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Clinical and Diagnostic Laboratory Immunology, November 2004, p. 1094-1099, Vol. 11, No. 6
1071-412X/04/$08.00+0 DOI: 10.1128/CDLI.11.6.1094-1099.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Center for Comparative Medicine,1 Comparative Pathology Laboratory,2 Department of Medical Pathology and Laboratory Medicine, University of California, Davis, California3
Received 25 May 2004/ Returned for modification 8 July 2004/ Accepted 30 July 2004
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ABSTRACT |
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INTRODUCTION |
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At present, three types of diagnostic assays are used for the detection of Helicobacter infection: isolation and identification by bacterial culture, enzyme-linked immunosorbent assay (ELISA), and DNA amplification by PCR. For the detection of serum or plasma antibodies, the current ELISA protocols use bacterial membrane extracts as antigens. Membrane extract is not sufficiently sensitive for the detection of low levels of antibody in the early stage of infection (6, 7, 12). In addition, ELISA with membrane extract shows limitations in specificity, frequently leading to cross-reactivity among Helicobacter species (20). DNA amplification by PCR is sensitive and specific, particularly for diagnosis of early-stage infection. However, this method is prone to yield false-negative data because of PCR inhibitors in blood and/or fecal samples. Therefore, diagnostic assays with improved sensitivity and specificity are needed for testing animal samples. Once developed, accurate and high-throughput serodiagnostic assays for the detection of infection by H. bilis and other murine Helicobacter species may also be useful for diagnosing infection in other animal species, including humans.
We recently identified P167, an immunoreactive recombinant protein of H. bilis that was utilized as an antigen for testing antibody in mouse sera by ELISA (4). Among five overlapping peptides of P167 that were generated, only P167C and P167D were reactive to the H. bilis immune serum (4). P167C and P167D peptides were expressed in Escherichia coli, purified, and tested by ELISA for antigenicity against sera from mice infected with H. bilis and H. hepaticus. In this small trial, both P167C and P167D proved to be more sensitive and specific antigens than H. bilis membrane extracts (4).
In the present study, a multiplex serodetection assay was developed to detect Helicobacter infection in mouse serum samples by using the recombinant antigens P167C and P167D of H. bilis. This assay utilizes instrumentation and microbeads developed by Luminex Corp. (Austin, Tex.). The multiplex capability of this system is based on unique sets of fluorescent polystyrene microbeads (5.6 µm in diameter) (10, 15). Individual bead sets are coded by internal labeling with a specific ratio of red and orange fluorophores. Each bead set is conjugated with one of several target analytes (e.g., an antibody or antigen) and assembled into one reaction container (2). Thus, multiple analytes can be simultaneously detected in a single sample. In contrast, the conventional ELISA format is limited to detection of a single analyte. In addition to P167C and P167D, the multiplex assay also included H. bilis bacterial membrane extract for comparison and H. hepaticus membrane extract as a control for specificity. The multiplex assay not only conserved the sample but also reduced assay time and labor. Results described here also show that in the multiplex assay format, purified recombinant H. bilis antigens P167C and P167D are superior in sensitivity and specificity to bacterial membrane extracts as antigens for serodetection.
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MATERIALS AND METHODS |
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The University of California, Davis, laboratory animal care program is fully accredited by the Association for Assessment and Accreditation of Laboratory Care, and this study was reviewed and approved by the Institutional Animal Care and Use Committee. All procedures and the use of mice were in compliance with the Public Health Service Guide for the Care and Use of Laboratory Animals.
Bacteria and native bacterial antigens. H. bilis (ATCC 51630) and H. hepaticus (ATCC 51448) were obtained from the American Type Culture Collection (Gaithersburg, Md.) and cloned by 3x limiting dilution (9). The species identity of each clonal population was verified by PCR amplification with species-specific primers (9). Membrane antigen extracts of H. bilis or H. hepaticus were prepared as previously described (12).
