Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells

Abstract

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis

“…Since immunodiagnostics are considered reliable and specific methods for detection of tularemia ( 15 – 17 ), a routinely implemented immunoassay was evaluated in the modified cultures. Accordingly, supplemented and nonsupplemented Bactec Plus Aerobic blood cultures were spiked with bacteria of the SchuS4 virulent strain.…”

“…The importance of early diagnosis incentivized the development of rapid and sensitive approaches for the detection of F. tularensis directly from clinical samples. These are mainly based on immune-labeling ( 15 – 17 ) and genetic ( 9 , 18 , 19 ) assays.…”