GASTROEh’TEROLOGY

1991;101:77-83

A Novel Enzyme Immunoassay for Serodiagnosis of Helicobacter pylori Infection TOSHIRO SUGIYAMA, KOHZOH IMAI, HIROKIYO YOSHIDA, YOSHIKAZU TAKAYAMA, TSUYOSHI YABANA, KENJI YOKOTA, KEIJI OGUMA, and AKIRA YACHI Departments of Internal Medicine and Microbiology,

Helicobacter pylori has recently been implicated as an etiologic agent of gastroduodenal disorders. Comparing the antibody to If. pylori in the sera of patients with that of normal controls by Western blot analysis, a unique antibody was detected in the sera of patients, which reacted with the 25-kilodalton antigen of H. pylori. On the other hand, monoclonal antibody CP3 prepared in the authors’ laboratory also recognized the 2!%kilodalton antigen of H. pylori. Whether the serum antibody of the patient recognized the CP3 antigen purified by monoclonal antibody CP3 was then examined. Western blot analysis showed that the patient’s serum reacted strongly with the affinity-purified CP3 antigen. Using monoclonal antibody CP3, an enzyme-linked immunosorbent assay to detect CP3 antibody in sera was established. In patients with chronic gastritis and gastric ulcers, the titer of CP3 antibody was significantly higher than in normal controls and correlated with the histological grade of antral gastritis. The detection of CP3 antibody in sera is useful in the diagnosis of chronic gastritis and gastric ulcer associated with H. pylori infection and also in evaluation of the grade of gastritis.

S

ince the initial report of Warren and Marshall (1) in 1983, the association of Helicobacter pylori with gastritis and/or gastroduodenal ulceration has received much interest. Following their reports, many investigators (2,3) confirmed that H. pylori was detected frequently in the gastric tissue of patients. In addition, experimental attempts (4,5) to fulfill Koch’s bacteriological postulates have been reported. Now, H. pylori has been implicated as an important etiologic factor (6-8) of gastritis and gastroduodenal ulceration. To diagnose H. pylori infection, many useful methods (1,9,10) have been reported. In addi-

Sapporo Medical College, Sapporo, Japan

tion, a specific immunostaining method using antibody was recently reported (1 l-14). However, these methods require endoscopic examination for obtaining tissue specimens. As a noninvasive method, detection techniques of antibody in serum have also been developed using H. pylori or the extracts as antigens (15-19). However, Rathbone et al. (18) have pointed out that there is a common antigen between H. pylori and other Campylobacter genus organisms, and therefore the antibody in serum should cross-react with the other Campylobacter organisms. In our study, using Western blot analysis, a set of unique antibodies was exclusively detected in the sera of patients with H. pylori infection but not found in the sera of normal controls. Of the unique antibodies, an antibody against 25-kilodalton antigen was studied using anti-H. pylori monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA) system was established for the detection of serum antibody against the 25 kilodalton antigen of H. pylori. Materials and Methods Bacterial Strains and Culture Conditions H. pylori was supplied by ATCC (ATCC 43504; Rockville, MD). The culture of H. pylon’ was streaked on Brucella (Difco Laboratories, Detroit, MI)-10% horse blood agar plates and incubated at 37°C for 5 days in an anaerobic jar with microaerobic condition (Gas Pack System without catalist; BBL, Cockeysville, MD). The colonies grown were inoculated into 450 mL of Brucella broth-IO% horse serum, and incubated at 37°C for 5 days with microaerobic condition. The cells were collected by centrifugation at 6000 rpm

Abbreviation used in this paper: ELISA, enzyme-linked immunosorbent assay. o 1991 by the American Gastroenterological Association 0016~5085/91/$3.00

78 SUGIYAh4AET AL.

for 20 minutes, washed two times with 0.05 mol/L phosphate-buffered saline (PBS) (pH 7.41, and resuspended into 50 mL of 3.7% formaldehyde in 0.05 mol/L phosphate buffer (pH 7.4). After incubation at 20°C for 20 minutes, the cells were washed three times and resuspended into 50 mL PBS. The resultant cells were then sonicated as shown below. Campylobacterjejuni, Campylobacter coli, and Campylobacter fetus were obtained from the Japan Federation for Culture Collections (GIFU 8733, GIFU 8728, and GIFU 8429, respectively; Tokyo, Japan). Escherichia coli was obtained from ATCC (ATCC 25922).

