Serological test for detection of Anti-hpv antibodies and test kit

Abstract

The present disclosure relates to a serological test for detection of anti-HPV antibodies. The serological test uses antigens having a linear epitope which is reactive and is such that multiple or many antibodies, each of which is effective against HPV of a different type, can alternatively bind specifically thereto. The present disclosure further relates to a test kit (1; 1.1; 1.2; 1.3; 1.4) for carrying out the serological test.

Description

[0001] Serological test for the detection of antibodies against HPV and test kit

[0002] Technical area

[0003] The present disclosure relates to a serological test for detecting antibodies against HPV and a test kit for such a serological test. In particular, the present disclosure comprises a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid.

[0004] Serological tests utilize in vitro antigen-antibody reactions. These reactions can be used, for example, to identify endogenous antibodies in a blood sample that are specific to certain antigens and target pathogens such as viruses and bacteria. These antibodies are specifically produced by a person's immune system in response to exposure to these antigens in order to protect the body.

[0005] Serological tests are used, among other things, to detect antibodies against human papillomaviruses, abbreviated to HPV. One such test is known from the publication WO 2012 / 097788 A1 and is used as the basis for and described in a method for therapy monitoring, for example, in the publication WO 2018 / 108957 A1. Reference is made here to these publications for the purpose of supplementing the disclosure, with the note that these publications may assign a meaning to identical terms that differs from the present meaning. The known test enables the identification of antibodies that are specifically effective against HPV type 16.

[0006] Type 16 is one of the HPV types known as high-risk types (high-risk HPV). We now know that high-risk types play a significant role in the development of precancerous lesions (dysplasias) and the resulting carcinomas, particularly in the cervix. We also now know that only 25% of HPV-caused precancerous lesions and carcinomas are caused by HPV type 16. However, 75% of such HPV-caused precancerous lesions and carcinomas are caused by the other high-risk types. This significant proportion is not taken into account in current tests.

[0007] There is therefore a need to use a serological test to detect antibodies that are effective against HPV types other than, for example, HPV type 16. This is based on the expectation that detecting as many HPV types as possible will lead to further improvements in predicting the formation of HPV-caused precancerous lesions and carcinomas based on a serological test.

[0008] Concepts

[0009] Fundamental improvements in detection are offered by a serological method for detecting antibodies against HPV, in which antigens with a linear epitope, in particular at least one linear epitope or exclusively one linear epitope, are used, wherein the linear epitope is preferably reactive and is such that alternatively several or many antibodies bind specifically to it or can bind specifically to it, each of which is effective against HPV of a different type.

[0010] The term "serological method" in the present disclosure means or includes a serological test or a serological test method or an assay or an immunoassay. The method is preferably an in vitro method, i.e. a method carried out in vitro.

[0011] The term "in vitro" in the present disclosure is to be understood in particular to mean that the method is carried out outside a living organism, i.e. is not carried out on a person. In order to carry out the method in vitro, at least one sample, for example of body fluid, is taken from the person concerned. For example, one drop (approx. 25 pL) is sufficient to carry out the method.

[0012] The term "antigen" in the present disclosure refers to proteins of any kind which trigger an immune response in the immune system of a person confronted with them. The term "antigen" in the present disclosure also includes a virus-like particle (VLP). The term "virus-like particle" or "VLP" in the present disclosure refers to a particle which consists of or comprises viral capsids but does not contain any nucleic acids. A VLP is therefore suitable for presenting the linear epitope described above without itself being capable of replication.

[0013] The term "specifically binding" in the present disclosure is to be understood in particular as meaning that an antibody binds exclusively to a specific binding site of an antigen. The antibody can therefore only bind to the antigen via such a binding site. With regard to the several or many antibodies that can bind to the linear epitope of the antigens, it is to be understood that the antibodies considered here are those that bind or can bind exclusively to this linear epitope.

[0014] By utilizing the specific linear epitope, a single serological test can be used to determine the presence of precancerous lesions and carcinomas caused by an entire group of HPV types. This is based on the surprising commonality that these HPV types share the same amino acid sequence. The improved method exploits this commonality by presenting the specific linear epitope of the antigens with the aforementioned amino acid sequence.

[0015] The linear epitope also offers further advantages. The antigens do not have to be kept in a physiologically active liquid to maintain the reactivity of the linear epitope. The antigens can also be dried or in solid form. Furthermore, no refrigeration is required to store the antigens. The antigens can be stored at room temperature, for example, without any loss of effectiveness, or at least without any significant loss of effectiveness. These are key differences to the known test that uses antigens with a conformational epitope. In order to maintain the reactivity of the conformational epitope, the antigens must be kept refrigerated in a physiologically active liquid.

