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Abstracts of Oak Ridge Posters |
(Departments of1 Pharmaceutical Sciences and2 Chemistry "G. Ciamician", University of Bologna, Bologna, Italy;3 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G. Caporale", Teramo, Italy;
aaddress correspondence to this author at: University of Bologna, via Belmeloro 6, I-40126 Bologna, Italy; fax 39-051-343398, e-mail aldoroda{at}unibo.it)
Up to 80 million cases of food-borne illness occur yearly in the US. Approximately 30% of these cases are caused by bacteria and their related toxic products (1)(2). The bacteria species that are most frequently responsible for food contaminations are Salmonella typhimurium (meat, milk, and eggs), Escherichia coli O157:H7 (meat), Staphylococcus aureus (milk, cream, and meat), Clostridium perfringens (sausages, preserved food), Campylobacter jejuni (poultry, eggs), Vibrio parahaemolyticus (shellfish), Yersinia enterocolitica (meat, milk), and Listeria monocytogenes (dairy products) (3).
Conventional microbiological methods for the identification of pathogenic bacteria are labor-intensive (several enrichment steps) and time-consuming (2 to 3 days to obtain results) (4). Currently, new rapid procedures based on immunological, DNA hybridization, or biosensing methods have been proposed (5)(6). However, many of these rapid tests are expensive, laborious, and all of them are based on cultural enrichment to enhance sensitivity and selectivity before analysis. Simpler, faster and more sensitive diagnostic techniques are required to improve food safety (food production, processing, storage and distribution) and to screen for potential bacterial infections in humans. Several immuno-based detection methods for infectious agents have been developed recently (7)(8). Such methods are of great diagnostic importance because they offer unique detection specificity and sensitivity, with relatively simple and low-cost assay formats. Most commercially available immunological methods for the detection of bacteria are based on conventional sandwich immunoassays, which involve the formation of an immunocomplex with an immobilized antibody, the target bacteria, a second labeled antibody, and the separation of free and bound antibody fractions by washing steps. Unfortunately, these separation steps reduce the assay production, so alternative approaches are welcome.
In general, flow-assisted immunoassays offer advantages over conventional microtiter solid-phase formats, mainly related to easier automation and faster kinetics of the immunological reaction (9)(10), which takes place in a pseudohomogenous phase so the antibody is not immobilized on a bulk surface.
Field-flow fractionation (FFF) is an analytical technique suited for the separation of nano- and microsized dispersed analytes (11). Separation is achieved through a relatively simple device within an empty capillary channel by combining the action of a transporting laminar flow and a field applied perpendicularly to the flow. Analyte particles are distributed according to their physical features (size, density, surface properties), at different positions within the parabolic flow profile and are thus swept down the channel at different velocities and eluted at different times (12). Recently, we proposed a flow-assisted, competitive immunoassay format based on flow FFF with chemiluminescent (CL) detection of microsized beads with immobilized antibodies (13). This new format does not require washing steps to separate free and bound tracer, which are quantified from void-peak and retained-peak area measurements, respectively.
We report the implementation of gravitational FFF (GrFFF), the simplest FFF variant, which employs gravity as the applied field in an innovative whole-cell CL enzyme immunoassay format for the detection of pathogenic microorganisms in biological samples. The method combines the high sensitivity of CL detection with the GrFFF capability for efficient separation of the bound bacterium-antibody complexes from the free antibodies. The immunoassay employs a horseradish peroxidase (HRP)-labeled monoclonal antibody (MAb) produced against the target bacterium Y. enterocolitica, chosen as a model analyte. The immunological reaction takes place as a separate phase on the whole bacterial cells.
The anti–Y. enterocolitica MAb was produced by immunizing balb/c mice with heat-inactivated and sonicated American Type Culture Collection reference strains (Rockville, MD) (14)(15). We determined the MAb isotype with a Pierce Immuno-Pure monoclonal antibody isotyping reagent set. The antigen-independent method was used. Microtiter plates were coated with a goat antimouse antibody, and the hybridoma supernatant was added to the plate wells. After incubation and washing, we added subclass-specific antimouse immunoglobulins (IgG2a, IgG2b, IgG3, IgA, and IgM) in separated wells and then added an HRP-conjugated, goat antirabbit IgG that we detected by spectrophotometry. The MAb was screened by immuno-Western blotting (16), purified by affinity chromatography, and coupled to HRP with the periodate method (17). Using an indirect ELISA procedure, we evaluated MAb specificity against other isolated strains of the target bacteria and different bacteria species (see the Supplemental Data that accompanies the online version of this Oak Ridge Poster abstract at http://www.clinchem.org/content/vol52/issue10). As a negative control to exclude cross-reactivity, we used IgG MAbs against E. coli O157:H7, S. thyphimurium, and L. monocytogenes.