Culfivafion

test ofH. pylori

Biopsies of two tissue specimens were performed using endoscopy both from disease and control groups. One specimen was cut in half and used for both culture and urease test. Another specimen was fixed with formalin for histopathology. These procedures were performed within 3 hours after biopsy. The homogenized preparations were streaked on Brucella-10% horse blood agar plates supplemented with polymyxin B (2.5 IU/mL), vancomycin (10 t_tg/mL), and trimethoprim (5 pg/mL) and incubated at 37°C for 5 days in an anaerobic jar under microaerobic conditions (Gas Pack system without catalyst). The cells grown were characterized by Gram’s stain and by catalase, oxidase, and urease tests. Urease test was performed by inoculating the cells into the Christensen urea medium. Urease Test for Tissue Specimen and Immunostaining by Monoclonal Antibody Urease testing of the sampled tissue specimens was performed using a Minitek disk purchased from BBL. The urea disk was dispensed into a well of a Minitek plate, and 200 ~.LLof Minitek broth was added. After a specimen was placed into the broth, the plate was incubated for 5-18 hours at 37°C. Immunostaining by a monoclonal antibody CPl was performed as described elsewhere (20, 21). Western Blot H. pylori was sonicated with an ultrasonicator (model 185; Branson, Danbury, CT) for 15 minutes, then 60 I&lane was electrophoresed in sodium dodecyl sulfate (SDS) gel and transferred to nitrocellulose membrane according to the method described previously (21). The membrane was reacted with serum (1:lOO diluted) and with I:300 diluted peroxidase-labeled anti-human immunoglobulin antibody (DAKO, Japan, Kyoto, Japanland then was developed with 4-chloro-l-naphthol (0.5 mg/mL) solution containing H,O,.

GASTROENTEROLOGYVol. 101, No. 1

polyethylene glycol2000, suspended in RPM1 1640 containing 10% fetal calf serum, and then distributed to microculture plate at 5 x lo5 cells per well. Hybridomas that produced antibodies reacting with the sonicated antigen of H. pylori were cloned by the limiting dilution method. Enzyme-Linked

Immunosorbent

Assay

Enzyme-linked immunosorbent assay was used for screening the production of anti-H. pylori antibodies and analyzing the specificity of antibodies (23). One hundred micrograms of sonicated antigen (H. pylori, C. jujuni, C. coli, C. fetus, and E. coli) was fixed to the ELISA plate (Becton Dickinson, Oxnard, CA). The supernatants of the hybridomas were reacted with the antigen for 2 hours at room temperature. Peroxidase-labeled anti-mouse immunoglobulin (Ig) antibody (1:2000 diluted; DAKO) was added. After washing, the substrate solution containing o-phenylenediamine (0.4 mg/mL) and H,O, was added to each well and developed. Antigenic Profile of H. pylori Monoclonal Antibody

Defined by

profile of H. pylori defined by anti-H. antibodies, designated CPl, CP2, and CP3, was determined by Western blotting. Sonicated H. pylori antigen (40 &lane) derived from the ATCC strain and from gastric tissue of patients was electrophoresed in 10% SDS-gel and electrotransferred to nitrocellulose membrane. It was reacted with each anti-H. pylori monoclonal antibody (10 pg/mL). An antigenic

pylori monoclonal

Purification of the Monoclonal Antibodies and H. pylori Antigen The peritoneal cavity of the BALB/c mice was innoculated with hybridoma-producing anti-H. pylori monoclonal antibody and the ascites were aspirated. The monoclonal antibodies, CPl (IgG2a), CP2 (IgG3), and CP3 (IgM), were purified from the ascites with 33% (for CPl and CP2) and 50% (for CP3) ammonium sulfate precipitation followed by DEAE (DE52; Whatman, Maidstone, England) for CPl and CP2 and gel filtration methods using a Sephacryl S-300 (Pharmacia, Uppsala, Sweden) for CP3. Each antibody was then coupled to CNBr-activated Sepharose 4B beads (Pharmacia). Crude H. pylori antigen derived from ATCC strain, which had been sonicated and dissolved, was applied to the above-mentioned anti-H. pylori monoclonal antibody CP3 affinity column and eluted by 0.2 mol/L glycine-HCl buffer (pH 2.5). The resulting solution was called purified CP3 antigen.

Hybridomas The fixed H. pylori (1 x 10")was injected four times into the peritoneal cavity of g-week-old female BALB/c mice. After the spleen was taken out of the immunized mouse, the splenocytes were fused with mouse myeloma cells (X63-Ag8.653) as described by Imai et al. (22). Briefly, splenocytes and myeloma cells (1O:l) were fused in 30%

Anti-H.

pylori An tibody in Sera

A FAST plate (Becton Dickinson) was coated with purified CP3 antigen (1 &mL). Sonicated antigen obtained from E. coli was used as a negative control. After blocking with 3% bovine serum albumin solution, I:300 diluted serum was added and incubated for 2 hours at room

SERODIAGNOSIS OF H.!XKOBAC7E~ PYLORIINFECTION

July 1991

temperature. Peroxidase-labeled anti-human IgG antibody (1:lOOO diluted; DAKO) was then added and incubated for 1 hour at room temperature.

