WO2023281160A1

WO2023281160A1 - Unified gastropanel in one microplate

Info

Publication number: WO2023281160A1
Application number: PCT/FI2022/050480
Authority: WO
Inventors: Osmo Suovaniemi; Tapani Tiusanen
Original assignee: Biohit Oyj
Priority date: 2021-07-09
Filing date: 2022-06-30
Publication date: 2023-01-12
Also published as: FI20215799A1

Abstract

According to an example aspect of the present invention, there is provided an improved method for screening or examining a symptomless subject or a subject having symptoms of an autoimmune disease, wherein the method and relating quantitative measurement of relevant bio markers is carried out in one microplate set-up.

Description

UNIFIED GASTROPANEL IN ONE MICROPLATE

FIELD

[0001] The present invention relates to an improved method and test for comprehensive diagnosis of Helicobacter pylori (Hp), atrophic gastritis (AG) and gastric output as well as for screening of gastric- and esophageal cancer risks, wherein the test and quantitative measurement of the relevant biomarkers is carried out in one microplate instead of conventional multiple microplate experiment set-up. BACKGROUND

[0002] Gastric cancer (GC) continues to be one of the most common cancers and causes of global cancer mortality; nearly one million new cases and 736.000 annual cancer deaths worldwide in 2018. In addition to the common-type risk factors (e.g. smoking, diet), there are two specific risk factors that far exceed in importance of all the others in pathogenesis of GC: 1) Helicobacter pylori (Hp) infection and 2) atrophic gastritis (AG)(1).

As early as in 1994, the International Agency for Research on Cancer (IARC, Lyon; a WHO agency) concluded that the accumulated scientific evidence is sufficient to declare Hp as a human carcinogen class I (2). In addition to being the key risk factor of GC, Hp-infection also plays a causative role in the development of peptic ulcer disease (5). [0003] Although Hp itself is not directly carcinogenic, AG is the single most potent risk factor of GC (1,3-5). Based on the Updated Sydney System classification (USS), AG is classified by its topographic location in the stomach (antrum, corpus, or both) as AGA, AGC or AGpan, respectively (6). The main histological type of GC (intestinal type) develops in atrophic mucosa (AG) through various degrees of dysplasia, often accompanied by intestinal metaplasia (IM). This pathogenetic chain of events is known as the Correa cascade (3). Indeed, the risk of GC increases in parallel with the severity of AG: compared with healthy stomach, the risk is 2-5 times higher in those with only chronic Hp gastritis but up to 90-fold in patients with severe AG both in the corpus and antrum (1,3,4). Importantly, both AG and Hp can be responsible for the symptoms known as dyspepsia; organic or functional (7). [0004] The diagnosis of AG has traditionally been made using histological biopsies on gastroscopy. However, gastroscopy is an invasive diagnostic tool, which requires expensive equipment and considerable professional skills. Gastroscopy is also a subjective diagnostic method, which is not suitable for population-based screening of GC (4). As to the diagnosis of Hp-infections, unfortunately, the two most widely used Hp tests; the ¹³C-Urea Breath Test (UBT) and Stool Antigen test (SAT), both have some limitations that hamper their diagnostic utility leading to false positive and -negative results in a substantial proportion of tests (8,9).

[0005] Because of these known obstacles in diagnosis of AG and Hp-infection, the need to develop an alternative diagnostic test increased in parallel with the increasing understanding of the importance of Hp and AG as the key risk factors of GC (1,3-6). To meet this increasing demand, the GastroPanel® test was designed in the late 1990’s by Bio hit Oyj (Helsinki, Finland), representing the first non- invasive diagnostic test for stomach health and disease (10,11). With GastroPanel®, both Hp and AG can be identified in a simple blood test, and the biomarker panel is equally applicable as the first-line diagnostic test for patients with dyspeptic symptoms as well as in screening of the risk conditions of GC (1,4,10,11).

[0006] With GastroPanel®, it has become possible for the first time to diagnose Hp- infection and AG by a non- invasive testing with a panel of four bio markers: pepsinogen I (PGI), pepsinogen II (PGII), gastrin-17 (G-17) and Hp IgG-antibodies (1,10,11). GastroPanel® test is based on stomach physiology in both health and disease. Accordingly, pepsinogen levels and their ratio is decreased in corpus atrophy (AGC), accompanied by elevated G-17b (basal). G-17b level also sensitively responds to gastric acid output, being low with high acid output and high when the stomach is achlorhydric. In severe antrum atrophy (AGA), G-17b is low and, importantly, does not respond to a protein stimulation (G-17s), because the antral G-cells have atrophied (1,10,11).

