WO1999041611A1

WO1999041611A1 - Assaying of antibodies directed against one or more antigens of helicobacter pylori in biological liquids by a heterogeneous immunologic method of the reverse type

Info

Publication number: WO1999041611A1
Application number: PCT/IT1999/000032
Authority: WO
Inventors: Alessandro Zuccato
Original assignee: Consortia Laboratories S.R.L.
Priority date: 1998-02-13
Filing date: 1999-02-12
Publication date: 1999-08-19
Also published as: IT1299312B1; AU2545999A; ITVR980010A1

Abstract

Method for the determination of the presence or absence of immunoglobulins directed against one or more Helicobacter pylori antigens, in animal biological liquids, which is carried out using an immunobiologic heterogeneous competition test commonly referred to as 'reverse' test. The presence or absence of specific immunocomplexes is assessed either in a direct fashion, which is when the reaction signal is directly carried by the antigen or the antigens of Helicobacter pylori, or in an indirect fashion, which is when the immunologic reaction signal is obtained by adding one or more tracing substances that bind to the antigen or to the antigens of Helicobacter pylori that were previously captured by the immunoglobulins. By comparision with the methods currently used in the diagnostic field, this method has the following main features: a) use of undiluted biological liquid in the diagnostic test, because the test is of the competitive type with respect to the solid phase used; b) determination of the presence of antibodies directed against one or more antigens of Helicobacter pylori in biological liquids, by a quantitative assessment of the antigens bonded to the solid phase; c) use of standard solutions made up of mixtures containing variable concentrations of anti-Helicobacter pylori immunoglobulins and immunoglobulins that do not react with the antigens used in the assay.

Description

"ASSAYING OF ANTIBODIES DIRECTED AGAINST ONE OR MORE ANTIGENS OF HELICOBACTER PYLORI IN BIOLOGICAL LIQUIDS BY A HETEROGENEOUS IMMUNOLOGIC METHOD OF THE REVERSE TYPE"

TECHNICAL FIELD

The present invention relates to a process for the qualitative or quantitative determination in biological liquids (plasma, serum, blood), of immunoglobulins directed against one or more Helicobacter pylori antigens. The process according to the invention is utilised in the field of serology for the detection of antibodies directed against Helicobacter pylori antigens.

This process can be used for the diagnosis of an infection, for monitoring of a patient after an eradicating therapy or in order to verify the positive onset of an immunisation triggered by a vaccination. STATE OF THE ART

According to techniques known in the art the detection of antibodies in biological liquids directed against Helicobacter pylori antigens is carried out using some quantitative or qualitative methods of the immunologic type. The methods currently in use are basically of three types:

a) indirect heterogeneous immunoenzymatic assay (ELISA); thanks to this method a qualitative and/or quantitative assessment of the presence of mύ-Helicobacter pylori antigens that occur in a biological liquid can be made; b) immunochromatographic test on strip or pad (STRIP TEST); this test makes it possible to rapidly determine the presence or absence of anύ-Helicobacter pylori immunoglobulins in a biological liquid; c) Latex Agglutination Test (LAT); this test makes it possible to rapidly determine the presence or absence of anύ-Helicobacter pylori immunoglobulins in a biological liquid.

The methods listed above are all of an immunologic type, in other words they exploit the specific reaction between antigen and antibody in order to detect the presence of immunoglobulins directed against Helicobacter pylori antigens in a biological liquid.

Generally speaking, the skilled in the art categorise immunodiagnostic methods into two types: homogeneous and heterogeneous. When the reaction between an antigen and an antibody is carried out in a liquid solution and the formation of the immunocomplex directly determined in it, the method is then defined as being of the "homogeneous" type. When one of the two immunologic counterparts is immobilised by binding it to a solid matrix, the immunologic method is instead of the "heterogeneous" type. A matrix insoluble in aqueous systems to which one of the two immunologic counteφarts has been either covalently linked or simply adsorbed, or else another substance which is able to immobilise either the antigen, or the antibody contained in a liquid solution is defined as "solid phase".

In so far as the present specification is concerned heterogeneous methods are categorised into "indirect" or "direct" methods. If the reaction between antigen and antibody is detected by using a further substance that binds to the immunocomplex, the method is of the indirect type, whereas measurements of the immunocomplex by conjugation of one of the two immunologic counteφarts with substances that are carriers of a signal are defined as being of the "direct " type.

