Introduction

Helicobacter pylori is a causative agent of gastritis and gastric ulcer diseases which leads to the development of gastric cancer (Bühling et al. 2004; Algood and Cover 2006). It is gram-negative microaerophilic, flagellated and spiral-shaped bacilli (Vaucher et al. 2000; Menaker et al. 2004). More than 50 % of the world population is colonized by H. pylori, but few of them suffer from active disease because of different factors such as age, gender, crowding, hyperacidity, smoking habit and poor socioeconomic status (Ghose et al. 2007; Bielanski et al. 2006; Amjad 1997). Developing countries had a prevalence rate of (80 %) as compared to developed countries (20–50 %) (Suerbaum and Michetti 2002; Torres et al. 1998). Agewise prevalence rate of developed countries increases between the ages 60 and 69 years (48 %); however, prevalence rate is also linked with age in developing countries although a higher prevalence (80–90 %) is found in younger patients (Dehesa et al. 1991). H. pylori infection persistent is resulted in the development of local and systemic inflammatory response (Torres et al. 1998) along with the secretion of large amounts of extracellular products. Humoral and cellular responses against H. pylori infection are unable to eliminate H. pylori and maintain systemic immune response with production of high IgG antibody titers. Along with other factors, the emerging antibiotic resistance and improper diagnostic facilities are also responsible for high incidence of H. pylori infections (Amjad 1997). H. pylori is diagnosed by different invasive tests such as microbiological culture, histological examination and rapid urease tests, while nowadays H. pylori is diagnosed through a noninvasive test such as PCR, urea breath test and enzyme immunoassays. The invasive test requires upper gastrointestinal endoscopy, while UBT and PCR need an expensive instrumentation with a specialized technician to operate it. ELISA is simple, inexpensive assay and has a less burden for the patient; these qualities make ELISA more attractive as compared to other noninvasive test (Attallah et al. 2004). It is better and reliable assay for the detection of H. pylori serum antibodies to evaluate prevalence in large communities (Torres et al. 1998). The natures of antigen play an important role in the improvement of ELISA sensitivity and specificity (Best et al. 1992). H. pylori have a diverse protein profile (Mégraud and Lehours 2007) with different candidates of antigens which can use as putative components of vaccines such as whole cell surface, sonicate, flagellar with partially and highly purified antigens (Amjad 1997; Aebischer et al. 2005). H. pylori surface whole cell antigen has a high probability of being neutralized and prevents infection as compared to intracellular components (Khafri et al. 2005; Watanabe et al. 2001). In-house ELISA based on the H. pylori local strain can give better results as compared to commercial ELISA (Khanna et al. 1998; Marassi et al. 2005). H. pylori strains from Asian countries have different antigenic properties as compared to western countries (Leung et al. 1999). We develop an in-house ELISA based on H. pylori surface whole cell antigen by using H. pylori local isolates. ELISA based on local H. pylori strain provides a better and reliable sero-diagnostic and sero-epidemiological tool for the diagnosis of H. pylori infection in Karachi, Pakistan (Hoang et al. 2004).

Materials and methods

Patients and gastroendoscopy

All gastric patients were undergone through gastroendoscopy and examination in the Civil Hospital Karachi, Pakistan. Tissue biopsies were obtained from the antral and corpus part of the stomach during gastrointestinal endoscopy along with 214 blood samples from consenting patients. Gastric patient’s blood samples were characterized as chronic gastritis and gastric ulcer. All the samples were transported to Immunology & Infectious Diseases Research Laboratory (IIDRL) for further processes and frozen at −20 °C until tested. All gastric samples collected from the patients and the research protocols were in accordance with the Karachi University’s Ethical Committee.

Preparation of H. pylori surface whole cell antigen

Tissue biopsies were processed and cultured on Colombia agar (CA) plates containing 7 % lysed horse blood and antibiotics (amphotericin B, trimethoprim, cefsulodin and vancomycin). The CA plates were incubated for 4–5 days under microaerophilic conditions at 37 °C, and identification was carried out by different conventional and molecular methods. Multiple H. pylori strains were inoculated in the BHI broth, incubated at 37 °C for 5 days in microaerophilic environment. H. pylori heavy growth was treated with 0.5 % formalized saline and kept overnight at 4 °C. Centrifuged at 10,000 rpm for 10 min and washed 3 times with 0.2 % formalizes PBS (pH 7.2). Finally, the concentration with carbonate buffer (pH 9.8) was adjusted and stored at 4 °C. Protein concentration was measured by Coomassie blue assay (Bio-Rad, Hercules, CA, USA), bovine serum albumin used as a standard.

