WO2003093818A2

WO2003093818A2 - Method for pre-treating stool samples

Info

Publication number: WO2003093818A2
Application number: PCT/EP2003/004571
Authority: WO
Inventors: George Wengler
Original assignee: George Wengler
Priority date: 2002-05-02
Filing date: 2003-04-30
Publication date: 2003-11-13
Also published as: DE10219741A1; WO2003093818A3

Abstract

The invention relates to a method for pre-treating stool samples, whereby Helicobacter pylori antigens are separated from bonds comprising endogenous antibodies. Said pre-treatment is carried out before the assay of Helicobacter pylori antigens in the stool, using immunological techniques.

Description

Process for the pretreatment of stool samples

description

The invention relates to a method for the treatment of stool samples according to claim 1.

State of the art

H. pylori is a gram-negative bacterium with occurrence in the gastrointestinal area, which is mainly responsible for chronic gastritis and peptic ulcer. Infection, among other things, an important risk factor in the development of gastric cancer.

Infection can be diagnosed using a biopsy taken during gastroscopy. Infection can also be diagnosed using non-invasive methods that save the patient the inconvenience of endoscopic examination. The detection of specific antibodies in the serum, the breath test and the detection of faecal antigens are less common. Compared to each other, each of these methods has positive and negative aspects. Serology is economical, but it has the fundamental problem that antibodies can still be present after healing and therefore their detection can be the cause of false positive results. The breath test shows increased sensitivity and specificity, but is time-consuming for the patient and also requires expensive equipment.

The detection of antigens, bacteria or bacterial DNA fragments of H. pylori in stool appears to be particularly interesting because it does not stress the patient and, in contrast to the detection of antibodies, is a direct expression of a acute infection. There are different techniques for the detection of an H. pylori infection known from stool samples. Cultivation studies did not yield any promising results. PCR is an efficient method for the detection of bacterial DNA in biological samples, but its application to stool samples is hindered by the substances that inhibit the reaction. Numerous attempts have been made to remove the inhibitors. The results are contradictory.

It is an object of the present invention to provide a method for the pretreatment of stool samples with which it is possible to cleave endogenous antibodies from H. pylori facal antigens and thus to improve the sensitivity and specificity in immunological assays for the diagnosis of H. pylori infections ,

This problem is solved by the features of claim 1.

Accordingly, a pretreatment method of stool samples prior to an immunological test on the stool is created in order to detect or isolate H.pylori antigens, the pretreatment reducing or eliminating interactions between endogenous antibodies and H.pylori antigens when the test is carried out.

In claim 16, a kit for pretreating stool samples is also defined as an independently tradable object.

In claim 17, a kit for the immunological detection of H. pylori antigens in stool samples is also defined as an independently tradable object. Detailed description

The present invention is concerned with pretreatment of stool samples to remove or eliminate interactions of endogenous antibodies in a subsequent immunological test, and isolation of antigens from H. pylori from the stool.

The pretreatment is based on the use of antigen-antibody binding methods well known to those of skill in the affinity chromatography field who are concerned with breaking bonds between H. pylori antigens and endogenous antibodies in stool samples. The pretreated sample is intended to be tested for H. pylori antigens in a subsequent immunoassay or to isolate antigens from Helicobacter pylori. The release of antigens from endogenous antibodies, obtainable as a result of the pretreatments according to the invention, facilitates the recognition of the fecal antigens by antibodies on which the immunological method is based and thereby improves the efficiency of the said system.

Some preferred pretreatments according to the invention are described below.

1. Pretreatment of stool samples, comprising the following steps: a) bringing the stool sample into contact with a liquid with chemical properties, suitable for any. To completely or partially dissociate antigen-antibody immune complexes.

2. Pretreatment of stool samples, comprising the following steps: a) bringing the stool sample into contact with a liquid with chemical properties, suitable for any. To completely or partially dissociate antigen-antibody immune complexes; b) complete or partial separation of H. pylori antigens from endogenous antibodies.