In "direct" heterogeneous methods the immunologic counteφart which is used to determine the occurrence of the antigen or antibody in a certain biological liquid is conjugated with substances which are capable of giving a signal (tracers). For example, in immunoenzymatic methods the antibody or antigen are conjugated with enzymes ; the transformation of the specific substrate is bound to yield to a signal of the optical, electrical, chemiluminescent, fluorescent type, according to the system used (El A, Enzyme Immunoassay).

The immunologic method in which antibodies or antigens conjugated with fluorescent substances (e.g. fluorescein) are used is defined by the skilled in the art as Fluorolmmunoassay (FIA) whereas, if these are conjugated with radioactive isotopes, the method is of the Radioimmunoassay type (RIA) or Immunoradiometric type (IRMA).

A variant of the methods described above consists in the conjugation of one of the two immunologic counteφarts with aptens or low molecular weight molecules that do not directly yield to a measurable signal by which the immunocomplex formation can be quantified.

However these conjugates can react afterwards or simultaneously to the formation of antigen-antibody complexes with a suitable tracers, obtaining a direct conjugation as a direct effect of one of the two immunologic counteφarts.

The antigen or antibody can for example be covalently conjugated with biotin which can itself react with a specific tracer made for example of strep tavidin conjugated with an enzyme (streptavidin-enzyme; the hyphen indicates the bond between the two compounds). An immunologic method of the "indirect" type is used in diagnostic serology for assays directed towards the detection of antibodies that are effective against pathogenic agent antigens. In this type of immunologic assay, solid phases made of plastic matrices (generally tubes or microtitre plates with 96 wells) are used, which are coated with one or more antigens of a pathogenic agent. After removing the reaction liquid, tracers that specifically recognise the antibody are added, for example anti- immunoglobulins conjugated with an enzyme or Protein A-enzyme, protein G-enzyme, Fc -receptor -enzyme (examples of immunoenzymatic methods).

The "indirect" heterogeneous immunologic assay is surely the most commonly used method in general diagnostic serology, and in particular for Helicobacter pylori serology.

In this context, documents WO-A-9601273, J07294530, EP-A-638175, EP-A- 582672 relate to the preparation of Helicobacter pylori antigens for the production of solid phase and their subsequent utilization in immunologic assays of the "indirect" type. A variant of the "indirect" method, used in serologic tests, consists of preparing a solid phase which is made of a matrix insoluble in aqueous solutions (e.g. plastic) which is coated with one or more monoclonal antibodies directed against Helicobacter pylori antigens. Afterwards a solution containing one or more Helicobacter pylori antigens is made to react on the solid phase. The final result is that of obtaining a solid phase that only consists of the antigens that are recognised by the monoclonal antibodies used. The subsequent carrying out of the serologic test is identical to that of the "indirect" method. This type of assay is called "antigen capture" by the skilled in the art. By using the method defined as "immunochromatography", rapid diagnostic tests have been developed for the detection of anύ-Helicobacter pylori immunoglobulins in biological liquids.

The method is still of the immunologic type, but the reaction between antigen and antibody takes place on a chromatographic paper strip or a strip made of another hydrophilic material on which the antigen or the antigens of Helicobacter pylori have been coated in a permanent manner in a very well defined zone. -An agent that is capable of capturing immunoglobulins, is also coated on a zone different from the former one. In an area which is close to the deposition point of the biological liquid sample there is a certain quantity of anti-immunoglobulins which are coated onto microspheres and recognise the immunoglobulins in the sample.

When the liquid sample is deposited on the chromatographic phase, by capillarity a migration takes place. Immunoglobulins present in the sample react with the anti-immunoglobulins which are already on the strip. Therefore, when the immunocomplexes formed by the immunoglobulins coated onto the microspheres and specific immunoglobulins of the sample, during the chromatographic migration, reach the area with the antigen they react with it concentrating over a very well defined area of the strip. On continuing their migration along the chromatographic strip, the unbound microspheres subsequently reach the immunoglobulin capture area, in so doing yielding to the formation of a second chromatographic band which aims at confirming the correct execution of the test and the efficiency of the reagents. This type of tests can be normally carried out in 5 to 10 minutes. Documents WO-A-9518378, WO-A-9517676 and WO-A-9516207 disclose immunochromatographic methods of the above-described type.