Rabbit immunization with H. pylori antigen

Adult female rabbits were immunized intravenously with the increasing amount of antigen (3 × 108 cells/ml) on the days of 1, 3, 5, 7 and 17. Blood was collected from a marginal ear vein and by cardiac puncture after the slide agglutination test. Pre-immunization sera were used as a negative control to standardization the ELISA based on surface whole cell antigen (wELISA).

Requirement for acceptance of ELISA plates

The ELISA plate was examined by different negative and positive serum samples. All reference serum samples were obtained from IIDRL, Department of Microbiology. Serum from rabbit, sheep and healthy human (1:100) were used as a negative reference, while gastritis patient serum was used as a positive control after confirmation by commercial ELISA Kit (Equipar, Italy). Different components of wELISA such as H. pylori antigens, PBS, blocking agent, enzyme conjugate and substrate were also used separately as negative control. The acceptance level of ELISA plate was OD >0.5 and <0.20 for positive and negative control, respectively. ELISA value was calculated as (EV = OD of test sample/OD of calibration unit) by taking 5 Uarb/ml (OD450nm = 0.723) as a calibration unit (5 Uarb/ml used to discriminate between negative and positive population). Data were present as mean ± SEM.

Development of in-house ELISA for H. pylori surface whole cell antigen (wELISA)

In order to develop wELISA, each component was separately optimized including antigen concentrations, enzyme conjugates, test serum and incubation times. Details of the wELISA standardization and development were indicated in the “Result” section.

H. pylori titer optimization and application

In order to optimize primary antibody titer, we were tested anti-H. pylori sera between the range of 10−1 and 10−15 at constant antigen concentration (20 μg/ml). Titer range was determined relative to the negative values which were greater than cutoff values. This optimized range was applied on a gastric patient serum to analyze the H. pylori titer status.

Quality control test of wELISA

In order to check the comparable results of serological assay, it is necessary to execute the quality controls of the developed else. Repeatability was determined by comparing coefficients of variation (CV) of strong positive (highest OD value) and weakest negative (lowest OD value) serum samples of the plate. Coefficient variation of each well was calculated by using the coefficient of variation of the plate as

$$ {\text{CV}} = \frac{{{\text{standard}}\;{\text{deviation}}\;{\text{of}}\;{\text{ODs}} \times 1 0 0}}{{{\text{mean}}\;{\text{of}}\;{\text{ODs}}}} $$

Application of wELISA

Developed wELISA was applied to gastric patient’s serum as; a 96-well plate (Sero-Wel, UK) was coated with 20 μg/ml (100 μl) of H. pylori surface whole cell antigen in coating buffer (Na2CO3, NaHCO3, pH 9.6) with overnight incubation at 4 °C. Next day, it was washed five times with PBS-Tween20 (0.05 %) (pH 7.2) and blocked free active sites by 3 % BSA. After washing, 100 μl (1:100 diluted) of patient serum was added into consecutive wells along with negative and positive controls and incubated at 37 °C for 1 h. After extensive washing, HRP-conjugated goat antihuman IgG antibody (1:2,500) was added and incubated for 1 h at 37 °C. Later, 100 μl of substrates (TMB One solution G7431, Promega, USA) was added and kept for 30 min at room temperature in dark place. The reaction was stopped by adding 0.3 M H2SO4, and color intensity was measured at 450 nm by an ELISA reader (STAT FAX-2100).

Cutoff value estimation

Receiver operating characteristic (ROC) curves [plots of sensitivity against (1−specificity)] were plotted to estimate the optimal cutoff value of different values of sensitivity (Se) and specificity (Sp). Area under the ROC curve (AUC) was estimated (DeLong et al. 1988) by two-tailed test with an accuracy value of 0.5 as significance level. The perfect diagnostic test would appear as close as possible to the upper left-hand corner, where both sensitivity and specificity are 100 %. We select a cutoff value of higher sensitivity with minimum false-negative value and high specificity with minimum false positive (Griner et al. 1981) and also measure by the mean optical density of the known negative samples plus two or three times the standard deviation.

