous microorganisms. Suspensions with a concentration of 1.0·10 9 m.c./ml were prepared from bacterial cultures. The reaction was carried out on glass Petri dishes heated to 37 °C with visual recording of the results using four cross system. Results and discussion . The latex agglutination reaction is based on agglutination of microbial cells (B. pseudomallei and B. mallei) with monoclonal antibodies that recognize the epitopes of the glycoprotein capsule of pathogenic burkholderia. 14 monoclonal antibodies of different class and epitope orientation were checked. The determining factors for antibody selection was their specificity in agglutination reaction with strains of melioidosis and glanders agents, as well as preservation of agglutinating activity after immobilization on a polymer carrier. As a result, monoclonal antibodies of class G immunoglobulin to capsule glycoprotein epitopes (AG 8) of the melioidosis agent were selected as an agent recognizing the target cell antigen. As a result, after mixing the samples with the diagnosticum, in samples containing B. pseudomallei and B. mallei bacteria, visible agglutinate generates in 10-15 min. Samples with closely related and heterologous microorganisms lacked agglutinate and were registered as negative. The obtained diagnosticum is characterized by high specificity.