Sera. Immune sera for H. bilis and H. hepaticus were generated in two steps as previously detailed (4, 9). They were used as positive controls for each species. Briefly, both Helicobacter strains were grown under microaerobic conditions in brucella broth for 3 days at 37°C. The bacteria were adjusted to 108 CFU/ml, and 0.1 ml of bacterial suspension (per mouse) was inoculated intraperitoneally into C3H/Smn.CIcrHsd-Prksdscid mice. After infection was established (4 to 8 weeks postinoculation) and confirmed by PCR amplification of fecal samples, the mice were euthanized. Livers were collected and homogenized in 10 ml of brucella broth. The liver homogenates (0.25 ml per mouse) were inoculated by gavage into C3H mice to produce immune sera to either H. bilis or H. hepaticus. The infection status of the inoculated C3H mice was monitored weekly by PCR amplification of fecal samples and culturing. At 6 months postinoculation, blood was collected and immune serum samples were harvested from infection-positive mice.
C3H mice, naturally infected with H. bilis or H. hepaticus and confirmed to be free of infection by other Helicobacter spp., were obtained from commercial sources. Eighteen mice, infected with only H. bilis, were purchased from Charles River (Portage, Mich.) colony PO3. These included 9 weanlings (9 males) and 9 retired breeders (4 females and 5 males). In addition, 20 mice, infected with only H. hepaticus, were obtained from Harlan Sprague Dawley. These included 10 weanlings (10 males) and 10 retired breeders (5 females and 5 males). Helicobacter species (H. bilis or H. hepaticus) verification was performed for these two types of mice upon arrival. Fecal samples from all 38 mice were subjected to PCR using both sets of species-specific primers (9) and PCR with Helicobacter-specific primers followed by restriction fragment length analysis (16). Both methods confirmed that H. bilis-infected mice were infected with H. bilis and free of H. hepaticus and that H. hepaticus-infected mice were infected with H. hepaticus and were free of H. bilis. In addition, bacterial cultures of mucosal scrapings from the cecum and colon of these mice were analyzed to confirm infection by the respective Helicobacter strains (9, 16). All mice were necropsied, and serum samples were collected for testing.
Expression and purification of recombinant proteins. The two H. bilis-specific peptides, P167C and P167D, were cloned in pMX, a pGEX-2T vector, in frame with the glutathione S-transferase gene (Pharmacia, Piscataway, N.J.) containing a modified polylinker (3, 17). Recombinant proteins were expressed and purified on glutathione columns and freed of their glutathione S-transferase fusion partner by thrombin cleavage (3).
ELISA. Immulon flat-bottomed microtiter plates (ThermoLabsystems, Franklin, Mass.) were coated with recombinant P167C and P167D antigens or membrane extracts from H. bilis and H. hepaticus. Antigens were diluted to a final optimized concentration of 0.33 µg/ml in a coating buffer of Hanks balanced salt solution containing 0.375% sodium bicarbonate. Antigens (100 µl/well) were added to plates and incubated overnight at 4°C. The plates were washed with wash buffer (0.1% Tween 20 in phosphate-buffered saline [PBS], pH 7.4) and blocked with BLOTTO (5% nonfat powdered milk in wash buffer) for 1 h at room temperature. After blocking, the plates were washed, and 100 µl of mouse serum diluted 1:200 in BLOTTO was added. The plates were allowed to incubate at room temperature for 1 h and washed again with wash buffer. Detection antibody, biotinylated goat anti-mouse immunoglobulin G (IgG; Kirkegaard and Perry), was diluted 1:10,000 in BLOTTO and added to ELISA plates (100 µl/well). The plates were incubated with the detection antibody for 30 min at room temperature. After washing the plates again, 100 µl of Vectastain ABC detection reagent containing horseradish peroxidase H (Vector Laboratories, Burlingame, Calif.)/well was added according to the manufacturer's instructions. The plates were washed and developed by adding 100 µl of 3,3',5,5'-tetramethylbenzidine color development reagent (Sigma Chemicals, St. Louis, Mo.)/well. The plates were developed at room temperature for 10 to 12 min, and the reaction was stopped by adding 50 µl of 1 M sulfuric acid/well. The color development was read at 450 nm in an ELISA plate reader (Bio-Rad, Hercules, Calif.). Cutoff values were determined for all four antigens; an average of ELISA readings in three separate pools of normal mouse sera was calculated, and three times the standard deviation value was added to the average. The samples were analyzed in duplicate. A minimum of two separate assays were performed for each antigen. Sera were designated positive or negative for antibodies to H. bilis and H. hepaticus based on the ELISA cutoff value.