Evaluation of wELISA

The agreement between the wELISA and other serological assays was assessed by the κ statistic. Student’s t test (one sample and independent samples) was used to compare mean value. Linearity of correlation was calculated by the Pearson’s coefficient correlation method. p < 0.01 were considered as significant. The statistical software, namely SPSS 16.0, MedCalc 11.3.3 and Graph Pad Prism 5.03, was used for analyzing the data.

Characterization of H. pylori surface whole cell protein by SDS-PAGE

Tris–HCl buffer SDS-PAGE (Laemmli) was applied to separate the surface whole cell antigen and serum at 10–8 % with 5 % stacking gel by using Bio-Rad Minigel apparatus. Coomassie staining was performed with Coomassie brilliant blue solution [Coomassie brilliant blue (CBB)-R250]. Relative molecular weight (M r) of unknown protein was determined to calculate their R f values by using a standard protein marker (Bio-Rad 161–0318) with linear regression curve method (excel spreadsheet program) (www.tulane.edu/~wiser/methods/homework/HW5_key.pdf).

H. pylori whole cell sonicate antigen ELISA (sELISA)

A 96-well plate (Sero-Wel, UK) were coated with H. pylori sonicate whole cell antigen with overnight incubation at 4 °C; after blocking, 100 μl of patient serum was added into respected wells and incubated at 37 °C for 1 h. After washing, HRP-conjugated antihuman IgG antibody (1:2,000) dilution was added followed by the addition of 100 μl substrates (TMB one solution G7431, Promega, USA) for 30 min at room temperature. Color intensity was measured at 450 nm by an ELISA reader (STAT FAX-2100).

H. pylori commercial kit ELISA (kELISA)

Serological assessment of H. pylori infections was measured by a commercial ELISA kit (Equipar VA, Italy). Commercial ELISA kits were used to analyze H. pylori titer values according to manufacturer’s instructions.

Ethical clearance

This study was approved by the Karachi University’s Ethical Committee in Karachi, Pakistan.

Results

This study was designed to develop an in-house ELISA for the diagnosis of H. pylori infection in gastritis and gastroduodenal patients of Karachi, Pakistan. A total of 214 blood samples were collected from patients reporting at Civil Hospital Karachi with the symptoms of gastritis and gastroduodenal problems. Blood collected from male or female patients (35 and 65 %, respectively) was processed in IIDRL (Immunology & Infectious Diseases Research Laboratory), Department of Microbiology, Karachi.

Requirement for acceptance of ELISA plates

The reference negative and positive control values were found to be in the range of assay limit. Negative controls showed the mean OD values between the range of 0.046 ± 0.535 to 0.139 ± 0.037, EV = 0.058–0.192, and positive controls showed values in the range of 1.074 ± 0.084 to 1.092 ± 0.1223, EV = 1.485–1.510. This result showed the correct validity of the controls with an acceptable level of ELISA value.

Rabbit anti-H. pylori serum valuation

Different parameters were optimized to develop wELISA. We found EV = 1.485 of anti-H. pylori sera against H. pylori surface whole cell antigen, which was sixteen times more than the pre-bleed rabbit serum EV = 0.063 and was considered as positive. The slide agglutination test was also shown significant agglutinates; hence, the batches of rabbit anti-H. pylori sera were used for the development of wELISA.

Developed wELISA parameters

The optimized condition for wELISA was performed as we tested H. pylori antigen concentrations by checkerboard method from 5 mg/ml to 5.96 × 10−7 which was followed by selecting five optimal antigen concentrations of 40, 30, 20, 10 and 4 μg/ml (Fig. 1a). The optimal antigen concentration for wELISA was chosen as 20 μg/ml concentration with 1:10−2 working serum dilution (Fig. 1b). The primary antibody titer range was found to be optimum between 10−1 and 10−9 of diluted serum at which the serum gives a positive result (Fig. 1c). An enzyme conjugate dilution of 1:2,500 was arbitrarily chosen and was subsequently used for detection of anti-H. pylori antibody (Fig. 1d).