We obtained standard bacterial samples from Y. enterocolitica cultures. For the isolation of bacteria, colonies were transferred from selective solid agar (Herellea Agar, Biolife) to 5–7 mL Luria-Bertani broth (Bacto® LB BROTH, LENNOX, Difco Laboratories) and were grown for 12–18 h at 37 °C. A 2-mL aliquot of cell suspension in Luria-Bertani broth was centrifuged at 3000g for 10 min, the supernatant was carefully removed, and the pellet was washed twice with phosphate buffered saline (PBS). Washed bacteria were serially diluted in PBS to obtain concentrations of 1012–107 CFU/L. Unsterilized fecal samples were first assayed to verify the absence of Y. enterocolitica and then artificially contaminated with the bacteria. The samples (25 g) were inoculated with the appropriate amount of bacteria (final concentration 0 to 10 CFU/g), homogenized in a stomacher bag, and incubated with 225 mL of buffered peptone water for 2 h at 37 °C. Subsequently, 1 mL of this solution was removed and incubated with 9 mL Yersinia enrichment broth (Sigma) for 6 h at 37 °C with shaking. For each sample, we determined the actual bacteria concentration (CFU/mL) by colony counting on selective medium agar plates.
The GrFFF system was assembled as described elsewhere (18). The separation device was a home-made, empty, and flat capillary channel. The channel volume was cut out from a thin plastic foil that was sandwiched between 2 plastic walls, the top wall made of polycarbonate and the bottom wall of polyvinylchloride. Channel dimensions were 2.0 cm wide, 0.014 cm thick, and 30 cm long. Sample injection was performed by a Rheodyne model 7125 valve equipped with a 20-µL PEEK loop. The carrier liquid was PBS, pH 7.4, containing 0.05g/L (w/v) bovine serum albumin (BSA). The carrier flow was delivered by a peristaltic pump (Gilson Miniplus 3) at 0.3 mL/min, and the channel outlet was connected to a flow-through luminometer (modified Berthold Detection Systems FB12 luminometer). To generate the CL signal, an enhanced luminol-based CL substrate (Pierce SuperSignal ELISA Femto) was used. In previous work, this substrate gave low background (19)(20). The substrate was diluted 1:5 in the carrier, and postchannel, was injected at 0.03 mL/min by a syringe pump (KdScientific, KDS100) through a low-volume "tee" reactor. The injection of the substrate was synchronized with the elution flow.
A schematic representation of the GrFFF-CL immunometric method is shown in Fig. 1A
. For the assay, 100 µL MAb-HRP was added to 100 µL standard or fecal sample; the mixture was vortex-mixed for 30 s, and 20 µL was injected in the GrFFF channel. After injection, the flow was stopped for 30 min, during which sample relaxation and the immunological reaction between bacterial cells and MAb took place. After relaxation, elution flow was restored. The free and bacterium-bound MAb fractions were eluted in 
15 min, with the MAb-bacterium immunocomplex eluted in a relatively broad band with a retention time of 8.3 min. The excess of unbound labeled MAb was eluted within the void volume. The resulting CL fractogram consisted of 2 resolved peaks: a void peak and a retained peak corresponding to the free and bound MAb fractions, respectively. The activity of the HRP-labeled bound fraction, which was proportional to the bacterial concentration, was measured from the entire area of the eluted immunocomplex band.
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The calibration curve (Fig. 1B
) obtained for Y. enterocolitica was linear between 108 and 1010 CFU/L, and the detection limit of the method was 109 CFU/L (P = 95%). We performed analytical validation of the method with enriched human fecal samples. Bacterial concentration in fecal samples was determined by interpolation from the calibration curve. Results are reported in Table 1
. Precision, determined by analyzing replicates (n = 6) of enriched samples, was satisfactory at <15%. We evaluated method performance by comparing the results with those obtained with a conventional culturing method on selective media and noticed a concordance between the 2 methods. To prevent a specific protein binding to the plastic wall and to improve reproducibility and recovery, channel plastic walls were also saturated with BSA by overnight flushing with a solution of 0.5 g/L BSA in isotonic PBS (18). To avoid system cross-contamination, a cleaning procedure was performed after each working day by flushing with a solution of 30 g/L ethanol and 0.5 g/L sodium dodecyl sulfate in water. Under these conditions, no run-to-run carryover was observed, and sample recovery was 90%–120%.
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Because of the MAb specificity, the method enabled recognition of all the wild strains of the target bacteria without cross-reactivity with other species. In addition, the analysis of bacteria mixtures confirmed that no cross-reaction occurred. No CL signal was detected for negative mixtures, but it was possible to detect the target bacteria in the mixture even at a low relative concentration.
For clinical validation, we evaluated a total of 15 fecal samples (10 positive and 5 negative) for the presence of Y. enterocolitica and compared the results with those obtained with a conventional microbiological method, and we achieved a 100% agreement.
This GrFFF-assisted, whole-cell, noncompetitive immunoassay shows an analytical performance similar to a microtiter plate sandwich-type format and offers many advantages. Because our method is a flow-assisted immunoassay, in which the antibody is not immobilized and only a single MAb is required for the detection of 1 bacterial species, costs and analysis time are decreased. The method is also suitable for automation. Moreover, multiplexed immunoassays can be developed for simultaneous analysis of bacterial mixtures because pathogenic bacteria present in the same sample with different morphological properties can be efficiently fractionated by GrFFF, detected, and quantified in a single run with a mixture of bactera-specific MAbs.
References
The following articles in journals at HighWire Press have cited this article:
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  N. J. Parham, F. J. Picard, R. Peytavi, M. Gagnon, G. Seyrig, P.-A. Gagne, M. Boissinot, and M. G. Bergeron Specific Magnetic Bead Based Capture of Genomic DNA from Clinical Samples: Application to the Detection of Group B Streptococci in Vaginal/Anal Swabs Clin. Chem., September 1, 2007; 53(9): 1570 - 1576. [Abstract] [Full Text] [PDF]
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