Coupling proteins to Luminex microbeads. Microbeads were purchased from Luminex Corp. The beads were coated with various proteins by chemical cross-linking according to the manufacturer's instructions. The bead stock was resuspended by vortexing and sonication (15 to 30 s). An aliquot of 2.5 x 106 beads was removed and centrifuged at 12,000 x g for 2 min. Beads were resuspended in 80 µl of activation buffer (100 mM monobasic sodium phosphate, pH 6.3) by vortexing and sonication (15 to 30 s).
To activate the beads for cross-linking to proteins, 10 µl of 50-mg/ml sulfo-N-hydroxysuccinimide (Pierce, Rockford, Ill.) was added, and the beads were mixed by vortexing. Then 10 µl of 50-mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce) was added, and the beads were mixed again by vortexing. The bead mixture was shaken on a rotator at room temperature for 20 min and centrifuged at 12,000 x g for 2 min. The beads were washed twice with 1 ml of 50 mM morpholineethanesulfonic acid (MES) buffer (pH 6.0). To coat with proteins, pelleted beads were resuspended in the relevant protein solution, diluted in 50 mM MES buffer (pH 6.0). Optimization of the antigen coating on microbeads was performed by coating different microbead sets with a protein concentration of each antigen at 1 to 200 µg/ml. These microbead sets were then used to test against standard mouse sera positive for antibodies to either H. bilis or H. hepaticus. Microbead sets that provided the strongest signal specific for each antigen against the standard mouse sera were selected. The optimized protein concentrations for each antigen are as follows: 100 µg/ml for P167C, P167D, and H. bilis membrane extract and 25 µg/ml for H. hepaticus membrane extract. Beads were also coated with a control antibody (biotin-conjugated goat IgG, 100 µg/ml) and bovine serum albumin (BSA) as a control protein (100 µg/ml) diluted in 50 mM MES buffer (pH 6.0). The mixtures of activated beads and proteins were incubated by shaking on a rocker for 2 h at room temperature for coupling. After being coated with proteins, the beads were washed twice with wash buffer (0.1% Tween 20 in PBS, pH 7.40) and resuspended in 1 ml of blocking buffer (1% BSA, 0.1% Tween 20 in PBS [pH 7.4], 0.05% sodium azide). Blocking was performed by shaking on a rocker at room temperature for 30 min. After blocking, the beads were washed twice in 1 ml of blocking buffer. Finally, antigen-coated beads were resuspended in 1 ml of blocking buffer and stored at 4°C for up to a week. For long-term storage, the beads were kept frozen at –70°C for several months.