[0007] The results of GastroPanel® test are interpreted by a specially designed software application (GastroSoft®), identifying several diagnostic marker profiles (Figure 1). Of those, four (profiles 1,2,3 and 8) represent purely functional disorders (of acid output), while three others specify structural abnormalities (profiles 5,6, and 7 for AGC, AGA, and AGpan, respectively). The remaining (profile 4) is typical to Hp-infection, with three possible outcomes: a) active Hp-infection, b) successful eradication, and c) failed eradication (1,10). By this approach, the patients can be stratified into three groups with a different risk: a) normal (healthy) mucosa membrane, 2) Hp-infection, and 3) AG (1,10,11,13,14). This stratification has important clinical implications in patient management.

[0008] During the past 10 years, GastroPanel® has been validated in an increasing number of studies both in clinical and screening settings (1,12), and its excellent clinical performance was validated in a recent meta-analysis by an independent Italian group (12). To illustrate the exceptional diagnostic performance of the most recent version of GastroPanel® test, enclosed are two figures (Figures 2A and 2B) from two recently published studies, depicting the results of ROC analysis for PGI in diagnosing moderate/severe AGC (AGC2+)(13) as well as GastroPanel IgG Hp ELISA in diagnosis of biopsy-confirmed Hp in the corpus (14). In the former setting, AUC approaches 0.950, while in the latter, AUC=0.882. These results clearly warrant the statement that GastroPanel® is a highly accurate non-invasive test for GC risk factors: AG and Hp (1,3,10-14).

[0009] In addition to accurately disclosing the GC risks in a single blood test, the GastroPanel® test results have also excellent longitudinal predictive values (NPV & PPV) for developing an incident GC during a long-term follow-up. This has been firmly confirmed in longitudinal population-based cohort studies in different populations, both Caucasian and Asian (15,16). In both studies, GastroPanel profiles (Figure 1) indicating AGC were significant predictors of an increased risk of developing GC during a long-term (>10 years) follow-up.

[0010] The problem with the population-based analysis of the GastroPanel® biomarker profiles signifying functional disturbances is the lack of a suitable reference test (17). In a recently completed analysis of 500 randomly selected population-derived subjects, reflux symptoms were reported by 35.2% and use of PPI medication by 36.8% of the study subjects. Biomarker profile 2 (high acid output) was the second most common GastroPanel® profile in this cohort; 31.2%, second only (33.6%) to profile 1 (healthy stomach). Hp- infection was detected in 25.0% of the subjects. Profiles related to use of PPI (low acid output, PPI effect) were found in 7.4% of the cases. Because derived from the population with the highest frequency of dyspepsia, these data have widespread implications in the diagnostic and screening practices at the population level (17).

[0011] Gastrin- 17 is a biomarker of antrum, but also controlled through a negative feedback by the gastric corpus (1,10,11). High acid output down-regulates G-17 resulting in very low serum levels of G-17b, whereas the contrary is true when acid- free stomach upregulates G-17b to reach very high levels (1,10,1 l)(Figure 1). Another potential cause for low G-17b levels is atrophic antrum gastritis (AG A), which is characterized by the failure to respond to protein stimulus (low G- 17s). It is a common knowledge that G-17 regulates the function of the lower esophageal sphincter, low levels being associated with gastroesophageal reflux disease (GERD) that increases the risk of esophagitis, Barrett’s esophagus and esophageal cancer (EC)(18). This was neatly demonstrated in a recent study, where GERD patients (with erosive esophagitis and with Barrett’s esophagus) had statistically significantly lower levels of G-17 as compared with the dyspeptic patients (19). The diagnostic accuracy of G-17 to detect GERD non-invasively was as high as 94.3% (19). From the patients’ point of view, these results are particularly important and further extend the scope of gastric diseases and functional disorders detectable by GastroPanel® test (U0,ll,19).

