Detection of High Titers of Antibody against Helicobacter Cysteine-Rich Proteins A, B, C, and E in Helicobacter pylori-Infected Individuals

Recombinant expression of HcpA, HcpB, HcpC, and HcpE.Hcp proteins were expressed, refolded, and purified under conditions similar to those reported previously for HcpA and HcpB (10, 11). Briefly, the ORFs HP0211, HP0235, HP0336, and HP1098 were amplified by PCR from the genomic DNA of H. pylori strain 26695 (American Tissue and Culture Collection), and the amplification products were inserted into a pTFT74 expression vector. The PCR also introduced a His₆ tag at the C-terminal ends of the proteins. Competent Escherichiacoli BL21(DE3) cells were transformed, and the cells were grown at 37°C. Three hours after induction with 1 mM isopropyl-β-d-thiogalactopyranoside, cells were harvested and broken up by passing the resuspended pellet over a French press.

Inclusion bodies were collected by centrifugation (15 min, 20,000 × g, 4°C), and the soluble fraction was discarded. The pellet was washed two times with buffer A (0.1 M Tris-HCl, 20 mM EDTA [pH 6.8]) and subsequently with buffer B (0.5 M GdnCl [guanidine hydrochloride] in buffer A). Inclusion bodies were solubilized in buffer C (5 M GdnCl, 0.2 M Tris-HCl, 0.1 M dithiothreitol, 10 mM EDTA [pH 8.0]), and insoluble material was removed by centrifugation. Solubilized inclusion bodies were dialyzed overnight against buffer D (5 M GdnCl, 0.1 M acetic acid). Hcp proteins were refolded by immobilizing the solubilized inclusion bodies on Ni-nitrilotriacetic acid-agarose (Qiagen). The column (5 to 10 ml) was washed with 50 ml of buffer E (5 M GdnCl, 0.1 M Tris [pH 8.0]), and Hcp proteins were refolded by replacing buffer E immediately with buffer F (50 mM Tris-HCl, 150 mM sodium chloride, 5 mM glutathione [pH 8.0]) and washing the column with 50 ml of buffer F at a flow rate of 1 ml/min. The protein was eluted with buffer G (250 mM imidazole, 50 mM Tris-HCl, 150 mM sodium chloride, 5 mM glutathione [pH 7.0]).

Protein-containing fractions were pooled and dialyzed against 1,000 ml of buffer H (40 mM sodium acetate, 1 mM EDTA [pH 5.5]). Buffer H was also used for gel-permeation chromatography. After concentrating the protein in a Centriprep (Millipore), 1 mg of refolded Hcp proteins was loaded onto a Superdex 75 HR 10/30 column (Amersham Pharmacia) and eluted as single peaks at a flow rate of 0.5 ml/min.

Investigation of patients.Thirty patients who were serologically positive for H. pylori were selected for this study. Thirteen patients were men (mean age, 49.1 years; range, 19 to 78 years), and 17 patients were women (mean age, 44.5 years; range, 18 to 78 years). The mean age of all patients was 46.8 years (range, 18 to 78 years). Identical serologic analyses were performed on samples from six serologically negative H. pylori patients (3 women and 3 men). The mean age was 50.9 years (range, 15 to 71 years). One sample was taken from each patient.

All serologic assays were performed in the laboratory of Clinical Immunology, University Hospital Zurich, which is accredited by the Swiss Federal Office of Metrology and Accreditation, Swiss Accreditation Service. Anti-H. pylori antibodies were measured with a commercially available enzyme immunoassay (Synelisa H. pylori [IgG], Abs; Pharmacia and Upjohn, Freiburg, Germany) by following the guidelines of the manufacturer.

