Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis

Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Conflict of interest statement

RLH and HC are employed by a commercial company, InBios International. DPA has a patent submitted for the detection of B. pseudomallei capsular polysaccharide. Both of these competing interests do not alter our adherence to all PLOS NTDs policies on sharing data and materials.

Figures

Figure 1. Calculation of mAb 3C5 affinity for CPS.

A BIAcore X100 instrument was used to determine the affinity of mAb 3C5 for CPS. Biotinylated CPS was immobilized on the surface of a streptavidin sensor chip. Samples (two-fold serial dilution of mAb 3C5 [333–5.2 nM]) were injected over the sensor surface for 60 s, after which the mAb was allowed to passively dissociate for 120 s (left panel). The dissociation constant (KD) was determined using the steady-state model in BIAevaluation software (right panel).

Figure 2. Detection of purified CPS by antigen-capture ELISA.

mAb 3C5 was used in the capture phase of the ELISA at the concentrations listed. Following a wash and blocking step, purified CPS was serially diluted across the microtiter plate at the concentrations listed. The wells were then washed and HRP-labeled mAb 3C5 was used in the indicator phase to detect captured CPS. The ELISA was performed in triplicate and mean values are plotted.

Figure 3. Prototype Active Melioidosis Detect (AMD) LFI.

(A) Schematic of LFI components. (B) B. pseudomallei strain Bp82 colony grown on an agar plate was picked and suspended in 2 drops of lysis buffer. The lysate was added to the sample pad followed by three drops of LFI chase buffer (top LFI). The LFI was imaged following a 15 min run time. The same test condition were used with a colony of E. coli (bottom LFI).

Figure 4. Determination of the LOD of the AMD LFI.

(A) Purified CPS was diluted in chase buffer at the indicated concentration and applied to the LFI sample pad. Results were photographed after 15 min. Purified CPS was also diluted in human control sera (B) and human control urine (C).

Figure 5. Prototype AMD LFI for detection of B. pseudomallei CPS in melioidosis patient samples.

(A) Preliminary testing of a variety of archived patient samples from Australia and Thailand. (B) Detection of CPS in melioidosis patient urine samples (filtered) listed in Table 2. Urine (50 µl) was combined with 100 µl of chase buffer and applied to the sample pad. Note that samples that were positive by antigen-capture immunoassay (Table 2) were also positive by LFI and the levels of CPS detected between both assays are congruent.