Fig. 1
figure 1

Optimization of different parameters of wELISA. a A checkerboard method to optimize the H. pylori surface whole cell antigen concentration. b Line graph shows the optimization of selected optimal antigen concentration at the constant serum 1:100 dilution; 20 μg/ml shows suitable optimum antigen concentration as compared to 40, 30, 10 and 4 μg/ml. Data of H. pylori antigens are represented as mean + SEM. c Line graph shows optimization of anti-H. pylori serum (10–15—10–10). The serum value (10–1—10–9) was found to be higher to the cutoff value. d Enzyme conjugate antibody dilutions (1:3,000–1:1,000) were optimized. The final concentration (1:2,500) was shown optimal concentration

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wELISA cutoff estimation

The diagnostic performance of a test was evaluated to discriminate between normal and diseases cases by using ROC curve analysis (Metz 1978; Zweig and Campbell 1993). In order to confirm the disease, we selected a cutoff value of 0.395 which has a higher sensitivity with negative predictive value and high specificity with a positive predictive value (Griner et al. 1981). The cutoff values by mean ± 2SD and mean ± 3SD was found to be as 0.397 and 0.424, respectively. The ROC plot closer to the upper left corner indicated the perfect separation and no overlapping of the ODs of gastroduodenal ulcer patients with highest overall accuracy of the ELISA test (Zweig and Campbell 1993). The area under the ROC curve equals to 1 with a 95 % confidence interval (CI) from 1.00 to 1.00 (Fig. 2a). Scatter plot showed an optimum cutoff value of 0.395 (39.5 %) with highest sensitivity and specificity of 100 % (95 % CI 97.5–100) and 100 % (95 % CI 85.0–99.7), respectively (Fig. 2b).

Fig. 2
figure 2

ROC analysis of wELISA based on present positivity of gastritis and gastroduodenal ulcer patients. a ROC curves for sensitivity and specificity obtained with the antigens based on H. pylori surface whole cell antigen from local H. pylori strain. b Scatter plot represents the cutoff value of 0.395

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Quality control test of wELISA

In order to assess the real variability of wELISA, we run the quality control test and determine the variation between the intra- and inter-assay. The coefficient of variation of plate was found to be 12.47 % (in acceptance limit), indicated that each well of the plate was coated with equal antigen concentrations. Since the surety of the good quality and stable state of the antigen, it is necessary to run quality control test in the development of ELISA.

Analysis of H. pylori titer by wELISA

We found 29 % of patients have 1:10−9 anti-H. pylori titer, while 11 % showed 1:10−1 titer value. This result showed the inconsistency of H. pylori titer (1:10−9–1:10−1) in gastric patients (Fig. 3). There was significant difference between the mean values of each dilution. The nature of H. pylori antigen taken as component of variation between wELISA and (sELISA) (data to be published) and found a significant effect of H. pylori antigenic variation on H. pylori antibody titer (Table 1).

Fig. 3
figure 3

Comparison of anti-H. pylori antibody titers between different positive percentages of gastroduodenal ulcer patients by wELISA

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Table 1 Table showing wELISA mean value and analysis of variance in comparison with sELISA
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Age- and sexwise distribution of gastric patients by wELISA

In this study, 214 patients were enrolled between the age of 15 and 75 years (65 % of females and 35 % of males); 87 % of the patients were found to be H. pylori seropositive with agewise distribution as female (89 %) and male (84 %). We observed a high risk of H. pylori seropositive in middle-aged patients as compared to the old ages. Agewise distribution showed a high risk of infection between the ages of 35 and 45 years (40 %) as compared to ages of 15–25 years (36 %) have low risk of infections. Male patients (65–75 years) were shown an apparent decrease in the risk of infection (14 %). The highest risk of infection in the female population was found between the ages of 35 and 55 (52 %).

wELISA comparison with serological assay

Enzyme-linked immunosorbent assay based on surface whole cell antigen was evaluated by comparing with (kELISA) and sELISA (data to be published). We found a significant mean value variation of wELISA (1.387) with kELISA (2.144) as compared to sELISA (2.020) (p < 0.001). wELISA showed significant correlation between sELISA R—0.8179 (95 % CI from 0.76802 to 0.85792) as compared to kELISA R—0.77020 (95 % CI from 0.709 to 0.819), (p < 0.0001).

Evaluation of wELISA

In order to evaluate the authenticity of wELISA, we compare it with different serological assays. We found a strong association of wELISA with sELISA (κ value = 0.902–0.844) and kELISA (κ value = 0.863–0.649). Sensitivity of the assay can be increased just by altering the cutoff value. An assay is acceptable with good sensitivity and reduced specificity while sometime in reverse is required. The cutoff values of 0.397 and 0.395 were recommended as the optimal cutoff value in order to maximize the accuracy and minimize false negativity. On the basis of the accuracy, we can say that the diagnostic value of wELISA were to be excellent (90–100 % = excellent test) (Table 2).