3. Pretreatment of stool samples, comprising the following steps: a) bringing the stool sample into contact with a liquid with chemical properties, suitable for completely or partially dissociating antigen-antibody-Im uncomplex; b) Addition to the obtained facal suspension of a reagent which is able to completely or partially inhibit the reunification of H. pylori antigens with specific or cross-reactive endogenous antibodies.

4. Pretreatment of stool samples, comprising the following steps: a) contacting the stool sample with a liquid; b) Treatment of the suspension obtained according to 4a) with physical methods which are suitable for completely or partially dissociating immune complexes formed from H.pylori antigen and specific or cross-reactive endogenous antibodies of the organism from which the sample originates.

5. Pretreatment of stool samples, comprising the following steps: a) contacting the stool sample with a liquid; b) treatment of the suspension obtained according to 5a) with physical methods which are suitable for completely or partially dissociating immune complexes formed from H. pylori antigen and specific or cross-reactive endogenous antibodies of the organism from which the sample originates; c) complete or partial cleavage of Helicobacter pylori antigens from endogenous antibodies.

6. Pretreatment of stool samples, comprising the following steps: a) contacting the stool sample with a liquid; b) treatment of the suspension obtained according to 6a) with physical methods which are suitable for completely or partially dissociating immune complexes formed from H. pylori antigen and specific or cross-reactive endogenous antibodies of the organism from which the sample originates; c) Adding to the obtained facal suspension of a reagent which is able to completely or partially inhibit the reunification of Helicobacter pylori antigens with specific or cross-reactive endogenous antibodies.

7. Pretreatment of stool samples, consisting of a combination of two or more of the previous pretreatments.

Any treatment of stool samples which causes a breakdown of H. pylori antigens and endogenous anti-H. pylori antibodies or with H. pylori cross-reactive antibodies is part of the invention. As already mentioned, the methods for breaking antigen-antibody bonds can be chemical or be physical. A common method among chemicals is to expose the sample to a low or a high pH. According to a preferred embodiment, the stool sample is brought into contact with an acidic solution in order to obtain a fecal suspension with a pH below 4. One such, non-limiting example is a buffer solution with 100 mM glycine pH 2.5, as communicated with Examples 1 and 2.

Dissociation of fecal immune complexes using chemical methods can also be achieved by contacting the stool samples with a solution with increased ionic strength. A more commonly used physical method to break down immune complexes is exposure to heat. A typical example according to the invention is the preparation of a facal suspension in a solution and the subsequent heating to above 40 ° C. Another chemical method to break antigen-antibody bonds is to pretreat the stool sample with chaotropic ions, which destroy the structure of water molecules and reduce hydrophobic interactions (e.g. sodium hydride, potassium tiocyanate, magnesium chloride, trifluoroacetic acid). There are also substances that, when applied in high concentrations, are able to break down antigen-antibody bonds. An example is urea and guanidine HCL, which are able to denature proteins. Substances that are able to reduce the charge of the solution are also able to dissociate the immune complexes. Such as. Dioxin and ethylene glycol. In some cases, the use of high salt concentrations is able to break or at least weaken antigen-antibody bonds.

A second object of the present invention is the use of methods to separate the antigens from the endogenous antibodies after the dissociation of fecal immune complexes. The separation phase ensures that re-bind the endogenous antibodies to the antigens before or during which the immunological tests are carried out or the facal antigens are isolated. Another purpose is to avoid interactions between endogenous antibodies, whether in a competitive or non-competitive immunological reaction.

The separation of fakalantigens from endogenous antibodies after the splitting of the immune complexes can be carried out according to the present invention in three different ways.

In a preferred embodiment, the suspension of the stool sample, which has been pretreated according to one of the methods described above for the splitting of antigen-antibody complexes, is subjected to centrifugation. The supernatant, which contains free endogenous antibodies, is separated from the sediment. The fraction with the deposited antigens, separated from endogenous antibodies, is brought into contact with a liquid and then subjected to immunological examination methods. Alternatively, focal H. pylori antigens are isolated.

