Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine

Abstract

Summary.Immunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of BrucelZa abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibodycoated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma.CBA/H mice were infected with various doses [65-(65 x lo6) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).

Predicting Kala-Azar Disease Manifestations in Asymptomatic Patients with LatentLeishmania donovaniInfection by Detection of Antibody against Recombinant K39 Antigen

“…rK39 antigen was used to develop an in-house latex agglutination methodology according to the standard method of Limet et al (10) with minor modifications. In this method, the rK39 antigen at a concentration of 1 mg/ml in glycine-buffered saline (GBS) (glycine, 0.17 mol/liter; NaCl, 0.1 mol/liter; NaN 3 , 0.1 g/liter [pH 8.2]) was resuspended in equal volume of latex beads (0.9-m diameter) (Sigma manual, Sigma Chemical Co.).…”

Brucella Sp. Antibodies in Polar Bears From Svalbard and the Barents Sea

“…To use the indirect Protein-G ELISA designed for detecting anti-Brucella sp. antibodies in cattle (Limet et al, 1988) and previously used for several marine mammal species together with the classical brucellosis tests (Tryland et al, 1999), we checked the affinity between polar bear immunoglobulin molecules to Protein-G and Protein-A. Microtiter plates were coated with polar bear plasma and cattle serum and incubated with biotinylated Protein-G and Protein-A.…”

“…Microtiter plates were coated with 100 l of Brucellergene OCB (batch 86W221; 0.5 mg/ml) and blocked with 3% bovine serum albumin (BSA) in PBS. Polar bear samples were diluted 1/50 and the assay was performed as described previously (Limet et al, 1988), except that a Protein A-HRPO conjugate (Nycomed Amersham, Buckinghamshire, UK) was used instead of a Protein-G conjugate. Nine of the 16 individuals classified as having anti-Brucella sp.…”

Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115

“…These were determined by direct latex agglutination immunoassay of the culture supernatants. Latex particles were coated with rat monoclonal anti-mouse IgG1, IgGZa, IgG2b, IgG3, IgM and polyclonal anti-mouse IgA according to a protocol similar to that described by Limet et al (1988).…”