Table 2 Table showing statistical analysis of wELISA with their comparative assays at different cutoff values
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Characterization of H. pylori surface whole cell protein

Helicobacter pylori protein characterization showed quantitative and qualitative differences between the normal and gastritis patient serum. Normal human serum revealed thirteen protein bands between 83 and 17 kDa (83, 71, 63, 54, 46, 43, 40, 38, 36, 30, 25, 21 and 17), while gastritis patient serum showed 14 protein bands between 102 and 17 kDa (102, 96, 89, 85, 81, 76, 69, 66, 63, 59, 51, 43, 36 and 17). Tris–HCl buffer resolves the H. pylori surface whole cell antigen into eight numbers of proteins with the size of 13 to 42 kDa (42, 34, 31, 28, 22, 21, 17 and 13), while their respective rabbit serum showed protein bands between the size of 11 and 68 kDa (68, 58, 45, 34, 28, 24, 21, 17 and 11) (Fig. 4).

Fig. 4
figure 4

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of H. pylori surface whole cell antigen (lane 2) (separated by 10 %) and their respected serum (lane 3) with human gastroduodenal ulcer diseases (lane 4) and normal human serum (lane 5) (separated by 8 %)

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Discussion

Helicobacter pylori is a noninvasive bacterium which stimulates the immune response by releasing different immunogenic proteins and lipopolysaccharides (Islam et al. 2007). Serological analysis of H. pylori infection is a noninvasive, less expensive method (Rahman et al. 2008). There are many serological assays for H. pylori detection which differs on the basis of their sensitivity (Amjad 1997; Ricci et al. 2007). ELISA performance is mainly based on the nature of antigen and H. pylori strain (Rahman et al. 2008). H. pylori have different type of antigens such as formaldehyde- or heat-treated whole bacteria, sonic extract, acid glycine extract. Whole cell lysate is the best choice of antigen as compared to a purified antigen which unable to recognize various type of antibodies present in the population. A different population may harbor different H. pylori antigen natures (Sunnerstam et al. 1999). H. pylori local strain antigen is useful with enough influence on the diagnostic properties of serological assay. In-house ELISA based on H. pylori local strain need to optimize different parameters to achieved better sensitivity and specificity (Leung et al. 1999). In order to study the seroprevalence of H. pylori in the population of Karachi, Pakistan, we investigate the immune response in gastric patients with developed in-house wELISA from the local strain of H. pylori.

Despite the simplicity of ELISA, the quality of the ELISA is mainly depending upon its optimum level. To develop the in-house ELISA, we need to optimize and adjust different parameters according to their local environment and population (Leung et al. 1999), such as antigen concentration, test serum and the enzyme conjugate dilution. In wELISA development, low antigen concentrations (1.218 × 10−3–5.96 × 10−7 mg/ml) were showing very low titer because of the unavailability of enough antigens to capture the antibody. However, high antigen concentrations (5–0.07 mg/ml) were shown very high titer and could produced signals even in the negative serum control indicated the presence of an adequate amount of antigen and availability of nonspecific antibody in the systems results in nonspecific binding reaction (Fig. 1a). From five selected optimal concentrations, 40 and 30 μg/ml of antigen concentration showed a high titer, even in negative control. The antigen concentration of 10 and 4 μg/ml gave the lowest titer which revealed that assay have very low specificity and sensitivity, while 20 μg/ml of antigen showed optimum titer even in the negative control which mean antigen was present in optimum concentration to capture the antibody with negligible nonspecific binding; 4 μg/ml of antigen concentration gave the negligible difference of optical density between the test sample and negative control, showed that there was no binding occur because of not enough antigen present in reaction to the capture antibody (Fig. 1b). The test serum working dilution of 1:10−3 showed low absorbance as compared to 1:10−2 serum dilution because of the too diluted concentration. The primary antibody titer was optimized by making serial dilution up to 10−15. The 10−10 diluted serums showed low absorbance value as compared to 10−9 diluted serum which showed a high absorbance to cutoff value. The 10−1–10−9 diluted serum range was found to be optimum at which the serum gives a positive result (Fig. 1c); 1:1,000, 1:1,500 and 1:2,000 dilutions of conjugate were shown very high detection level as compared to the positive control, while 1:3,000 gave very low detection value almost near to the value of negative control. The enzyme conjugate dilution of 1:2,500 showed optimal antibody detection level (Fig. 1d). The higher conjugate concentration increased the nonspecific reading, while lower conjugate levels decreased the specific absorbance measurement but the nonspecific binding was remain unchanged. The cutoff values were validated from the pool of reference sera of H. pylori local isolates, which was helpful to constant the specificity because of the diverse H. pylori antibody profile (Mégraud and Lehours 2007). Blecker et al. used the cutoff value derived from ROC curve analysis (Sunnerstam et al. 1999). The low coefficient of variation (13 %) of wELISA indicated repeatability was in the agreement of the international recommendation for CV of replicating samples (Dawo and Mohan 2007).