Patients and the Degree of Gastritis In all patients, diagnoses were made by endoscopic examination and tissue specimens were obtained by biopsy. H. pylori infection was judged as positive when results of either the cultivation test, the urease test, or the immunoperoxidase staining of biopsy specimens using monoclonal antibody CPl were positive. The serum was collected from H. pylori-infected patients and normal controls for the antibody assay. The degree of gastritis (mild, moderate, or severe] was graded according to the extent of inflammatory infiltrate on the formalin-fixed biopsy tissues stained with H&E as described (24). Briefly, when neutrophils infiltrated into the intraepithelial layer, the grade was judged as “severe.” “ Mild” or “moderate” was judged according to the degree of inflammatory cells in the lamina propria.

79

6

5

WOK)

Statistic al Analysis Wilcoxon’s nonparametric test was used to compare the titer of serum antibody of patients with that of normal controls.

Results Profile of Serum Antibody

Against H. pylori

We compared the profiles of serum antibody against sonicated H. pylon’ antigen in patients and normal controls by Western blotting. Figure 1 shows the representative pattern of serum antibody against H. pylori. Many positive bands were observed, and polyreactive antibody was detected in the sera of gastric ulcer patients as well as chronic gastritis patients. In the case of the serum of normal controls, a few positive bands were observed. Interestingly, the antibodies against the llO-, 36-, and 25kilodalton antigens of sonicated H. pylori showed remarkably high incidences in sera from patients of gastric ulcer and chronic gastritis compared with the sera of normal controls (Table 1). Therefore, we decided to focus on these antibodies for serodiagnostic usage. Monoclonal Antibodies Thirty mouse monoclonal antibodies were selected with ELISA using sonicated H. pylori antigen. Among them, three monoclonal antibodies, CPl, CP2, and CP3, strongly reacted with sonicated H. pylori by ELISA but did not react at all with C. jejuni, C. coli, C. fetus, or E. coli (Table 2). The reactivity of these monoclonal antibodies with H. pylori was confirmed by immunoperoxidase staining of tissue sections (photographs not shown).

Figure 1. Western blotting analysis of antigens detected by anti-H. pylori antibodies in sera. Many positive bands were observed in the sera of gastric ulcer patients (lanes 1 and 2) and in that of chronic gastritis patients (lanes 3 and 4) compared with sera of normal controls (lanes 5 and 6).

Western Blotting Analysis Western blotting was carried out for the analysis of those antigens recognized with monoclonal antibodies CPl, CP2, and CP3. Monoclonal antibody Table 1. Antibody Profile Against H. pylori in Sera of Patients and Normal Controls Antigen

(kilodaltons) 180 150 110 60 36 25

Gastric ulcer (n = 5) 5 5 5 5 5 5

Chronic gastritis (n = 4) 4 4 4 4 4 4

Normal control (n = 7) 5 5 2 7 2 0

80

SUGIYAMA ET AL.

GASTROENTEROLOGY Vol.

Table 2. Reactivity of Monoclonal Antibodies to H. pylori

and Other Bacteria Detected by Enzyme-Linked ImmunosorbentAssay Monoclonal antibodies (subclass) CPl

Antigen

(IgG2al

H. pylori C. jejuni C. coli C. fetus E. coli

++ -

CP2 (IgG3) ++ -

-

-

-

-

CP3 (IgM) ++ -

-

-

+ +, Strongly positive; -, negative.

reacted with a 36-kilodalton molecule of sonicated H. pylori, CP2 with 60- and 180-kilodalton molecules (data not shown), and CP3 with a 25kilodalton molecule (Figure 2). In addition, monoclonal antibody CP3 reacted with a 25-kilodalton molecule of H. pylori obtained from the gastric tissue of a patient with gastric ulceration (Figure 2). CPl

101. No. 1

body. This measurement was repeated three times, and similar results were obtained. As negative controls, the sera of high school students who showed negative results for either the cultivation test of H, pylori, the urease test, or immunostaining were used. As positive controls, sera from patients with gastric ulcers who had shown positive results for the methods of detecting H. pylori from gastric tissue were used. In 30 healthy controls, the mean antibody level was 33.4 EU and the standard deviation was 17.8. The proposed cut-off value was taken to be 69.0 EU (mean + 2 SD). Sensitivity and specificity of the assay were 96.9% and 100% respectively. Positive and negative predictive values or the test were 1.00 and 0.94, respectively. As shown in Figure 5, the sera of H. pylori-infected (i.e., either test positive) chronic gastritis patients as well as gastric ulcer and duodenal ulcer