[0016] The antigens used in the improved method can be multimeric, i.e., present as a multimer. The term "multimer" in the present disclosure refers to a molecule or molecular complex in which the linear epitope described above occurs at least twice.

[0017] Alternatively, the antigens can be monomeric, i.e., present as a monomer. The term "monomer" in the present disclosure refers to a molecule in which the linear epitope described above occurs only once.

[0018] In particular, it is intended that labeled antibodies be used which are designed in such a way that they specifically bind or can specifically bind to the linear epitope of the antigens. The labeled antibodies are therefore antibodies directed against HPV types with an amino acid sequence which is presented or represented by the linear epitope of the antigens. These antibodies are preferably labeled, for example, using methods known per se.

[0019] In particular, the labeled antibodies are designed, preferably by means of the label, such that in the presence of a reactant they cause, for example, a measurable and / or user-perceptible reaction or, in the case of spatial accumulation, in particular spatial accumulation of bound labeled antibodies, they cause, for example, a measurable and / or user-perceptible change. The labeled antibodies thus enable the identification of antibodies against HPV contained in body fluid. The reaction or change can be measured photooptically.

[0020] According to a first embodiment, the improved method can be a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid and in particular comprise or consist of the steps of providing a carrier medium, such as a test strip, on which a predetermined amount of the labelled antibodies is presented in a mobile manner and a predetermined amount of the antigens is present at a predetermined location, in particular is presented immobilely, for example is fixed; for bringing a preferably predetermined volume of body fluid together with the labelled antibodies, i.e. the labelled antibodies present on the carrier medium; for carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis of the carrier medium, for example for a change preferably in the region of the predetermined location.

[0021] In particular, in this case, the labeled antibodies are designed in such a way that, for example, in the case of binding, i.e., binding to the linear epitope of the antigens, they result in a measurable and / or user-perceptible change if there is a spatial clustering, i.e., a spatial clustering of labeled antibodies. For example, the labeled antibodies are labeled with gold particles and / or silver particles.

[0022] The step of bringing the body fluid together with the labelled antibodies particularly includes bringing the body fluid together with the labelled antibodies directly. It also particularly includes bringing the body fluid together with the labelled antibodies indirectly. This can be done via the carrier medium. For example, the body fluid is applied to the carrier medium at a point spatially spaced from the labelled antibodies. For example, in this case the body fluid is brought together with the labelled antibodies by the body fluid flowing on the carrier medium towards the labelled antibodies. Additionally or alternatively, a capillary effect can be used. For example, the carrier medium has or consists of a material which is designed to exert a capillary effect on a liquid.

[0023] This embodiment is based on a competitive approach known per se, in which any antibodies against HPV contained in the body fluid compete with the labelled antibodies for binding to the linear epitope of the antigens. The improved method according to this embodiment can be carried out in just a few steps. The body fluid can be applied immediately to the carrier medium. A preceding step is not necessary with regard to the body fluid. The carrier medium used, with its mobile labelled antibodies and the immobile antigens, makes it possible to provide a rapid test in which a change occurs in a reaction zone that can be quickly measured and / or perceived by a user. The improved method can, after a second,alternative embodiment, a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid and in particular comprise or consist of the steps of providing a predetermined amount of the antigens; providing a carrier medium, such as a test strip, on which a predetermined amount of the labelled antibodies is provided in a mobile manner and a predetermined amount of capture molecules is present at a predetermined location, in particular immobilely provided, for example fixed; bringing together a preferably predetermined volume of body fluid with the provided antigens; bringing together the body fluid and the provided antigens with the labelled antibodies,i.e. the labelled antibodies present on the carrier medium; for carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis of the carrier medium, for example for a change preferably in the area of ​​the specified location.

[0024] Bringing the body fluid and the antigens together with the labelled antibodies particularly includes bringing the body fluid and the antigens together with the labelled antibodies directly. It also particularly includes bringing the body fluid and the antigens together with the labelled antibodies indirectly. This can be done via the carrier medium. For example, the body fluid and the antigens are applied to the carrier medium at a point spatially separated from the labelled antibodies. For example, in this case the body fluid and the antigens are brought together with the labelled antibodies by the body fluid and the antigens flowing on the carrier medium towards the labelled antibodies. In addition or alternatively, a capillary effect can also be used.For example, the carrier medium comprises or consists of a material which is designed to exert a capillary effect on a liquid.