The "indirect" heterogeneous immunologic method is very sensitive, however it has the considerable drawback to require a dilution of the biological liquid sample (serum or plasma) before carrying out the test (dilutions generally range between 1:50 and 1 :200 v/v), on account of the fact that the total amount of immunoglobulins is very high. Furthermore the test reference curves (standard curves) are not generally straight lines; in other words there is not a linear dose/response relationship within the range of immunoglobulin concentrations to be determined.

DETAILED DESCRIPTION OF THE INVENTION The present invention aims at overcoming the disadvantages and drawbacks typical of the prior art, by proposing a method for the quantitative or qualitative determination of immunoglobulins that are directed against one or more Helicobacter pylori antigens in biological liquids. This method can be directly carried out on a biological liquid at its undiluted state, and makes it possible to obtain a constant dose/response ratio within the range of the immunoglobulin concentrations to be determined.

The above aim has been accomplished by proposing a method whose features are detailed in the main claim.

The presence or absence in biological liquids of immunoglobulins of the G, M, or A immunoglobulin classes which are directed against Helicobacter pylori antigens, is determined by an immunologic reaction, using Helicobacter pylori antigens marked with substances that directly or indirectly indicate the formation of antigen-antibody complexes.

The term biological liquid is meant to categorise a liquid solution produced by a human or an animal, and this includes urine, saliva, serum, plasma, and whole blood. In its preferred form, the biological liquid sample is made of animal serum or plasma.

According to the invention, the process schemes can be described as follows, according to the "direct" or "indirect" detection method used. "Direct" method: 1) applying a biological liquid sample to assay onto an area defined as being a

"capture" area, composed by a solid matrix that has been coated with a substance which is capable of capturing immunoglobulins (a capture area is by definition a well defined area of a solid phase, which is also separated from other capture areas, and which is capable of binding specific substances present in a solution that is in contact with the solid phase);

2) reacting the immunoglobulins that are present in the biological liquid with the solid phase; 3) separating the biological liquid from the capture area, e.g. by suction of the liquid, or washing of the solid phase;

4) applying a liquid solution containing one or more Helicobacter pylori antigens conjugated with one or more substances onto the capture area; 5) reacting the antigen or antigens described at 4) with the immunoglobulins that were previously captured by the solid phase; 6) washing the solid phase;

7) determining the presence or absence of antigens bound to the immunoglobulins captured by the solid phase, by a suitable detection system "Indirect" method:

1) applying a biological liquid sample to assay onto an area defined as being a "capture" area, composed by a solid matrix that has been coated with a substance which is capable of capturing immunoglobulins (a capture area is by definition a well defined area of a solid phase, which is also separated from other capture areas, and which is capable of binding specific substances present in a solution that is in contact with the solid phase);

2) reacting the immunoglobulins that are present in the biological liquid with the solid phase;

3) separating the biological liquid from the capture area, e.g. by suction of the liquid, or washing of the solid phase;

4) applying a liquid solution containing one or more Helicobacter pylori antigens conjugated with one or more substances onto the capture area; the antigen or antigens may be in their natural form or conjugated with one or more substances;

5) reacting the antigen or antigens described at 4) with the immunoglobulins that were previously captured by the solid phase;

6) adding a substance carrying a signal and capable of binding to the antigens or the substances bound to the antigens as described at 4). This step can be performed either directly or after removing the solution containing one or more of the antigens detailed at 4); 7) reacting the substance carrying a signal with the antigen or antigens described at 4);

8) washing the solid phase; 9) determining the presence or absence of antigens bound to the immunoglobulins captured by the solid phase, by a suitable detection system

The method according to the invention further comprises the quantitative determination of those immunoglobulins that are directed against Helicobacter pylori antigens.

Quantitative determinations are carried out in the test using one or more solutions containing a known quantity of anti-Helicobacter pylori immunoglobulins (standard solutions) that react with the antigens used in the immunologic test.

The standard solutions are prepared by gradually diluting (for example to 1 :2, 1:4, 1:8, 1: 16 v/v) a solution containing a high concentration of anti-Helicobacter pylori immunoglobulins with solutions containing immunoglobulins that do not react with the Helicobacter pylori antigens.