In a second preferred embodiment, the separation of free antigen from endogenous antibodies after pretreatment is achieved by means of one of the methods described above for the dissociation of antigen-antibody complexes by means of filtration, using filters which retain the antigen fraction, while at the same time they let pass parts contained endogenous antibody. The filter-treated fraction containing antigen is brought into contact with a liquid and then subjected to an immunological test method. Alternatively, focal H. pylori antigens are isolated. In a third preferred embodiment, free antigens are separated from endogenous antibodies after pretreatment using one of the methods described above for dissociating antigen-antibody complexes by means of technical chromatography. The antigen fraction purified by means of technical chromatography is brought into contact with a liquid and then an immunological test method. Alternatively, focal H. pylori antigens are isolated.

That the phase of separation of the anti-H. pylori antibody from the antigens after the step of dissociating the fecal immune complexes is an important aspect of the invention, although it is not a mandatory sequence. In fact, the present invention also relates to a pretreatment which only comprises a separation phase of the immune complexes, as already illustrated in the first and fourth methods described above. According to the present invention, the pretreatment methods for stool samples can be applied to all immunological tests which are concerned with the examination of H. pylori antigens in stool. These immunological test methods are well known to the person skilled in the art. They can be of a competitive or non-competitive nature and they usually employ polyclonal and / or monoclonal antibodies or fragments thereof that are specific for certain H. pylori antigens.

In a preferred embodiment, the present invention is applicable to immunological diagnostic assays of H. pylori infection as described in the patents cited above.

Patent WO 9824885 claims immunological methods which detect H.pylori infection by means of tests for an antigen with an apparent weight of 16 +/- Determine 2kDa. According to a further preferred embodiment, the present invention is applicable to the dissociation of fecal immune complexes, which consist of antigens as described in patent WO 9824885 and endogenous antibodies which are present in the sample. A typical application of the invention, which is in no way intended to be limiting, relates to the examination of the antigen which is described in patent WO 9824885 and in example 1. The pretreatment methods of stool samples according to the invention are also applicable to methods which use methods which are well known to the person skilled in the art, to use antigen-antibody reactions for the separation of fragments and / or H. pylori bacterial cells, from which bacterial DNA is subsequently extracted in order to carry out an analysis of the specific nucleotide sequence of H. pylori. The pretreatment methods of stool samples according to the invention are also applicable to methods which use antigen-antibody reactions for the separation of H. pylori from the stool for the isolation of bacterial cultures.

The following examples illustrate the invention. Such examples are by no means to be interpreted in a manner that limits the general invention or the following claims.

EXAMPLE 1:

Pretreatment of stool samples using chemical methods. Use of a non-competitive immunoassay based on monoclonal antibodies.

Patients: The stool samples used were from 20 patients who had undergone gastroscopy. Based on a histological examination and a urea breath test, 15 of them were infected with H. pylori, another 5 were not infected. Pretreatment of the stool samples: The stool samples of each patient were pretreated according to the following protocol: 100-200 mg of the stool sample were transferred to a conical test tube, model Eppendorf 1.5 ml. 1 ml of glycine buffer 100 mM pH 2.5, which contains 0.5 mol / 1 sodium chloride (dissociation buffer), was added. After the stool sample had dissolved under vortexing, the sample was centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. 0.9 ml of the supernatant was transferred to a new Eppendorf 1.5 ml tube and centrifuged for a further 20 minutes at 14000 rpm. After the supernatant had been removed, the pellet was taken up in 0.3 ml of a buffer containing 0.2 M Tris pH 8.5 and 3 mol / 1 potassium chloride. This suspension is used for the subsequent immuno-enzymatic assays.

Non-pretreated stool samples: 100-200 mg of the stool sample are transferred to a conical test tube, model Eppendorf, 1.5 ml. 1 ml of phosphate buffered saline (PBS) is added to the sample. After the sample has been vortexed, the test tube is centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. The supernatant is used for the subsequent immunological enzymatic assays.