ELISA based on surface whole cell antigen were developed to determine the presence of H. pylori infection with a high degree of specificity and sensitivity. We compared wELISA with other immunoassay which were applied in our laboratory to demonstrate the variability in sensitivity, specificity and other evaluation parameters. Sensitivity can be effect by many factors such as concentration of reactants, the capacity of the solid phase, the concentration of the detector, the assay speed, incubation temperature and ELISA reader model. The high concentration of enzyme conjugate can increase the binding rate but have an effect on the sensitivity by elevating nonspecific binding. The elevated and long incubation temperature result in the improvement of sensitivity and speed of the assay, but the dissociation rate and intra-assay variation can also be high. Commercial ELISA kits are so expensive and have no surety of high specificity and sensitivity because of the H. pylori strain variations. H. pylori heterogenicity and cost of commercial ELISA kit limited its application, while in-house ELISA has no such problems. We found high sensitivity (95–93 %), specificity (100 %) and accuracy (95–94 %) of wELISA in comparison with kELISA and sELISA. Our results showed comparable values with those from other published papers; for example, Krajaejun et al. (2002) reported the 100 %sensitivity with 95.07 % specificity of in-house ELISA. Ghasemian et al. found sensitivity of 90 % with 88 % specificity (Safaei et al. 2005). Laheij et al. reported the disparity between western countries ELISA (sensitivity 93 % and specificity 94 %) and Asian countries ELISA (50 % and specificity 72 %). According to Khanna and Stone et al., these disparities are caused by the H. pylori strain heterogeneity from different parts of the world (Marassi et al. 2005). The perfect association of wELISA was found between kELISA (κ value = 0.863) and sELISA (κ value = 0.902). wELISA showed significant correlation with sELISA (R—0.8179) as compared to kELISA (R—0.7702) (p < 0.0001). False-positive test results may be due to the presence of cross-reacting bacterial antigens (Safaei et al. 2005) which can reduce by adsorption assay with H. pylori closely related species. In wELISA found no false-positive result; hence, no absorption step was considered necessary (Sunnerstam et al. 1999). At least 36 H. pylori serology kits are commercially available, but most of the kits do not have the sensitivity and specificity values greater than 90 % (Xia et al. 2000). Our study showed high sensitivity, specificity and accuracy to detect H. pylori titer in patients with gastritis and gastroduodenal ulcer diseases.

Antibody titer represents the meaningful data to verify the disease status and useful for antibody screening. ELISA can be used to detect various classes of H. pylori antibodies to characterize between current or past infection. As compared to IgA and IgM, the level of IgG is remaining constant elevated for 2 years (Oderda et al. 1988; Vaira et al. 1988). The antibody titer represents the antibody concentration which is strongly influenced by the total immunoglobulin concentration (Hayashi et al. 1998). H. pylori antibody is the indication of gastric colonization which may be used as a tool to screen large number of gastric patients. Previous results from our laboratory showed high H. pylori prevalence rates in male (59.36 %) as compared to female (53.95 %) (Amjad 1997), while this study showed overall high prevalence rate while indicated a high prevalence rate of H. pylori infection in females (89 %) as compared to male (84 %). The agewise prevalence rate of H. pylori infection was highest among the age group of 35–45 (40 %) as compared to ages 15–25 (36 %). Female population showed high risk of infection between the ages of 35 and 55 (52 %). Similar high prevalence rate of age group 35–45 was previously reported by our laboratory and the other researcher of the world (Dooley et al. 1989; Graham et al. 1998). In our study, overall prevalence rate of H. pylori infection in Karachi, Pakistan, was found to be more in female (79 %) as compared to males (74 %) population. Our results showed a comparable prevalence with those from other (Tygat et al. 1990; Satti et al. 1990) parts of the world including Brazil, Korea, Japan, Canada and Turkey between the range of 31 and 78 %. This H. pylori incidence rate indicates a quite wide range from different parts of the world. In developed countries, 40 % of the adults are colonized by H. pylori as compared to children (Perez-Perez et al. 1990). In France, less than 1 % of children were infected (>6 years), 5 % (6–12 years) and 15 % (15–18 years) as compared to developing countries like Thailand which have 18 % (5–9 years), 15 % (15–18 years) and 55 % (30 years), while Vietnam have about 40 % of the infection rate in the teenagers (Perez-Perez et al. 1990; Mégraud et al. 1989). Some specific age groups show high frequency and acquisition toward H. pylori infection (Klein et al. 1991).