For example a human hyper-immune serum or a pool thereof may be used as a source of anti -He bcøbαcter v/orz immunoglobulins and as a diluting solution a serum free from anti-Helicobacter pylori antibodies or a pool thereof.

The immunologic method according to the present invention may also be defined as being an antibody capture assay of a competitive type. In fact the amount of capturing agent-which occurs on the solid phase and which is capable of capturing the immunoglobulins that are present in the biological liquid is in large defect with respect to the amount of the antibodies that occur in the biological sample.

Therefore the anti-Helicobacter pylori immunoglobulins compete for a binding site on the solid phase with the unreactive immunoglobulins of the biological sample.

In other words, the amount of anti-Helicobacter pylori immunoglobulins that are due to bind to the solid phase depends on the ratio between the amount of anti-Helicobacter pylori immunoglobulins and the total amount of immunoglobulins that are in solution.

Therefore, according to the method disclosed in the present invention, the amount of antigen bonded to the antibodies captured by the solid phase is bound to be proportional to the amount of anti-Helicobacter pylori immunoglobulins that have been captured by the solid phase.

Therefore, using solutions which contain known quantities of specific anti- Helicobacter pylori immunoglobulins diluted in solutions containing fixed amounts of aspecific immunoglobulins, it is possible to work out the concentration of specific immunoglobulins in a certain sample, by comparison with the standard solutions used. According to a preferred form of embodiment of the present invention, standard solutions can be incubated prior to the diagnostic test carried out on the capture area which has been described above. Once the capture reaction has gone to completion, the solid phase is washed, dried and kept in a suitable environment that is capable of keeping the bond between the solid phase and the immunoglobulins integer. This new solid phase, which contains the previously adsorbed standard solutions, may be used for a subsequent assay. According to this form of embodiment of the present invention, the distribution and incubation of the standard solutions can be avoided, thus remarkably simplifying the operational steps of the immunologic tests.

With respect to the methods for the detection of immunoglobulins directed against one or more Helicobacter pylori antigens which are currently in use in the diagnostic field, the method according to the present invention bears the following innovative features:

a) use in the diagnostic test of undiluted biological liquid: b) determination of the occurrence of antibodies directed against one or more Helicobacter pylori antigens in biological liquids, by dint of a quantitative assessment of the antigens bonded to the solid phase; c) use of standard solutions made up of mixtures containing anti-Helicobacter pylori immunoglobulins and aspecific immunoglobulins, at variable concentrations' ratios. First of all, these characteristics make it possible to substantially simplify the execution of the immunologic assay, with respect to current methods, on account of the fact that biological liquids are not diluted before the test.

Furthermore, it is worth noting that a better precision and accuracy of the analytical data obtained is achieved, to be accounted for by the test results not risking being affected by errors occurring during the distribution of the biological liquids onto the capture area, as the test result is only affected by the ratio of anti-Helicobacter pylori immunoglobulins and aspecific immunoglobulins. Finally, a better assessment of the amount of those immunoglobulins that are directed against the Helicobacter pylori antigen or antigens of the biological liquid is possible, as the dose/response ratio is a straight line over a large range of specific immunoglobulin concentrations. DESCRIPTION OF ONE FORM OF EMBODIMENT

In animal biological liquids, the occurrence or absence of immunoglobulins directed against one or more Helicobacter pylori antigens is determined according to the present invention availing of a competitive heterogeneous immunologic test which is commonly referred to as "reverse" test by the skilled in the art. The occurrence or absence of specific immunocomplexes is ascertained in a

"direct" way (the reaction gives a signal that is directly carried by the Helicobacter pylori antigen or by its antigens) or in an "indirect" way (the immunologic reaction gives a signal that is obtained by adding one or more tracing substances that bind to the Helicobacter pylori antigen or antigens which have been captured by immunoglobulins).