[0012] GC is a disease with peculiar geographic distribution. In general, the incidence of GC closely parallels the incidence of Hp-infection in the population, being highest among Asian populations. Similar to Hp, also prevalence of AG is subject to wide geographic variation. With the declining trend of Hp in the Western countries, also AG has become less common, particularly among younger generations. According to conservative global estimates, some 500 million people might be affected by AG, whereas significantly more people are infected with Hp. Although not yet systematically implemented in any country, the screening for GC has enormous prospects against this background. As the only non- invasive diagnostic test on the market for this purpose, GastroPanel® is the prime option whenever such a screening program is being designed. Given that some 20-40% of the people do present with dyspeptic symptoms during their lifetime, GastroPanel® also has a practically limitless potential as the only non-invasive diagnostic tool for the first-line clinical assessment of these dyspeptic patients (1,10,11).

[0013] There is thus a need for a novel method and test for carrying out the analysis of the specific biomarker panel (GastroPanel®) in a quicker, easier and more cost-efficient manner, yet maintaining the high accuracy and even decreasing error margins. SUMMARY OF THE INVENTION

[0014] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0015] According to an aspect of the present invention, there is provided a method for screening or examining a symptomless subject or subject having symptoms of an autoimmune disease, wherein the method specified biomarkers are quantitatively measured in one microplate.

[0016] According to a further aspect of the present invention, the method provides population-based, non- invasive screening of subjects for Helicobacter pylori infection, atrophic gastritis and high acid-output and their risk of gastric- and esophageal cancer.

[0017] This and other aspects, together with the advantages thereof over known solutions are achieved by the present invention, as hereinafter described and claimed.

[0018] The method of the present invention is mainly characterized by what is stated in the characterizing part of claim 1. [0019] Considerable advantages are obtained by means of the invention. For example, the present concept enables early diagnosis of Helicobacter pylori infection, atrophic gastritis and high acid output and their risk of gastric- and esophageal cancer. Instead of traditional time-consuming four separate biomarker analyses, the present invention enables quantitative measurement of the relevant biomarkers in one microplate and during the same time as it takes to analyse one distinct GastroPanel® marker. This novel method reduces measurement mistakes and saves both time and costs for the laboratory.

[0020] Next, the present technology will be described more closely with reference to certain embodiments. EMBODIMENTS

[0021] The present technology provides means to screen and examine subjects/patients having a risk for Helicobacter pylori infection, atrophic gastritis and high acid-output and their risk of gastric- and esophageal cancer.

[0022] The objective of the present invention is to carry out quantitative measurement of the relevant biomarkers in one microplate, which enables quicker, less- laborious and more cost-efficient and accurate analysis of the possible disease.

[0023] The Biohit Unified GastroPanel® is an excellent clinical test for detecting gastritis on the mucosa of the stomach. It is intended as the first-line diagnostic test for dyspeptic complaints to detect Helicobacter pylori infection and atrophic gastritis (mucosal atrophy) caused by helicobacter or autoimmune disease, to disclose the associated risks like gastric and esophageal cancer as well as malabsorption of B12 vitamin, calcium, magnesium, zinc, iron and certain medicines.

[0024] Unified GastroPanel® is highly optimized for patient, clinician and laboratory in mind. It is a simple patient-friendly blood test. It gives lot of information for the clinician about the structure and function of the stomach of the patient. Although the test composes of four ELISA tests, the assay can be run as easily as one single ELISA assay. Further, as all the markers provides very linear response, one could even maximize cost per test not losing performance substantially.

[0025] Although the Unified GastroPanel® have been designed automation in mind, the one plate setup makes it very attractive even for small laboratories running ELISA tests by manual means.

[0026] The GP20 concept uses Unified GastroPanel® tests set up as ready-made one plate GastroPanel® test using reduced number of calibrators to provide easy-to-use cost- effective full GastroPanel® analyses on one plate intended for both manual as well as automated use.

[0027] This invention describes for example on how the GP20 has been set up from the Unified GastroPanel® as highly optimized one plate test for the laboratory.

[0028] Unified GastroPanel® is composed of four (4) ELISA tests - Helicobacter pylori IgG (HPG), Gastrin- 17 (G17), Pepsinogen I (PG1) and Pepsinogen II (PG2) - all run at the same time from the same patient sample. All the reagents, except was buffer, are ready to use. Every individual test apply the same timing scheme (60/60/30); 60 minute incubation time for the samples and conjugates, following 30 minutes incubation time for the substrate. All this enables to set up a plate for the test as shown in the Figure 3.

