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WO2003093818A2 - Procede de pretraitement d'echantillons de selles - Google Patents

Procede de pretraitement d'echantillons de selles Download PDF

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Publication number
WO2003093818A2
WO2003093818A2 PCT/EP2003/004571 EP0304571W WO03093818A2 WO 2003093818 A2 WO2003093818 A2 WO 2003093818A2 EP 0304571 W EP0304571 W EP 0304571W WO 03093818 A2 WO03093818 A2 WO 03093818A2
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WO
WIPO (PCT)
Prior art keywords
stool
pretreating
stool samples
pylori
pretreatment
Prior art date
Application number
PCT/EP2003/004571
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German (de)
English (en)
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WO2003093818A3 (fr
Inventor
George Wengler
Original Assignee
George Wengler
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by George Wengler filed Critical George Wengler
Publication of WO2003093818A2 publication Critical patent/WO2003093818A2/fr
Publication of WO2003093818A3 publication Critical patent/WO2003093818A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Definitions

  • the invention relates to a method for the treatment of stool samples according to claim 1.
  • 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.
  • 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.
  • a kit for pretreating stool samples is also defined as an independently tradable object.
  • kits for the immunological detection of H. pylori antigens in stool samples is also defined as an independently tradable object.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the stool sample is brought into contact with an acidic solution in order to obtain a fecal suspension with a pH below 4.
  • an acidic solution 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).
  • chaotropic ions which destroy the structure of water molecules and reduce hydrophobic interactions (e.g. sodium hydride, potassium tiocyanate, magnesium chloride, trifluoroacetic acid).
  • 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.
  • 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.
  • 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.
  • focal H. pylori antigens are isolated.
  • 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.
  • focal H. pylori antigens are isolated.
  • 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.
  • focal H. pylori antigens are isolated.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Pretreatment of stool samples using chemical methods Use of a non-competitive immunoassay based on monoclonal antibodies.
  • 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.
  • 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.
  • PBS phosphate buffered saline
  • 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.
  • A524A monoclonal antibody of Class IgG directed against the H. pylori antigen with the apparent weight of 16 kDa.
  • the microwells of the ELISA plate (NUNC maxisorp) were mixed with 100 ⁇ l of the monoclonal antibody AM524A in a con concentration of 10 ⁇ g / 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 ⁇ l PBS containing 0.5% bovine serum albumin (BSA) for 16 hours at room temperature. The wells are then washed twice with PBS. Then 100 ⁇ l of the wells are pretreated in the double batch of the fakal suspension and not pretreated, as described above.
  • BSA bovine serum albumin
  • TMB 3, 3 ', 5, 5' tetramethylbenzidine
  • H 2 0 2 100 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 ⁇ l of a 2N HCL. Optical density is determined with a 450 nm microplate spectrometer using a 620 nm filter.
  • the pretreated samples often show a higher optical density compared to non-pretreated samples.
  • the diagnostic sensitivity and specificity of the immuno-enzymatic test increases.
  • 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 ⁇ l of the anti-H. pylori. Rabbits in unglobulin at a concentration of 10 ⁇ g / 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 ⁇ l PBS containing 0.5% bovine serum albumin (BSA) for 16 hours at room temperature. The wells are then washed twice with PBS.
  • BSA bovine serum albumin
  • the wells are pretreated and 100 ⁇ l each in the double batch of the fakal suspension not pretreated as described above.
  • 100 ⁇ l of a biotinylated, polyclonal, peroxidase-coupled rabbit immunoglobulin in a concentration of 2 ⁇ g / 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 ⁇ l PBS.
  • the pretreated samples often show a higher optical density compared to non-pretreated samples.
  • the diagnostic sensitivity and specificity of the immuno-enzymatic test increases.
  • 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.
  • test tube 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 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.
  • 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.
  • Pretreatment 100-200 mg of the stool sample are transferred to a conical test tube, model Eppendorf, 1.5 ml.
  • 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.
  • the test tube is vortexed for 4-5 seconds and centrifuged at 14000 rpm for 20 minutes.
  • the pellet is resuspended with 0.3 ml of a 0.2 M Tris buffer pH 8.5, containing 3 mol / 1 potassium chloride.

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  • Health & Medical Sciences (AREA)
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Abstract

Procédé de prétraitement d'échantillons de selles pour détacher par clivage des antigènes de H. pylori de liaisons avec des anticorps endogènes, ledit prétraitement étant effectué avant l'analyse des selles à la recherche de H. pylori à l'aide de techniques immunologiques.
PCT/EP2003/004571 2002-05-02 2003-04-30 Procede de pretraitement d'echantillons de selles WO2003093818A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002119741 DE10219741A1 (de) 2002-05-02 2002-05-02 Verfahren zur Vorbehandlung von Stuhlproben
DE10219741.5 2002-05-02

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WO2003093818A2 true WO2003093818A2 (fr) 2003-11-13
WO2003093818A3 WO2003093818A3 (fr) 2004-02-26

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Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5716791A (en) * 1996-05-09 1998-02-10 Meridian Diagnostics, Inc. Immunoassay for H. pylori in fecal specimens
EP1125130B1 (fr) * 1998-10-29 2006-07-26 DakoCytomation Denmark A/S Determination de la presence de micro-organismes resistant aux acides dans des selles
TWI237695B (en) * 1999-12-14 2005-08-11 Joy Biomedical Corp Helicobacter pylori antigens in blood
DE10006432A1 (de) * 2000-02-14 2001-08-16 Ganzimmun Inst Fuer Ganzheitli Verfahren zum Nachweis von Helicobacter pylori in Stuhl- und Speichelproben
DE10043161A1 (de) * 2000-09-01 2002-03-14 Connex Ges Zur Optimierung Von Lösung zur Aufbereitung von Stuhlproben für diagnostische Zwecke

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WO2003093818A3 (fr) 2004-02-26

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