Multiplex detection of antibodies in mouse serum. Immunoreactions were set up in 96-well, filter-bottom plates designed for high-throughput separations (1.2-µm-pore-size MultiScreen; Millipore Corporation, Bedford, Mass.). Typically, up to 2,500 beads for each individual bead set, coated with a known protein, were added per well. For example, for a six-target assay, 2,500 beads from each set coated with a known antibody were mixed to provide a total of 15,000 beads. Mouse serum was diluted 1:200 in 5% BLOTTO, and 50 µl of diluted serum was mixed with the six-target bead mixture per well, in a 96-well plate. The mixture of beads and serum was then incubated on a shaker for 1 h at room temperature. After incubation, liquid was drained from the bottom of the plate in a vacuum manifold designed to hold 96-well plates (Millipore Corporation). The beads were washed three times successively by adding 100 µl of wash buffer per well and draining under a vacuum. For detection of mouse IgG, biotinylated goat anti-mouse IgG was used (Vector Laboratories) at a 1:1,000 dilution in wash buffer, and 100 µl was added per well. The beads were mixed as before and incubated at room temperature for 30 min. Following incubation with the secondary antibody, the beads were washed three times as before. To detect biotinylated IgG, 100 µl of streptavidin conjugated to R-phycoerythrin (streptavidin-PE; CalTag, Burlingame, Calif.) was added at a dilution of 1:500 in wash buffer. The contents of each well were mixed and incubated at room temperature for 15 min. The beads were washed three times with wash buffer. The washed beads were finally resuspended in 100 µl of wash buffer per well and analyzed in a Luminex-100 instrument equipped with an XY-Platform for automatic reading of a 96-well plate.
Luminex-100 instrument operation and data analysis. The Luminex-100 instrument was manufactured by Luminex Corp. and purchased from Upstate USA (Lake Placid, N.Y.). The instrument was used at default settings, set by the manufacturer for routine applications, as directed by the user's manual. Data were acquired by use of the Luminex Data Collection software (version 1.7). This software package allowed routine operation of the instrument, data acquisition, and data analysis. Calibration beads supplied by the manufacturer were used to adjust the instrument settings for bead set identification and for the detection of reporter (phycoerythrin). Events were gated to exclude doublets and other aggregates. A minimum of 100 independent gated events were acquired for each bead set. The median fluorescence intensity (MFI) of 100 events (beads) was used as a measure of antibody detection. After acquisition by Luminex software, the data were further processed with Microsoft Excel software. For each assay, cutoff values were determined for all four antigens conjugated at optimal protein concentration. An average MFI for each bead set in three separate pools of normal mouse serum was calculated, and three times the standard deviation value was added to the average. For routine analysis, each experiment also contained a positive (immune) control serum sample. The samples were analyzed in duplicate, and a minimum of two separate multiplex experiments were performed. Serum samples were designated positive or negative for antibodies to H. bilis, based on the MFI readings.
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RESULTS |
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The results of the multiplex analysis of the sera tested by ELISA are shown in Table 1. The microbead set coated with BSA served as a control for nonspecific serum reactivity. For all mouse sera tested, these beads displayed MFI values of 15 to 20. The microbead set coated with biotinylated goat IgG served as a positive control for the detection reagent, streptavidin-PE; these beads displayed MFI values of 3,500 to 4,000. The multiplex results demonstrated a good correlation between serodetection and infection status. For H. bilis antibody detection, there were two clear differences between the two assay formats. First, there was an overall improvement in sensitivity and specificity in the multiplex microbead assay with P167C and P167D peptide antigens compared to that with the H. bilis membrane extract. Second, purified recombinant antigens in the ELISA yielded a higher sensitivity than H. bilis membrane extract in the multiplex microbead assay. For example, in weanling mice that might have low antibody levels (1596-3 to 1596-11), microbeads coated with either one of the two recombinant antigens, P167C or P167D, detected H. bilis antibodies in all nine sera (Table 2). On the other hand, two weanling mice (1596-8 and 1596-9) and two retired breeders (1597-8 and 1597-10) that were found by ELISA to be positive for H. bilis antibodies against H. bilis membrane extract were negative for a reaction to microbeads coated with the same antigen (Table 1). In addition, when microbeads coated with H. bilis membrane extract did detect H. bilis antibodies, the sensitivity was much lower than that of the beads coated with P167C and P167D (Table 1). The simplest explanation for this lower sensitivity in the multiplex assay with membrane extract is that microbeads have a much smaller surface area than ELISA wells, so the number of specific antigen molecules per bead is also lower. As discussed below and shown in Tables 2 and 3, purified recombinant antigens P167C and P167D were both highly sensitive (94 and 100%, respectively) and specific (100 and 95%, respectively) compared to H. bilis membrane extract (78% sensitivity and 65% specificity) in the detection of serum antibodies to H. bilis.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, the multiplex microbead methodology was used to test mouse sera for antibodies to four different Helicobacter antigens and to detect simultaneously antibodies to two different species of Helicobacter. To validate results obtained by the multiplex microbead assay, ELISA was performed on all the serum samples. A summary of the results from the two assays is presented in Tables 2 and 3. Mice infected with H. bilis (18 mice) and H. hepaticus (20 mice) were used. In each category, one half of the mice were weanlings, representing animals likely to have had a shorter exposure to bacterial infection or containing maternal antibodies, and the other half were retired breeder mice, which were likely to have had a longer exposure to bacterial infection (Table 1).