ELISA conditions.Anti-HcpA, -HcpB, -HcpC, and -HcpE IgG antibodies were detected by enzyme immunoassay as follows. The antigens (0.2 μg/ml) (HcpA, HcpB, HcpC, and HcpE) in 0.1 M carbonate bicarbonate buffer (pH 9.6) were coated overnight at 4°C onto 96-well microtiter plates (Immulon 2; Dynatech Labs, Chantilly, Va.). The plates were blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS) at room temperature for 60 min. Negative and low- and high-positive controls from Pharmacia as well as serum samples were diluted 1:101 in PBS-0.05% Tween 20. After adding the samples, the plates were incubated at room temperature for 60 min. After four washes, horseradish peroxidase-conjugated rabbit anti-human IgG (DAKO Diagnostics A/S, Copenhagen, Denmark) diluted 1:2,500 in PBS-0.05% Tween 20 was added and incubated at room temperature for 60 min. After four washes, o-phenylendiamine hydrochloride solution was added and the color was allowed to develop at room temperature in the dark. The reaction was stopped with 4 M sulfuric acid, and the optical density (OD) was analyzed at a wavelength of 492 nm with a Tecan SLT enzyme-linked immunosorbent assay (ELISA) reader. All ELISAs were done in duplicate. Results were expressed as OD₄₉₂ values. To assess the variation among subsequent experiments, the sera of six H. pylori-positive individuals were analyzed against HcpA on all ELISA plates. The variation between subsequent ELISAs was between 3 and 8%. For samples where the absorption was saturated, values were truncated at 4.0 OD₄₉₂ units. For statistical analysis, Student's t test function, assuming unequal variances, was applied.

Detection of anti-HcpA, -HcpB, -HcpC, and -HcpE IgG antibodies.Anti-HcpA, -HcpB, -HcpC, and -HcpE IgG antibodies titers were analyzed by immobilizing the purified proteins to ELISA plates, incubating the plates with sera obtained from 30 H. pylori-infected and 6 uninfected individuals, and detecting bound IgG antibodies by standard ELISA techniques. Expression and refolding of more Hcp family members was attempted, but so far only HcpA, HcpB, HcpC, and HcpE were obtained in sufficient quantities. As shown in Fig. 1, the recombinant Hcp proteins were pure enough to rule out unspecific binding to impurities that might have been present in all Hcp protein preparations. The mean ODs for the anti-Hcp IgG titers in infected and uninfected individuals are given in Table 1. In the group of H. pylori-positive individuals, the average anti-HcpA, -HcpC, and -HcpE IgG titers were 1.11, 1.35, and 1.61, whereas in the control group, they were 0.306, 0.292, and 0.620, respectively. Although high values for the standard deviations indicated significant variations of IgG titers among individuals, the presence of anti-HcpA, -HcpC, and -HcpE IgG antibodies is expected, with probabilities below 5 × 10⁻⁴. For HcpB, the difference between the mean titers for infected and uninfected individuals is three times smaller and the P value is 100 times higher than the titers for HcpA, HcpC, and HcpE.

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FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of recombinant HcpA (lane a), HcpB (lane b), HcpC (lane c), and HcpE (lane d). Ten micrograms of protein was loaded per lane. Higher-molecular-weight impurities represent Hcp dimers that formed during sample preparation.

Correlation between anti-HcpA, -HcpB, -HcpC, and -HcpE IgG titers.As indicated by the values given in Table 2 and the line-plots shown in Fig. 2, there is a good correlation between anti-HcpA, -HcpC, and -HcpE IgG titers. The correlations for the pairwise comparisons are all above 0.7, indicating a clear trend. For HcpB, this trend is much less significant, as indicated by correlations below 0.4. No correlation was detected between titers of IgG measured by the commercial serological test to validate the infection state and the titers of anti-Hcp IgG (correlation < 0.15 for anti-HcpA, -HcpB, and -HcpC in the group of H. pylori-positive individuals).

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FIG. 2.

Line plots to indicate the correlations between HcpA and HcpC (a), HcpA and HcpE (b), and HcpC and HcpE (c).

There is substantial variation between titers of anti-Hcp IgG in the group of infected individuals. In some cases the titers of IgG are as low as in the group of H. pylori-negative individuals, but in some cases, the titers are 10 times larger. These fluctuations cannot be addressed as measurement errors but reflect the individual response of the host.