[0025] Bringing the body fluid together with the antigens and bringing the body fluid and the antigens together with the labelled antibodies can be understood as two, preferably separate steps, or as one step. Bringing the body fluid together with the antigens and bringing the body fluid and the antigens together with the labelled antibodies can, for example, be understood as taking place at separate times. In particular, the body fluid is brought together with the antigens first, followed by the body fluid and the antigens being brought together with the labelled antibodies. In particular, the body fluid should incubate with the antigens for a predetermined period of time and / or a freely selectable period of time before this composition is brought together with the labelled antibodies.If antibodies that bind to the linear epitope of the antigens are present in the body fluid, interactions between these antibodies and the antigens can occur before the body fluid and the antigens are brought together with the labelled antibodies.

[0026] Bringing the body fluid together with the antigens and bringing the body fluid and the antigens together with the labeled antibodies can also be understood as simultaneous steps or as a common step. For example, in this case, the body fluid, the antigens, and the labeled antibodies are brought together simultaneously.

[0027] In particular, in this embodiment, the labeled antibodies are designed such that, for example, in the case of binding, i.e., binding to the linear epitope of the antigens, they result in a measurable and / or user-perceptible change if there is a spatial clustering, i.e., a spatial clustering of labeled antibodies. For example, the labeled antibodies are labeled with gold particles and / or silver particles.

[0028] For example, the predetermined amount of antigens is stored, in particular dissolved, in a preferably predetermined volume of a buffer liquid, in particular buffer solution. In this case, the buffer liquid containing the antigens is provided, and the predetermined volume of body fluid is brought together with it. This brought together volume, or a predetermined volume thereof, is then brought together with the labeled antibodies located on the carrier medium. In principle, the buffer liquid with the antigens it contains, the body fluid, and the labeled antibodies can also be brought together simultaneously.

[0029] This embodiment is also based on the known competitive approach, in which any antibodies against HPV contained in the body fluid compete with the labeled antibodies for binding to the linear epitope of the antigens. The improved method according to this embodiment can also be carried out in just a few steps. The carrier medium used, with its mobile labeled antibodies and the immobile capture molecules, also makes it possible to provide a rapid test in which a change that can be quickly measured and / or perceived by a user occurs in a reaction zone.

[0030] If the body fluid is brought together with the antigens in a prior step, any antibodies against HPV contained in the body fluid will come into close proximity to one another before this combination is brought together with the labelled antibodies. This brings the interactions between the antibodies in the body fluid and the antigens forward in time and thus takes precedence over any interactions between the antigens and the labelled antibodies. This promotes a rapid onset of a measurable and / or user-perceptible change if antibodies against HPV are present in the body fluid.

[0031] In one embodiment, the capture molecules are designed in such a way that they can specifically bind to the linear epitope of the antigens. As a result, those antigens that bind to the capture molecules are retained at the location of the capture molecules on the carrier medium. In this case, the antigens are multimeric, or exist as a multimer.

[0032] In another embodiment, the capture molecules are designed such that they can each specifically bind to a different binding site on the antigen than the linear epitope. In this case, the antigens are monomeric or exist as a monomer. The other binding site can in principle be an epitope. In this case, the capture molecules are designed such that they can each specifically bind to a different epitope on the antigen than the linear epitope. The other epitope can be a linear or a conformational epitope.

[0033] According to a third, alternative embodiment, the improved method can be a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid and in particular comprise or consist of the steps of providing a predetermined amount of the antigens, preferably on or at a carrier medium, such as a microtiter plate, in particular on or at.presented immobilely on the carrier medium, for example fixed; for bringing together a preferably predetermined volume of body fluid with the provided antigens; for bringing together a predetermined amount of the labelled antibodies with the body fluid and the provided antigens; for washing the carrier medium; for applying a predetermined volume of a reactant to the carrier medium; for carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis, preferably of the carrier medium, for example for a reaction.

[0034] Bringing the body fluid together with the antigens and bringing the labelled antibodies together with the body fluid and the antigens can be understood as two, preferably separate, steps or as one step. Bringing the body fluid together with the antigens and bringing the labelled antibodies together with the body fluid and the antigens can, for example, be understood as taking place at separate times. In particular, the body fluid is brought together with the antigens first, followed by the labelled antibodies coming into contact with the body fluid and the antigens. In particular, the body fluid should be incubated with the antigens for a predetermined period of time and / or a freely selectable period of time before the labelled antibodies are brought together with this composition.If the body fluid contains antibodies that bind to the linear epitope of the antigens, interactions between these antibodies and the antigens can occur before the labeled antibodies are added. In principle, the body fluid can first be brought into contact with the antigens, for example, by adding them to the carrier medium, and then, for example, after a predetermined period of time and / or a freely selectable period of time, the carrier medium can be washed. Furthermore, it can be provided that the labeled antibodies are only added after this.