[0029] FIGURE 1 is a chart showing different GastroSoft® biomarker profiles as GastroPanel® interpretation guide snapshot.

[0030] FIGURES 2A and 2B are charts showing GastroPanel® biomarkers in detecting biopsy-confirmed AG and Hp in ROC-analysis. Figure 2A relates to PGI in detecting AGC2+ and Figure 2B relates to lgG Hp EFISA in detecting Hp in corpus.

[0031] FIGURE 3 is a chart describing microplate ready set up with four three strips segment - each segment for different test.

[0032] FIGURE 4 combines 4 charts (. Helicobacter pylori IgG (EIU), Gastrin- 17 (pmol/1), Pepsinogen I (pg/l) and Pepsinogen II (pg/l) biomarkers) showing expected calibration curves when only two calibrators are used to construct the calibration curves.

[0033] According to one embodiment of the present invention, the present method includes screening or examining a symptomless subject or a subject having symptoms indicating an autoimmune disease, wherein the method comprises at least the following steps: quantitatively measuring concentration(s) of a) pepsinogen I (PGI), pepsinogen II (PGII), gastrin- 17 (G-17) and Helicobacter pylori antibody (HpAb) biomarkers, or b) pepsinogen I (PGI) biomarker, or c) PGI biomarker, PGII biomarker and PGI/PGII biomarker ratio, or d) G-17 biomarker, from a blood, serum or plasma sample obtained from said subject, and comparing obtained value(s) to a pre-determined reference range(s) and/or cut-off value(s), characterized in running at least one, such as four, analytical immunoassay(s) comprising said four biomarkers at the same time from the same sample in one microplate and measuring said biomarker concentrations during 3 hours, preferably in about 2,5 hours.

[0034] In a preferred embodiment of the present invention, the values are indicative for atrophic gastritis, if the PGI concentration in said sample is close to the lower limit or below the reference range or cut-off value, PGI/PGII ratio is close to the lower limit or below the reference range or cut-off value and G-17 concentration is close to the upper limit or above the reference range.

[0035] More precisely, according to one embodiment of the present invention the reference range for PGI value is 30 - 160 pg/l, for PGII 3 - 20 pg/l, for PGI/PGII ratio 3 or below 3, for G-17S (stimulated) value 5 - 30 pmol/1, for G-17B (fast) 2 - 10 pmol/1 and the reference range for HpAb 0 - 30 EIU.

[0036] Furthermore, the typical cut-off values for the biomarkers are selected from the group comprising: PGI 30 pg/l, PGI/PGII ratio 3, G-17S value 5 pmol/1, G-17B 2 pmol/1 and HpAb 30 EIU.

[0037] As to atrophic gastritis (AG) of the corpus, the typical PGI cut-off values are: 1. PGI below 30 pg/l for the severe AG, 2. PGI 30-50 pg/l for moderate AG and 3. PGI 50- 70 pg/l for mild AG.

[0038] In one embodiment of the present invention, it is enough to measure only gastrin- 17 (G-17) biomarker concentration from a blood, plasma or serum sample of a subject, and compare the obtained value(s) to the reference range(s) and/or cut-off value(s), in order to determine the presence of i) atrophic gastritis and/or ii) presence of acid free stomach.

[0039] According to one embodiment of the present invention, the method is characterized in that the analytical immunoassay i.e. immunoassay is selected from enzyme- linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA).

[0040] One embodiment of the present invention is an enzyme immunoassay test for screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating Helicobacter pylori infection and/or atrophic gastritis according to a method described herein above, wherein the test comprises purified H. pylori bacterial antigen adsorbed on a microplate and a detection antibody labelled with horseradish peroxidase.

[0041] A test kit comprising reagents for PGI, PGII, G-17 and H. pylori antibody biomarkers, and instructions for use also belongs to the scope of the present invention. The test kit is designed to be used in the method described herein above.

[0042] Typical and known GastroPanel® measurement is carried out in four different microplates, each measurement taking 2,5 hours, whereby the results are available after 10 hours. The test kit currently in use also consists of four packages, one for each biomarker reagent.

