To study the topology of Na+,K+‐ATPase monoclonal antibodies (MAbs) specific for membrane‐bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra‐ or intracellular portions of the α‐subunit. The extracellular location of peptide loop 804–841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween‐20), was shown to bind to the hydrophilic loop 868–945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887–904 and 810–825, respectively. The C‐terminus of the α‐subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity‐purified antibodies to the synthetic peptide corresponding to fragment 999–1008.
Approbation of the experimental series of latex agglutination and immune chromatography test-systems was carried out for identification of Bacillus anthracis spores and vegetative cells respectively. These test-systems are suitable for rapid diagnostics of anthrax and have a number of advantages over already-existing commercial tests.
Immobilization of anti-B. anthracis monoclonal antibodies (MAbs) on latex microparticles was studied, the optimal load of these MAbs was determined to be 20 µg for 50 µl of the stock latex suspension. The highest sensitivity of latex agglutination test was observed for 1E6 MAbs. Latex suspensions with immobilized MAbs were lyophilized. Their sensitivity and specificity were shown to be highly competitive with those of the stock liquid latex suspension. Latex diagnosticum for Bacillus anthracis spores detection was constructed on the basis of these lyophilized reagents, developed and approved was the regulatory documentation that included their application instructions and technical specifications. Carried out were inter-laboratory and commission tests of experimental prototypes of the designed latex diagnosticum. These preparations demonstrated high sensitivity (from 1•10 5 to 2•10 6 spores/ml and even more) and specificity (absence of cross-reactions with spores of different species of sporogenous bacilli at concentration of 10 8 spores/ml).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.