[0035] In the present disclosure, the term "washing" is to be understood in particular as meaning that the substances present on the carrier medium, with the exception of the antibodies specifically bound to the linear epitope of the antigens, are removed from the carrier medium. This removes substances contained in the body fluid, for example, which could undesirably interfere with the interactions of the labelled antibodies with the linear epitope of the antigens.

[0036] In particular, in this embodiment, the labeled antibodies are designed such that, in the presence of a reactant or the reactant, they cause a measurable and / or user-perceivable reaction. The reactant used in the improved method is preferably designed such that, through it, the labeled antibodies cause the measurable and / or user-perceivable reaction. This reaction can be a color reaction, for example, if the labeled antibodies are labeled with an enzyme and the reactant is a reactant of the enzyme.

[0037] This embodiment is also based on the known competitive approach, in which any antibodies against HPV contained in the body fluid compete with the labeled antibodies for binding to the linear epitope of the antigens. This embodiment offers the advantage that the incubation time and / or the temperature during incubation can be freely selected. Thus, in this embodiment, the improved method can be carried out by optimizing the setting of the incubation time and / or the incubation temperature so that only a relatively small amount of antigens is required.

[0038] According to a fourth, alternative embodiment, the improved method can be a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid and in particular comprise or consist of the steps of providing a predetermined amount of the antigens, preferably on or at a carrier medium, in particular presented immobilely on or at the carrier medium, for example fixed; bringing a preferably predetermined volume of body fluid together with the antigens; washing the carrier medium; applying a predetermined amount of labelled antibodies to the carrier medium; washing, in particular again or.washing the carrier medium again; applying a predetermined volume of a reactant to the carrier medium; carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis, preferably of the carrier medium, for example for a reaction.

[0039] In this case, the labelled antibodies are designed in such a way that they can specifically bind to human antibodies that can bind or specifically bind to the linear epitope of the antigens. The labelled antibodies are in particular designed in such a way that they can specifically bind to a binding site on the human antibodies other than a binding site that specifically binds or can be bound to the linear epitope of the antigens. Therefore, if such human antibodies are present in the body fluid and if these human antibodies are bound to the linear epitope of the antigens, binding to these bound human antibodies will occur after the labelled antibodies have been applied.

[0040] In particular, the labeled antibodies are such that, in the presence of a reactant or the reactant, they cause a measurable and / or user-perceivable reaction. The reactant used in the improved method is preferably such that, through it, the labeled antibodies cause the measurable and / or user-perceivable reaction. This reaction may be a color reaction, for example, if the labeled antibodies are labeled with an enzyme and the reactant is a reactant of the enzyme.

[0041] The improved method of this embodiment is based on a known sandwich approach due to the labelled antibodies used here, in which the labelled antibodies do not bind specifically directly to the antigens, but indirectly via the human antibodies. The improved method of this embodiment makes it possible to qualitatively and / or quantitatively determine any antibodies against HPV contained in the body fluid and, in addition, to make a statement about the type of antibody present. For example, it is possible to determine whether the identified antibodies are immunoglobulin A (IgA) or immunoglobulin D (IgD) or immunoglobulin E (IgE) or immunoglobulin G (IgG) or immunoglobulin M (IgM). For this purpose, the labelled antibodies only need to be based on an antibody which specifically binds to a human antibody which presents IgA or IgD or IgE or IgG or IgM.If the human antibodies present , for example , IgA and a measurable and / or user - perceptible reaction occurs , this means that the antibodies detected against HPV are antibodies of the IgA type .

[0042] Important information can be obtained by identifying the antibody type. For example, detected IgA antibodies indicate that a mucous membrane of the person from whom the body fluid was taken is affected. For example, detected IgM antibodies indicate that the infection is new. For example, detected IgD antibodies indicate an infection of the gastrointestinal tract.