An immunologic test of the "direct" type or else a test of the "indirect" type according to the present invention is carried out according to the following operational steps:

1) providing a solid phase which has been previously subdivided into capture areas and is made of a matrix insoluble in aqueous solutions which is coated with one or more substances that are capable of binding to the immunoglobulins that occur in a biological liquid sample;

2) providing a certain prefixed quantity of Helicobacter pylori antigen or antigens for the "direct" or "indirect" determination of the formation of specific immunocomplexes captured by the solid phase;

3) providing two or more anti-Helicobacter pylori immunoglobulin standard solutions for carrying out an immunologic test of the quantitative type;

4) as an alternative to the reagents described at the previous point, providing two or more solid phases containing different standard quantities of anti-Helicobacter pylori immunoglobulins, for carrying out an immunologic test of the quantitative type. These solid phases are prepared by using the solid phase described at point 1) onto which the immunoglobulins of the standard solutions of point 3) end up being 1 0

immobilised, by dint of the capture agent. Said solid phases are in their dried form and they are further ready for use;

5) providing one or more solutions containing the immunoglobulins directed against one or more Helicobacter pylori antigens to be used in the immunologic test as test quality control sample(s);

6) providing one or more solutions for the detection of immunocomplexes if the immunologic test is of the "indirect" type;

7) providing washing solutions;

8) providing a diagnostic kit for the determination of immunoglobulins directed against one or more Helicobacter pylori antigens.

According to the present invention the following operational steps are further carried out: a) adding and incubating a prefixed amount of biological liquid to the capture area of the solid phase; b) washing said capture area; c) adding and incubating a prefixed amount of the Helicobacter pylori antigen or antigens described above; d) determining the amount of specific immunoglobulins captured by the solid phase by measuring the amount of Helicobacter pylori antigen or antigens bonded to the specific immunoglobulins by a "direct" or "indirect" method. In the latter case a further liquid solution is added and incubated, containing one or more agents that by binding to the Helicobacter pylori antigens which are themselves bonded to the anti- Helicobacter pylori immunoglobulins on account of their molecular affinity with them, give a signal of their occurrence on the solid phase. EXAMPLE OF A PREFERRED DIAGNOSTIC KIT AND OF THE

OPERATIONAL PHASES OF THE ASSAY METHOD Preparation of the solid phase According to a preferred form of embodiment, the insoluble matrix in aqueous solutions is made of a polystyrene microtitre plate with 96 wells (in a second preferred form of embodiment plastic tubes are used) onto which a substance which is capable of binding to the immunoglobulins is adsorbed or covalently bonded.

According to a particularly advantageous form of embodiment of the present invention, Staphylococcus aureus Protein A is utilised. 1 1

Further forms of embodiment comprise the utilisation of Protein G, Protein B , specie-specific anti-immunoglobulins, fragments of anti-immunoglobulin antibodies, Fc receptors, complement factors. These substances can be natural, or else obtained by organic synthesis or else recombinant techniques and active fragments thereof. Preparation of the standard solutions and of the dried solid phases containing the solid phase references, solid phase that itself contains the references Solutions containing increasing proportions of anti-Helicobacter pylori immunoglobulins that do not recognise the bacterium antigens are added and incubated on the solid phase. For example human serum pool with a high concentration of anti-Helicobacter pylori immunoglobulins is diluted, to 1:2, 1:4, 1 :8, 1: 16, with a human sera pool that does not contain immunoglobulins directed against the Helicobacter pylori antigens, prior to carrying out the test

There are therefore found to be obtained four solutions that are respectively bound to contain 50%, 25%, 12.5%, 6.25% of the specific immunoglobulins with respect to the starting solution.

Those standard mixtures can be utilised simultaneously to the execution of the test aimed at drawing the method's reference curve or after washing and drying the solid phase it is possible to resort to these preset standards in a subsequent analytical session.

Preparation of the Helicobacter pylori antigen or antigens

Helicobacter pylori antigens are obtained from Helicobacter pylori cells by sonication of a bacterial cell suspension followed by a number of extraction steps by dint of aqueous solutions (the antigen or antigens may be recombinants that is obtained using molecular biology techniques and chemical synthesis or else they may be fragments thereof).

According to a form of embodiment of the present invention, in which the method used is of the "indirect reverse" type, one or more antigens are conjugated to biotin according to classical chemical conjugation methods. According to further forms of embodiment of the present invention, other aptens which are chemically bonded to the Helicobacter pylori antigens, like for example digoxygenin, are used, these being capable of being recognised after their reaction with the immunoglobulins of the biological liquid sample by a second 1 2

antibody conjugated with a tracer and from an animal species that is different from that which is being assayed.