The H. pylori responses detected by ELISA and immunoblot could provide the correct evidence of immunological reaction (Ricci et al. 2007). We found low molecular weight protein of H. pylori surface whole cell antigen between 13 and 42 kDa (42, 34, 31, 28, 22, 21, 17 and 13 kDa), while their respected serum (anti-H. pylori serum) showed the proteins between 11 and 68 kDa (68, 58, 45, 34, 28, 24, 21, 17 and 11 kDa) (Fig. 4). H. pylori low molecular weight antigen appears to be more reliable for the detection of H. pylori infection in children and adults (Andersen et al. 1995). Kimmel et al. reported different immunodominant antigens of H. pylori such as 44 kDa (translation elongation factor EF-Tu), 38 kDa (DNA-directed RNA polymerase), 28 kDa (conserved hypothetical protein), 25 kDa (3-oxoadipate coenzyme A transferase subunit A), 22 kDa (alkyl hydroperoxide reductase), 20 kDa (peptidoglycan-associated lipoprotein precursor (Omp18)), 17 kDa (biotin carboxyl carrier protein) and 14 kDa (ribosomal protein L7/L12) which nearest to our H. pylori whole cell antigen relative molecular weight (Kimmel et al. 2000). Park et al. reported H. pylori low molecular weight protein such as 43 kDa (hypothetical protein), 36 kDa (thioredoxin reductase), 22 kDa (3-oxoadipate co-A transferase subunit B) and 17 kDa (ribosomal protein S6) (Park et al. 2007). According to Andersen et al., H. pylori low molecular antigen protein has dominative role in H. pylori infection (Andersen et al. 1995). The conserved epitope characterization is beneficial for diagnostic and vaccine development (Cho et al. 2002). Haque et al. (1993) reported the immunogenic proteins with molecular weight of 61, 58 and 24 kDa from H. pylori whole cell extract. We found gastric patient serum proteins between the range of 102 and 17 kDa (102, 96, 89, 85, 81, 76, 69, 66, 63, 59, 51, 43, 36 and 17 kDa), while normal human serum showed protein between 83 and 17 kDa (83, 71, 63, 54, 46, 43, 40, 38, 36, 30, 25, 21 and 17 kDa) (Fig. 4). According to Andersen et al., H. pylori-positive serum has more significant numbers of protein than H. pylori-negative and normal sera (Andersen and Espersen 1992). According to Islam et al., 100 and 85 kDa proteins have higher immunogenic effects among H. pylori infected patients which could be used for vaccine preparation (Islam et al. 2007). A diversified high number of H. pylori proteins reflects the host response to H. pylori infection and indicates the importance of using a multicomponent antigen for detection of H. pylori antibodies. The crude antigen contains a large number of different proteins which may play a major antigenic role. The complex pathogenesis of H. pylori infection demands better approaches for the identification of novel immunogens that would give substantial protection. In-house wELISA is a multicomponent antigen assay which can used to detect H. pylori antibodies in the gastritis and gastroduodenal ulcer patients.

We conclude that in-house ELISA based on H. pylori surface whole cell antigen is better and reliable diagnostic assay as the various parameters were adjusted according to their native environment and conditions. The high accuracy suggests that wELISA can be used to detect anti-H. pylori antibodies in gastric and gastroduodenal ulcer patients. ELISA for H. pylori sero-diagnostic infection should be based on the local strain for better sensitivity and specificity. In-house ELISA can provide a reliable and a clinically useful method for the diagnosis of H. pylori infection in gastric patients of Karachi, Pakistan.