Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

Abstract

Background: Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay.

Methods: To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed.

Results: MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample.

Conclusion: Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.

Fig. 1

Antigen-binding properties of the generated Brucella LPS-specific mAbs. a Reactivity of the produced mAbs with extracted B. melitensis or B. abortus LPS in ELISA. b Western blot staining-patterns obtained with mAbs 10G1, 3D12 and 1E2 after SDS-PAGE of extracted B. melitensis and B. abortus LPS. c Indirect immunofluorescent staining of inactivated B. melitensis (16 M) cells by mAbs 3D12 and 10G1. The upper panel shows DNA staining with DAPI, the middle panel Alexa 568-specific immunofluorescence staining and the lower panel merged pictures of both stainings. d Reactivity of mAb 3D12 with gamma, formalin and heat inactivated B. melitensis (16 M) and B. abortus (544) cells in ELISA

Fig. 2

Comparative testing of mAb pairs in an antigen capture ELISA. To evaluate optimal antibody combinations, each of the 11 mAbs was used as a capture or detection (biotin-conjugate) antibody at a concentration of 10 μg/mL. Optical densities were measured for each antibody combination using gamma-irradiated B. melitensis (16 M) cells at a concentration of 10⁷ cells/mL

Fig. 3

Comparison of the sensitivity of the bead-based Luminex immunoassay (a) and the corresponding antigen capture ELISA (b). Assay sensitivities were determined by analysing serial dilutions of inactivated B. melitensis (16 M, γ), B. abortus (544, γ) and B. suis (1330, formalin) cells. Dashed lines indicate the assay dependent limit of detection (LOD) defined as mean blank (i.e. the no-antigen control) plus three times the standard deviation (SD)

Fig. 4

Multiplexed Luminex immunoassay for detecting potential bioterror agents, B. melitensis, B. anthracis, F. tularensis and Y. pestis. a Test samples contained B. melitensis 16 M (Bm, 5 × 10⁵ cells/mL), B. anthracis PXO1+ (Ba, 5 × 10⁵ cells/mL), F. tularensis 6223 (Ft, 5 × 10⁵ cells/mL) and Y. pestis CO92 (Yp, 5 × 10⁴ cells/mL) cells in PBS either alone or in combination. In (b), PBS and milk samples were spiked with all four bacterial species and used at a concentration of 2.5 × 10⁶ cells/mL. MAbs 3D12, MTA1, T14 and YPF19, coupled to distinct magnetic beads, were used as capture antibodies and the biotinylated mAbs 10G1, MTD6, FB11 and YPF19 were used for detection. Reporter dye fluorescence intensities measured for each bead set are shown. Dashed lines indicate the limit of detection (LOD) defined as mean blank plus three times the standard deviation