Immunological enzymatic assay: According to the instructions from patent WO 9824885 with the title "Helicobacter pylori antigen having an apparent molecular weight of 16 + / kDa, a specific antibody, and its use for the detection of said antigen", a monoclonal antibody of Class IgG (AM524A) directed against the H. pylori antigen with the apparent weight of 16 kDa. This was then used in a non-competitive immuno-enzymatic assay of the sandwich ELISA type as described below: The microwells of the ELISA plate (NUNC maxisorp) were mixed with 100 μl of the monoclonal antibody AM524A in a con concentration of 10 μg / ml in sodium carbonate buffer 50 mM pH 9.5 incubated overnight at room temperature. After the liquid was removed by aspiration, the wells were saturated with 200 μl PBS containing 0.5% bovine serum albumin (BSA) for 16 hours at room temperature. The wells are then washed twice with PBS. Then 100 μl of the wells are pretreated in the double batch of the fakal suspension and not pretreated, as described above. After a one-hour incubation period at room temperature in a microplate shaker, the wells are washed 5 times with PBS. 100 ul of a biotinylated monoclonal. Antibodies in the concentration of 1 μg / ml in PBS containing 1% BSA are distributed to the wells and incubated for 30 minutes at room temperature under rubble. Then 100 μl streptavidin, bound to peroxidase (Sigma), is added to the same wells in a concentration of 2 μg / ml in PBS containing 1% BSA. After an incubation time of 15 minutes under rubble, the wells are washed 5 times with 250 μl PBS. 100 ul of a solution containing 3, 3 ', 5, 5' tetramethylbenzidine (TMB) and H ₂ 0 ₂ are added to all wells and incubated for a further 10 minutes. The color reaction is blocked after 10 minutes with 50 μl of a 2N HCL. Optical density is determined with a 450 nm microplate spectrometer using a 620 nm filter.

Result: The optical density according to the ELISA test of the stool samples to be examined with and without pretreatment according to the present invention is shown in the following table: Table example 1

As can be seen from the table, the pretreated samples often show a higher optical density compared to non-pretreated samples. As a result, the diagnostic sensitivity and specificity of the immuno-enzymatic test increases.

EXAMPLE 2:

Pretreatment of stool samples using chemical methods. Use of a non-competitive immuno-enzymatic assay based on polyclonal antibodies.

Patients: The stool samples used were from 20 patients who had undergone gastroscopy. Based on a histological examination and a urea breath test, 15 of them were infected with H. pylori, another 5 were not infected.

Pretreatment of stool samples: Each patient's stool samples were pretreated according to the following protocol: 100-200 mg of the stool sample was transferred to a conical test tube, model Eppendorf 1.5 ml. 1 ml of glycine buffer 100 mM pH 2.5, which contains 0.5 mol / 1 sodium chloride (dissociation buffer), was added. After the stool sample had dissolved under vortexing, the sample was centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. 0.9 ml of the supernatant was transferred to a new Eppendorf 1.5 ml tube and centrifuged for a further 20 minutes at 14000 rpm. After the supernatant was removed, the pellet was taken up in 1 ml of a buffer containing 200 mM Tris pH 7.5. This suspension is used for the subsequent immuno-enzymatic assays.

Non-pretreated stool samples: 100-200 mg of the stool sample are transferred to a conical test tube, model Eppendorf, 1.5 ml. 1 ml of a 200 mM Tris buffer pH 7.5 containing 0.9% KC1 is added to the sample. After the sample has been vortexed, the test tube is centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. The supernatant is used for the subsequent immunological enzymatic assays.