Digerence Between a 25Kilodalton Molecule of H. pylori Defined by Patient Serum and Monoclonal Antibody CP3 To examine whether the 25-kilodalton molecule of H. pylori recognized by monoclonal antibody CP3 was the same as that recognized by the antibody in the serum of gastric ulcer and chronic gastritis patients, CP3 antigen was purified by affinity chromatography, which was prepared by Sepharose 4B coupled with monoclonal antibody CP3. Western blotting analysis was then carried out. The purified CP3 antigen was electrophoresed to 10% sodium dodecyl sulfate gel and transferred to nitrocellulose membrane and then reacted with the serum (1:lOO dilution) of a patient with gastric ulceration. As shown in Figure 3, the serum was found to react more strongly with the 25kilodalton molecule of sonicated C. pylori antigen (CP3 antigen) purified by monoclonal antibody CP3 than with the crude H. pylori antigen. On the other hand, the serum of a normal control failed to react with purified CP3 antigen (Figure 3). An ti-CP3 Antibody in the Serum

An ELISA system for detection of anti-CP3 antibody in the serum was established. For standardization of antibody titer in the serum, an ELISA unit was set tentatively according to the methods previously described by Evans et al. (25) (Figure 4). The serum of a patient in which anti-CP3 antibody had clearly been detected by Western blotting (T.M., female, gastric ulcer) was used as a standard anti-

Antigen

H. pylori E. coli H. pylori patient (ATCC43504)

Figure 2. Reaction of monoclonal antibody CP3 with H. pylori from the gastric tissue of a gastric ulcer patient and with H. pylori from long-term culture [ATCC 43504) was assessed by Western blotting. Monoclonal antibody CP3 reacted with the 33-kilodalton molecule of H. pylori derived both from ATCC and from gastric tissue, but it did not react with E. coli.

July

SERODIAGNOSIS OF HELICOBACTER

1991

PYLORI INFECTION

P

81

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60K

25K

Figure 3. Serum from a patient with a gastric ulcer reacted strongly with the 3Ucilodalton molecule (designated as CP3 antigen), which was purified by monoclonal antibody CP3, whereas normal control serum did not.

patients showed significantly than that of normal controls.

higher

Antigen lintibody

ELISA units,

The histological grade of antral gastritis was determined according to the modified Warren’s classification. As shown in Figure 6, the titer of anti-CP3 antibody in sera from patients with chronic gastritis significantly correlated with the grade of antral gastritis. Thus the detection of anti-CP3 antibody in the serum is diagnostic of H. pylori infection and also O.D. 2.0-l

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patient’s

serum

useful for evaluation gastritis.

purified CP3 Ag

crude CP Ag

purified CP3 Ag

normal

control

of the histological

grade of

Discussion

Association of An ti-CP3 Antibody and the Histological Grade of Gastritis

01

crude CP

dilution

Untt

Figure 4. Standard curve of ELISA for detection of CP3 antibody in serum. High-titer serum from a patient with a gastric ulcer was used as a standard. The ELISA unit was determined according to the reciprocal dilution of the serum.

The ELISA system is a simple and easy method for the detection of serum antibody to diagnose several infectious diseases, cancers, and so on. The critical point of ELISA is in the antigen used in the assay to ensure specificity and usefulness. For the diagnosis of H, pylori infection associated with gastroduodenal diseases, many ELISA systems have already been reported (15-18). Most of them, however, have used whole extracts of H. pylon’as antigen. A few recent papers (19,25) have reported the use of purified antigen of H. pylori to improve specificity. In this study, we chose two approaches to select a specific and diagnostic antibody in the serum against H. pylori. The first was comparative analysis of the antigen profile of serum antibody against H. pylori between H. pylori-infected patients and H. pylorinoninfected normal controls by Western blotting. The second was preparation of the monoclonal antibodies against specific antigens of H. pylori. These could also be useful for establishing ELISA to detect antibodies in the serum of patients. Through analysis of serum antibody, a set of unique antibodies against H. pylori was observed in the serum of H. pylori-infected patients. The antibody against the 25kilodalton molecule of H. pylori was detected exclusively in the

82

SUGIYAMA

GASTROENTEROLOGY

ET AL.

a unique 25kilodalton

p