Tracer preparation for the heterogeneous immunologic method of the "indirect reverse" type. The heterogeneous method of the "indirect reverse" type preferably avails of the

Helicobacter pylori antigen or antigens conjugated with biotin, whereas the tracer is made of a streptavidin-horseradish peroxidase conjugate obtained by chemical reaction.

According to further forms of embodiment of the present invention, the tracers used are made of streptavidin-beta-galactosidase, streptavidin-alkaline phosphatase, streptavidin urease, streptavidin-glucose oxidase.

Diagnostic kit The method according to the present invention can be carried out by means of a so called "diagnostic kit" that comprises a solid phase, conjugated antigen or antigens, tracers, standard solutions or solid phases ready to be used for a reference curve, washing solutions, control samples, possible substrates and chromogens for the immunoenzymatic methods, possible solutions for the stop of enzymatic reactions and instructions for carrying out the test.

Execution of the immunologic test according to an advantageous form of embodiment of the present invention 100 μl of biological liquid sample (preferably serum, plasma, whole blood) and standards solutions are added to the microtitre plate wells and incubated.

After washing the solid phase, 100 μl of the solution containing the antigen or the antigens conjugated with biotin is added, to all the wells.

After a time lapse ranging between 15 and 90 minutes, 100 μl of tracer (e.g. streptavidin-horseradish peroxidase) are added to all the wells.

After 5-30 minutes the solid phase is washed and to each well, 100 μl aliquots of a solution consisting of a specific substrate and chromogen for the enzyme used as tracer is added. In case the horseradish peroxidase enzyme is used, the preferred form of the solution contains tetramethylbenzidine (chromogen) and hydrogen peroxide (substrate).

After a time ranging between 5 and 20 minutes, the enzymatic reaction is stopped with a hydrochloric acid or sulphuric acid solution. In case tetramethylbenzidine is used, the 1 3

acidification of the solution causes a colour change of the solution that turns from blue to yellow.

By dint of a spectrophotometric detector for 96 well microplates it is possible to read the optical densities obtained by enzymatic reaction of the wells used for the biological liquid and the standard samples. By means of suitable calculations and graphic inteφolation it is therefore possible to calculate the amount of specific immunoglobulins of the samples under assay, by comparing them with the signal obtained in the standard curve wells. Generally, standard values are arbitrarily represented as being for example 100 units/ml for the standard solution at its highest antibody concentration, whereas the other solutions are bound to be fractions of it. Because of the competitive capture technique used, the samples and standards values obtained by this assay can be also represented as μg specific Helicobacter pylori immunoglobulins / mg total immunoglobulins.

Claims

14

CLAIMS Immunologic heterogeneous method of the direct reverse type for the quantitative or qualitative determination in biological liquids, for example plasma, serum, whole blood, of the occurrence of immunoglobulins of the IgG and/or IgM and/or IgA classes, directed against one or more antigens of

Helicobacter pylori, said method comprising the following steps:

a) providing a solid phase made of a matrix which is insoluble in aqueous solutions, which is coated with one or a number of substances which are capable of binding the immunoglobulins present in the biological liquid sample under assay; b) providing a solution containing a certain prefixed amount of Helicobacter pylori antigen or antigens, said antigen or antigens being natural or recombinant or obtained by chemical synthesis, which are conjugated with marker substances that reveal the bond with the anti-

Helicobacter pylori immunoglobulins ; c) providing one or more immunoglobulin solutions, referred to as standard solutions, and containing different amounts of both anti- He / cøbαcter >y/oπ immunoglobulins and immunoglobulins that do not bond to the antigens described at point b, to be employed when devising a reference curve valid for the immunologic process; d) adding and incubating the standard solutions of point c) and the biological liquid to assay on the solid phase of point a), distributing the different solutions in different capture areas; e) washing the solid phase; f) adding to the capture areas used in point d) and incubating the solutions containing a certain prefixed amount of conjugated Helicobacter pylori antigens of point b); g) washing the solid phase; h) in case these substances are of an enzymatic type, adding the specific substrate for the marker substances as of point b), and incubating them; i) determining the amount of anti-Helicobacter pylori immunoglobulins captured by the solid phase, by measuring the amount of Helicobacter 1 5

pylori antigen or antigens described at point b) that have reacted with the anti-Helicobacter pylori immunoglobulins which are present in the different capture areas of the solid phase.

