A turbidimetric latex inhibition immunoassay for detergent solubilized lipopolysaccharide: application to Brucella cells

Abstract

A turbidimetric latex agglutination-inhibition assay was developed for the estimation of the smooth lipopolysaccharide (S-LPS) content in Brucella cells. Proteinase K (PK)-digested Brucella cell lysates were distributed in flat-bottom multiwell plates and incubated with an anti-S-LPS monoclonal antibody (mAb). Unbound antibody was then titrated by agglutination of S-LPS-coated latex particles, in the presence of human rheumatoid factor (IgM anti-IgG) to enhance agglutination. The percentage of agglutinated particles was measured in a microplate spectrophotometer by monitoring the decrease of absorbance at 405 nm. The inhibitory effect of sodium dodecyl sulfate (SDS) present in the samples, was prevented by the addition of bovine serum albumin (BSA). Recovery of S-LPS was not influenced by the concentration of the other components of the bacterial lysate. Rough LPS (R-LPS) was note detected in contrast to O-polysaccharide (O-PS), which was effectively assayed. The intra-assay variation coefficient was lower than 5%. The range was suitable to show differences in the LPS content between clones of the same Brucella vaccinal strain. The same samples could be studied simultaneously by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).