Immunological enzymatic assay: The diagnostic procedure used is based on a sandwich assay, with immunoglobulin of a rabbit vaccinated with H. pylori. The procedure is as follows: The microwells of the ELISA plate (NUNC maxisorp) were filled with 100 μl of the anti-H. pylori. Rabbits in unglobulin at a concentration of 10 µg / ml in sodium carbonate buffer 50 mM pH 9.5 incubated overnight at room temperature. After the liquid had been removed by aspiration, the wells were saturated with 200 μl PBS containing 0.5% bovine serum albumin (BSA) for 16 hours at room temperature. The wells are then washed twice with PBS. Then the wells are pretreated and 100 μl each in the double batch of the fakal suspension not pretreated as described above. Immediately afterwards, 100 μl of a biotinylated, polyclonal, peroxidase-coupled rabbit immunoglobulin in a concentration of 2 μg / ml in PBS containing 1% BSA is added to all micro-wells and incubated for 60 minutes at room temperature under rubble. The wells are then washed 5 times with 250 μl PBS.

100 μl of a color solution containing TMB and H ₂ 0 ₂ are added to all wells and incubated for a further 10 minutes. The color reaction is blocked after 10 minutes with 50 μl of a 2N HCL. Optical density is determined with a 450 nm microplate spectrometer using a 620 nm filter.

Result: The optical density according to the ELISA test of the stool samples to be examined with and without pretreatment according to the present invention is shown in the following table:

Table example 2

EXAMPLE 3

Pretreatment of stool samples with chemical and immunological methods for a subsequent immuno-enzymatic assay on H. pylori facal antigens.

Pretreatment of the stool samples: 100-200 mg of the stool samples are transferred to a conical test tube, model Eppendorf, 1.5 ml. 1 ml of a Gycin buffer 100 mM pH 2.5 containing 0.5 mol / 1 sodium chloride (dissociation buffer) is added to the sample. After the stool sample has been dissolved under vortexing, the test tube is centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. 0.5 ml of the supernatant is transferred to a new test tube, model Eppendorf, 1.5 ml. 0.5 ml of a buffer solution containing 2.0 M Tris pH 8.5 and 3 mol / 1 potassium chloride and 20 mg / ml rabbit antiserum directed against human immunogloboline which had been preincubated with mouse immunoglobolin are added to the test tube. The test tube is vortexed for 4-5 seconds and incubated for 5 minutes at room temperature before this is carried out in the non-enzymatic assay.

Immuno-enzymatic assay: The immuno-enzymatic assay used corresponds to that described in Example 1, which uses monoclonal antibodies, or that according to Example 2, which uses polyclonal antibodies. AT S PI EL 4:

Pretreatment of the stool sample using physical methods for a subsequent immuno-enzymatic assay for focal H. pylori antigens

Pretreatment: 100-200 mg of the stool sample are transferred to a conical test tube, model Eppendorf, 1.5 ml. 1 ml 0.1 M Tris buffer pH 8.5 containing 0.5 mol / 1 sodium chloride is added to the sample. After the stool sample has been dissolved under vortexing, the test tube is centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. 0.5 ml of the supernatant is transferred to a new test tube, model Eppendorf, 1.5 ml. The test tube containing the sample is then heated to 60 ° C. for 5 minutes and then immediately centrifuged at 14,000 rpm for 20 minutes. After the supernatant has been removed by aspiration, the pellet is resuspended with the addition of 0.3 ml of a 0.2 M Tris buffer pH 8.5 containing 3 mol / 1 potassium chloride.

Immuno-enzymatic assay: The immuno-enzymatic assay used corresponds to that described in Example 1, which uses monoclonal antibodies, or that according to Example 2, which uses polyclonal antibodies.