2 . Process according to claim 1 , characterised in that said substances referred to in point a), which are capable of binding to the immunoglobulins present in the biological liquid sample under assay, comprise Protein A, Protein G, Protein B, specie-specific anti-immunoglobulins, Fc receptors, complement factors, peptides, all both natural or obtained by recombinant techniques or by chemical synthesis, and fragments thereof.

3 . Method according to the preceding claims, characterised in that said marker substances described in point b) comprise radioactive isotopes, bioluminescent or chemiluminescent molecules, fluorescent molecules, and enzymes.

4 . Immunologic heterogeneous method of the indirect reverse type for the qualitative or quantitative determination of immunoglobulins of the IgG and/or

IgM and/or IgA classes, directed against one or more antigens of Helicobacter pylori, in biological liquids like for example plasma, serum, whole blood, said method comprising the following phases:

a) providing a solid phase made of a matrix insoluble in aqueous solutions, which is coated with one or more substances capable of binding the immunoglobulins that are present in the biological liquid sample under assay; b) providing a solution containing a certain prefixed amount of Helicobacter pylori antigen or antigens, both natural and obtained by recombinant techniques or chemical synthesis, which are not modified or conjugated with substances, particularly aptens or molecules having a molecular weight which is lower than 2 kD; c) providing one or more immunoglobulin solutions, referred to as standard solutions, and containing different amounts of both anti- 1 6

Hebcøbαcter y/ør immunoglobulins and immunoglobulins that do not bond to the antigens described at point 4.b, to be employed when devising a reference curve valid for the immunologic process; d) providing one or more solutions containing a certain prefixed amount of a tracer made of a substance that specifically binds to the antigen or the apten molecule bonded to the antigen, referred to at point 4.b), conjugated with substances that are capable of giving a measurable signal of the antigen-antibody reaction; e) adding and incubating the standard solutions of point 4.c) and the biological liquid to be assayed on the solid phase of point 4. a), distributing the different solutions to different capture areas; f) washing the solid phase; g) adding and incubating the solutions containing a certain fixed amount of antigen or antigens described at point 4.b), on the capture areas used during step 4.e); h) directly, or after washing the solid phase, adding and incubating the solution containing a certain fixed quantity of substances of point 4.c); i) washing the solid phase; j) adding and incubating the substrate specific for the tracers of point 4.c), in case they are tracers of an enzymatic type; k) determining the amounts of the anti-Helicobacter pylori immunoglobulins captured by the solid phase, by measuring the amount of conjugated antigen or antigens of Helicobacter pylori that have reacted with the anti-Helicobacter pylori immunoglobulins present in the different capture areas of the solid phase.

Method according to claim 4, characterised in that the substances referred to at point 4. a) comprise protein A, Protein G, Protein G, Protein B, specie-specific anti-immunoglobulins, Fc receptors, complement factors, peptides, both natural and obtained by recombinant technology or chemical synthesis, and fragments thereof. 1 7

6 . Method according to anyone of claims 4 to 5, characterised in that the substances referred to at point 4.b) comprise biotin, digoxygenin, fluorescein, dinitrofluorobenzene .

7 . Method according to anyone of claims 4 to 6, characterised in that the conjugates of point 4.c) comprise compounds like streptavidin-enzyme, particularly streptavidin-peroxidase, or else streptavidin-alkaline phosphatase, or streptavidin-glucose oxidase, or streptavidin-urease, or streptavidin-beta- galactosidase, or else streptavidin marked with one or more radioactive isotopes, or streptavidin-chemiluminescent molecules, or streptavidin- fluorescent molecules, and or anti apten antibodies or antibodies of Helicobacter pylori antigen or antigens conjugated with enzymes, radioactive isotopes, bioluminescent or chemiluminescent molecules, fluorescent molecules.

8 . A diagnostic kit for the execution of a method according to anyone of the preceding claims, characterised in that it comprises a solid phase, a conjugated antigen or antigens, marker substances or tracers, standard solutions or solid phases ready for use for the determination of the standard curve, washing solution, control samples, possibly substrates and chromogens for the immunoenzymatic methods, solutions for the stop of the enzymatic reactions.