[0043] GastroPanel® test reveals Hp-infection and AG as well as high acid-output with their associated risks, including an increased risk of gastric (GC) and oesophageal cancer (EC). Particularly high risk of GC is associated with an increased exposure to carcinogenic acetaldehyde accumulated in acid-free stomach that may develop as a result of: 1) atrophic corpus gastritis, 2) long-term PPI medication, 3) stomach surgery.

[0044] Increased acetaldehyde exposure is associated with 1) gene mutation affecting acetaldehyde metabolism, 2) chronic Hp-infection producing acetaldehyde, 3) alcohol intake, 4) cigarette smoking, 5) acetaldehyde in foodstuffs/beverages.

[0045] Acetium® capsule (slow-release L-cysteine) that binds carcinogenic acetaldehyde into a harmless MTCA compound in the stomach, may reduce the risk of these cancers.

[0046] The most important causes of acid- free stomach are 1) atrophic gastritis (AG) in the corpus (Hp-induced or autoimmune type), and 2) chronic use of PPI, both being diagnosed by GastroPanel®. [0047] For AG, there is i) no medication, or ii) no vaccination available for the affected

500 million (conservative estimate) people worldwide.

[0048] Increased risk of GC (>1 million annual new cases worldwide) associated with AG is conveyed by exposure to carcinogenic acetaldehyde (IARC Class I) accumulating in acid-free stomach. [0049] Another Biohit innovation, Acetium® capsule (slow-release L-cysteine), effectively eliminates acetaldehyde in acid-free stomach by covalent binding to form a harmless MTCA compound. By protecting the stomach mucosa, Acetium® capsule is a potential preventive tool against the carcinogenic effects of acetaldehyde. [0050] Gastric (GC) and esophageal cancer (EC) are causes of significant global cancer burden; over one million and over 600.000 new cases and 768.000 and 544.000 annual cancer deaths, respectively, worldwide in 2020.

[0051] It is currently possible to diagnose the key risk factors of GC and EC: Helicobacter pylori, atrophic gastritis (AG) and GERD, by a non- invasive blood test based on a panel of serological biomarkers (GastroPanel®, Biohit Oyj).

[0052] With proven superior clinical performance confirmed in a wide variety of clinical settings and verified in recent meta-analyses, this innovative test detects with high accuracy the risk conditions of both GC and EC.

[0053] Unique to any test, also the disturbances in gastric acid output are disclosed, predisposing the patients e.g. to peptic ulcer, reflux disease, esophagitis and EC.

[0054] Apart from its use as a diagnostic test for symptomatic (dyspeptic) patients, GastroPanel® is equally well suited for population-based, non- invasive screening of asymptomatic subjects for their risk of GC and EC.

[0055] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed.

[0056] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below. [0057] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", that is, a singular form, throughout this document does not exclude a plurality.

EXAMPLES

The GastroPanel® test

The GastroPanel® is a user-friendly ELISA technique based on four biomarkers specific for the stomach mucosa: 1) Pepsinogen I (P-PGI), 2) Pepsinogen II (P-PGII), 3) Gastrin- 17 (P- G-17) and 4) H. pylori antibody (P-HpAb).

ELISA test for Pepsinogen I and Pepsinogen II

P-PGI is secreted solely by the chief cells (chief cell/mucous neck cells) of the corpus mucosa. AG of the corpus leads to loss of these cells and, as a result, the P-PGI level in circulation decreases. P-PGII is produced by the chief cells and mucous neck cells of the gastric mucosa, by pyloric glands in the gastric antrum and by Brunner’s glands in the proximal duodenum. The ratio of PGI to PGII concentration in the plasma of normal subjects is above 3.0.

ELISA test for Gastrin- 17

The P-G-17 ELISA method in the GastroPanel® is specific to amidated G-17 molecule. G- 17 peptide is the most important member of the gastrin/cholecystokinin- family which regulates the physiology of the upper gastrointestinal tract. This peptide is the biologically most active gastrin peptide, stimulating gastric acid secretion with 6-times higher potency than the biologically next most active gastrin, G-34. G-17 is secreted exclusively by the gastrin-cells (G-cells) in the antrum, representing a fraction of the total gastrin concentration in the circulation. The maximal secretion is achieved after physiological protein stimulation, or when the acid secretion in the stomach decreases, is low or absent. As a result of antral atrophy, the amount of G-cells decreases and, consequently, both the basal and post-prandial secretion of G-17 will decrease. ELISA test for Helicobacter pylori (HpAb ELISA)

The H. pylori infection is the most important cause of chronic gastritis. Another well-known cause for severe AG is the autoimmune mechanism, which can also be activated by H. pylori infection. GastroPanel® test for H. pylori is performed from the plasma samples. The test is based on an ELISA technique, with purified H. pylori bacterial antigen, adsorbed on a microplate, and a detection antibody labeled with horseradish peroxidase (HRP).