For H. bilis, the membrane extract produced consistent results in the ELISA, with 94% sensitivity (Table 2); this extract was less sensitive in the microbead assay (78%) (Table 3). Among the two recombinant antigens, P167D showed the highest sensitivity (100%) in both the ELISA (Table 2) and the microbead assay (Table 3). P167C demonstrated higher sensitivity in the microbead assay (94%) (Table 3) than in the ELISA (88%) (Table 2). In addition, both recombinant antigens demonstrated higher specificity than the H. bilis membrane extract in the ELISA as well as the multiplex microbead assay. In particular, the specificity for the H. bilis recombinant antigens was significantly higher (100% for P167C and 95% for P167D) than for the H. bilis membrane extract (65%) in the microbead assay (Table 3). In the ELISA, the specificities of H. bilis recombinant antigens P167C and P167D were both 75%, whereas H. bilis membrane extract showed 65% specificity (Table 2).
In both immunoassay formats, H. hepaticus membrane extract was the least sensitive (70% in the ELISA and 75% in the microbead assay) and specific (50% in the ELISA and 39% in the microbead assay) antigen in the detection of antibodies against H. hepaticus (Tables 2 and 3). Thus, recombinant antigens for H. hepaticus, like those for H. bilis (P167C and P167D), may be necessary for use in serodetection.
Taken together, the above results show that P167C and P167D recombinant antigens are better suited for the detection of H. bilis-specific antibodies in mouse sera than the membrane extract by both the multiplex microbead assay and the ELISA. The two recombinant antigens detected antibodies in all the H. bilis-infected retired breeder mice. More importantly, the two recombinant antigens together provided a highly sensitive assay for serodetection in animals with low levels of H. bilis antibodies, i.e., weanling mice. The added advantage of P167C and P167D recombinant antigens over the H. bilis membrane extract is a higher specificity in testing mice that may be coinfected with other species of Helicobacter. Thus, P167C and P167D together provide a highly sensitive and specific assay. Because the multiplex microbead method is ideally suited for testing the seroreactivities of multiple antigens simultaneously, the multiplex assay reported here will enhance the ability to reliably detect H. bilis infection in mice.
Purified recombinant proteins like P167C and P167D offer a significant advantage in the control of batch-to-batch consistency of antigen purity and yield. The importance of these properties in the development of a new assay is manifold: (i) assay robustness, (ii) ease of quality control, (iii) affordability, and (iv) shelf life. In contrast, whole-membrane extracts provide not only inconsistent purity but also a poor yield, making antigen preparation difficult at a small scale and production unfeasible at a large scale. Therefore, membrane extracts are not well suited for an assay intended for widespread use.
A main advantage of the multiplex technology is its ability to perform simultaneous detection of several analytes in a small volume of test sample. We estimate that under our present research laboratory conditions, approximately 300 to 400 serum samples can be analyzed per day. ELISA tests of 400 sera with four antigens would require several times more labor, time, and resources (e.g., plates and detection reagents). Because of the high-throughput capability of the multiplex microbead format, we will continue to expand this assay by adding capabilities for serodetection of additional Helicobacter species, e.g., H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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