EXAMPLE 5

Pretreatment of stool samples for the immunological extraction of Helicobacter pylori bacteria or fragments thereof

Pretreatment: 100-200 mg of the stool sample are transferred to a conical test tube, model Eppendorf, 1.5 ml. 1 ml 0.1 glycine buffer 100 mM, pH 2.5 contains 0.5 mol / 1 sodium chloride (dissociation buffer), is added to the sample. After the stool sample has been dissolved under vortexing, the test tube is centrifuged for 5 minutes at 3000 rpm in an Eppendorf microcentrifuge. 0.5 ml of the supernatant will be transferred to a new test tube, model Eppendorf, 1.5 ml. After 0.5 ml of the dissociation buffer has been added, the test tube is vortexed for 4-5 seconds and centrifuged at 14000 rpm for 20 minutes. After the supernatant has been removed by aspiration, the pellet is resuspended with 0.3 ml of a 0.2 M Tris buffer pH 8.5, containing 3 mol / 1 potassium chloride.

Immunological extraction of Helicobacter pylori bacteria or fragments:

20 μl of a suspension of magnetic microbeads (oxoid) covered with rabbit immunoglobolin, directed against H. pylori, is added to the pretreated facal suspension. The mixture is incubated for 1 hour in a shaker at room temperature. Using a magnetic aid, the microparticles are washed with 3 ml of a 20 mM sodium phosphate buffer pH 7.3, containing 150 mM sodium chloride.

Claims

Expectations

1. A method for pretreating stool samples in order to cleave H.pylori antigens from bonds with endogenous antibodies, said pretreatment being carried out using immunological techniques before testing for H.pylori antigens in the stool.

2. A method for pretreating stool samples according to claim

1, wherein the dissociation of the preferably endogenous antigen-antibody bonds is obtainable by contacting the stool sample with a chemical solution.

3. A method for pretreating stool samples according to claim

2, the chemical substances being selected from acids, salts, bases, detergents, organic solvents or multiple alcohols.

4. A method for pretreating a stool sample according to claim 1, wherein the sample is suspended in a liquid and wherein said suspension is heated to a temperature above 40 ° C.

5. A method for pretreating stool samples according to claim 4, wherein the suspension of the stool sample is heated by means of microwaves.

6. A method for pretreating the stool sample according to any one of the preceding claims, comprising one or more methods that prevent the re-formation of immune complexes between H. pylori antigens and endogenous antibodies.

7. A method for the pretreatment of stool samples according to any one of the preceding claims, wherein the complexation of H.pylori antigens with endogenous antibodies by adding gene of animal immunoglobolin, directed against the immunoglobolins of the host from which the stool sample comes, are inhibited.

8. A method for pretreating the stool sample according to any one of the preceding claims, which comprises, in addition to the process of dissociation, a process of separating H. pylori antigens from endogenous antibodies.

9. The method for pretreating stool samples according to claim 8, wherein the separation of H. pylori antigens from endogenous antibodies can be achieved by filtration.

10. A method for the pretreatment of stool samples according to any one of the preceding claims, wherein the separation of H.pylori antigens from endogenous antibodies can be achieved by centrifugation.

11. A method for pretreating stool samples according to any one of the preceding claims, wherein the pretreatment is preceded by an examination for H. pylori antigens using competitive or non-competitive immunodiagnostic methods.

12. A method for pretreating stool samples according to any one of claims 1 to 10, wherein the pretreatment is preceded by an extraction of bacterial DNA.

13. A method for pretreating stool samples according to any one of claims 1 to 10, wherein the treatment is preceded by isolation of H.pylori cultures from the stool.

14. A method for pretreating stool samples according to claim 11, wherein the H. pylori antigen sought is recognized in the stool by antibodies which are directed directly against the H. pylori antigen with the size of 16 +2 kDa, the Size was determined in an acrylamide electrophoresis under denaturing conditions.

15. A method for pretreating stool samples according to claim 1, comprising the following steps: a) bringing the stool sample into contact with a liquid with a pH value of less than 4; b) centrifuging the suspension; c) separation of supernatant and precipitation; d) resumption of precipitation in a liquid with a pH of> 4.

16. Kit for the pretreatment of stool samples, characterized by at least one of the features of claims 1 to 15.

17. Kit for the immunological detection of H. pylori antigens in stool samples, which has a pretreatment part according to claim 16 and a detection portion, which preferably uses a monoclonal antibody.