Sample collection for GastroPanel^® test

The person taking the blood sample shall fill the test request (remittance) form. A minimum of 2 ml EDTA plasma from a fasting blood sample is taken into an EDTA tube (e.g. Biohit Cat. no. 454235 Vacuette 4ml tube containing K2EDTA). Use of G-17 stabilizer 100id/2ml plasma (Biohit Cat. No. 601 050 or 601 051) allows a temporary storage of the sample at room temperature (20-25°C), before frozen.

Sample processing

The blood sample needs to be centrifuged within 30 minutes, at 1800-2000g for 10 minutes or as prescribed by tube manufacturer. Because not used for on-site testing, the EDTA plasma needs to be frozen instantly (-70°C). Using G-17 stabilizer enables a temporary storage in the refrigerator (at 2-8°C), for up to 3 days, but immediate freezing at -70°C is the preferred method of storage. This is most critical for G-17, to avoid decay at too high temperature. Once the sampling has been completed, the frozen plasma samples were delivered to the laboratory for analysis by GastroPanel® test. All samples were analyzed, following the instructions for use (IFU) of the test kits.

Stimulated G-17

Apart from the fasting sample, the study protocol also necessitates another blood sample from all subjects, following protein stimulation to analyze the level of stimulated G-17. The secretion of G-17 can be stimulated by the intake of a protein drink having average protein content of 77% [Biohit Cat. No. 601038 (50x20 g). This stimulation was not performed for patients who were sensitive to lactose (i.e., lactose intolerance or hypolactasia). To prepare the protein juice, 20g of protein (one foil bag of protein powder) is mixed to 150 ml of water. The stimulated blood sample must be taken 20 minutes after the intake of the protein juice. Evaluation of GastroPanel® results

The results of the GastroPanel® test were evaluated using the GastroSoft® interpretation software. GastroPanel® test is optimized for the Updated Sydney System (USS) for classification of gastritis, both including 5 diagnostic categories (see above for study endpoints). Of primary interest in this study was the presence/absence of atrophic gastritis (AG) in the antrum, corpus or both locations [48] The second point of interest is the presence of acid-free stomach due to non-AG-causes, i.e., chronic use of PPI-medication, which was also diagnosed by GastroPanel test [48] See also www. gastropanel.com.

Gastroscopy and biopsy procedures In a regular study setting of this type, all patients who test positive in GastroPanel, i.e., the result is suggesting AG, should be subjected to gastroscopy, providing the histological confirmation to be NORMALLY used as the gold standard examination to confirm the GastroPanel results.

However, under these exceptional circumstances of ongoing pandemic Coronavirus outbreak, it is not feasible to request gastroscopic confirmation of GastroPanel test results in these virus-debilitated patients. Accordingly, the GastroPanel confirmed AG can be considered as a surrogate of biopsy-confirmed AG and accepted as the dichotomous study endpoint (AG present/ AG absent). The same is true for the GastroPanel diagnosis of acid- free stomach in the absence of AG, which is also the normal practice, because gastroscopy biopsy is normal in these subjects with low acid output due to chronic PPI medication. The use of GastroPanel detected AG as a surrogate of biopsy-confirmed AG, because of the established extremely close correlation between GastroPanel and biopsy histology.

Biopsy protocols The optimal use of the USS system necessitates that the biopsy protocol follows an agreed systematic. As explained above, in this particular study setting, gastroscopy and biopsies are not needed, because it is not ethically acceptable to predispose these severely ill patients to this invasive diagnostic examination.

GP20 set up on one microplate

GP20 microplate has been ready set up as follows:

1. Strip location 1 to 3 is equipped with strips for HPG 2. Strip location 4 to 6 is equipped with strips for G17

3. Strip location 7 to 9 is equipped with strips for PG1

4. Strip location 10 to 12 is equipped with strips for PG2

Running the GP20: Before starting the assay, allow all the reagents to reach ambient temperature. Remember to mix all the reagents and samples well just before pipetting.

1. Dilute the wash buffer as 1:10.

2. After mixing, pipette 100 pL of the diluent buffer (blank), the calibrators (low and high), the control, and diluted patient samples into the wells, according to Figure 3.

The calibrators supplied with the kit contain following target concentrations

HP low 20 EIU HP high 440 EIU

G17 low 1 pmol/1 G17 high 30 mol/1

PG1 low 25 pg/l PGI high 200 pg/l

PG2 low 6,3 pg/l PGII high 50 pg/l 3. Incubate for 60 min at ambient temperature with shaking (750 rpm) 4. Wash the strips with 3 x 350 pL of the diluted washing buffer

5. Pipette 100 pL of the mixed test specific Conjugate solution into the wells; H. pylori conjugate to the strips 1-3, Gastrin- 17 conjugate to the strips 4-6, Pepsinogen I conjugate to the strips 7-9 and Pepsinogen II conjugate to the strips 10-12. 6. Incubate for 60 min at ambient temperature with shaking (750 rpm)

7. Wash the wells three times with 350 pi of the diluted washing buffer

8. Pipette 100 pL of the mixed Substrate solution into the wells

9. Incubate for 30 min at ambient temperature

10. Pipette 100 pL of the mixed Stop solution into the wells 11. Read at 450 nm within 30 minutes

12. Calculate the results as instructed by the corresponding IFUs; use linear fit for the Gastrin- 17, Pepsinogen I and Pepsinogen II and logarithmic for the H. pylori.

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Claims

CLAIMS:

1. A method of screening or examining a symptomless subject or a subject having symptoms indicating an autoimmune disease, dyspepsia or reflux symptoms, wherein the method comprises at least the following steps: quantitatively measuring concentration(s) of a) pepsinogen I (PGI), pepsinogen II (PGII), gastrin- 17 (G-17) and Helicobacter pylori antibody (HpAb) biomarkers, or b) pepsinogen I (PGI) biomarker, or c) PGI biomarker, PGII biomarker and PGI/PGII biomarker ratio, or d) G-17 biomarker, from a blood, serum or plasma sample obtained from said subject, and comparing obtained value(s) to a pre-determined reference range(s) and/or cut-off value(s) characterized in running at least one, such as four, analytical immunoassay(s) comprising said four biomarkers at the same time from the same sample in one microplate and measuring said biomarker concentrations during 3 hours.

2. The method according to claim 1, characterized in that the value(s) is/are indicative for atrophic gastritis, if the PGI concentration in said sample is close to the lower limit or below the reference range or cut-off value, PGI/PGII ratio is close to the lower limit or below the reference range or cut-off value and G-17 concentration is close to the upper limit or above the reference range.

3. The method according to claim 1 or 2, characterized in that the reference range for PGI value is 30 - 160 pg/l, for PGII 3 - 20 pg/l, for PGI/PGII ratio 3 or below 3, for G-17S (stimulated) value 5 - 30 pmol/1, for G-17B (fast) 2 - 10 pmol/1 and the reference range for HpAb 0 - 30 EIU.

4. The method according to any of the preceding claims, characterized in that the typical cut-off values for the bio markers are selected from the group comprising: PGI 30 pg/l, PGI/PGII ratio 3, G-17S value 5 pmol/1, G-17B 2 pmol/1 and HpAb 30 EIU.

5. The method according to any of the preceding claims, characterized in that the analytical immunoassay is selected from enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA).

6. An enzyme immunoassay test for screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating Helicobacter pylori infection and/or atrophic gastritis according to a method of any of claims 1 to 4, wherein the test comprises purified H. pylori bacterial antigen adsorbed on a microplate and a detection antibody labelled with horseradish peroxidase.

7. A test kit comprising reagents for PGI, PGII, G-17 and H. pylori antibody biomarkers, and instructions for use.

8. The test kit according to claim 7 for use in a method according to any of claims 1 to 5.

9. Use of PGI, PGII, Gastrin- 17 and Helicobacter pylori antibodies as biomarkers for revealing high gastric acid output and a consequent risk of peptic ulcer disease.