[0043] In the improved method it is particularly provided that the predetermined amount of antigens is smaller than the predetermined amount of labelled antibodies or corresponding to the predetermined amount of labelled antibodies. It has been shown that with such a quantitative ratio between the antigens and the labelled antibodies the change or reaction described above and brought about by the labelled antibodies is achieved to a desired extent in order to be able to carry out a qualitative and / or quantitative identification of antibodies against HPV. This is particularly the case if the improved method is based on one of the embodiments described above with a competitive approach. In principle the predetermined amount of antigens can also be greater than the predetermined amount of labelled antibodies.In the improved method, the predetermined amount of antigens and the predetermined amount of labelled antibodies are measured in relation to the volume of body fluid in such a way that, when the amount of antibodies contained in the body fluid and specifically binding to the linear epitope of the antigens is above a predetermined minimum amount, the labelled antibodies cause a measurable and / or user-perceptible reaction in the presence of a reactant or, when spatially concentrated, result in a measurable and / or user-perceptible change. In this way, the sensitivity of the method can be adjusted. Data from verified clinical diagnoses and / or from demonstrably healthy individuals can be used to adjust the predetermined minimum amount.

[0044] In particular, the predetermined minimum amount is set at a value which is above the amount of healthy individuals. The improved method is therefore adjusted in such a way that it does not work in healthy individuals. In particular, the predetermined minimum amount is set at a value which is above the amount of healthy individuals and below the amount of individuals infected with HPV. The improved method is therefore adjusted in such a way that it works in individuals infected with HPV.

[0045] In the present disclosure, the term "HPV-infected" is to be understood in particular as meaning that the affected person has or has had an HPV-related disease and as a result the person's immune system is producing or has produced antibodies against the HPV. In other words, "HPV-infected" is to be understood in particular as meaning that the affected person has antibodies, in particular endogenous antibodies, which are caused by an HPV-related disease.

[0046] In the improved method, the linear epitope of the antigens is particularly designed in such a way that the several or many antibodies which can bind alternatively to it are each effective against HPV of a different type from a class comprising at least two of the types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82. These HPV types form the high-risk group which play a significant role in the development of precancerous lesions (dysplasias) and the carcinomas resulting from them. If the linear epitope covers two of these HPV types, the linear epitope is based on an amino acid sequence which is present in the same way in both of these HPV types. The more of these HPV types are covered by a common amino acid sequence and this amino acid sequence is mapped to the linear epitope, the better the sensitivity of the improved method with regard to detecting precancerous lesions and carcinomas.

[0047] In the improved method, the linear epitope of the antigens is in particular such that the plurality or plurality of antibodies that can alternatively bind to it are effective against the L1 protein of the HPV. Alternatively, the linear epitope of the antigens can be such that the plurality or plurality of antibodies that can alternatively bind to it are effective against the L2 protein of the HPV. Alternatively, the linear epitope of the antigens can be such that the plurality or plurality of antibodies that can alternatively bind to it are effective against the E6 protein of the HPV. Alternatively, the linear epitope of the antigens can be such that the plurality or plurality of antibodies that can alternatively bind to it are effective against the E1 protein of the HPV. Alternatively, the linear epitope of the antigens can be such that the plurality or plurality of antibodies that can alternatively bind to it are each effective against a different one of the HPV proteins from the group L1, L2, E6 and E7.

[0048] The term "body fluid" in the present disclosure is to be understood in particular as meaning the water present in the various compartments of the human body, including the substances dissolved therein. The body fluid can be blood and / or lymph and / or saliva and / or urine and / or sweat. For example, the body fluid is whole blood or serum and / or plasma of whole blood. For example, the body fluid is serum and / or plasma from a blood sample obtained by venipuncture. The body fluid can also be capillary blood or whole blood from the fingertip of a person. In particular, the body fluid is a sample of body fluid.

[0049] The improvements in detection can be achieved with a test kit for detecting antibodies against HPV, comprising antigens with a linear epitope, in particular at least one linear epitope or exclusively one linear epitope, which is preferably reactive and is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it. In particular, the antigens are the antigens described above in relation to the improved method. The test kit enables the improved method to be carried out. In this respect, the advantages of the improved method can be achieved by means of the test kit.

[0050] Furthermore, due to the special antigens used, the improved test kit offers the advantage that no refrigeration is required for storage. The test kit can be stored, for example, at room temperature without any loss of effectiveness, or at least without any significant loss of effectiveness. The improved test kit also offers the advantage that it does not require a physiologically active liquid in which the antigens are stored to maintain their reactivity. The antigens remain reactive even when they are present, for example, in solid form and / or in a dry state and / or in a non-physiologically active liquid.

[0051] In particular, the improved test kit comprises labelled antibodies which are designed in such a way that they can specifically bind to the linear epitope of the antigens. In particular, the labelled antibodies are designed in such a way that in the presence of a reactant they cause, for example, a measurable and / or user-perceptible reaction or, in the case of spatial accumulation, in particular spatial accumulation of bound labelled antibodies, they result in, for example, a measurable and / or user-perceptible change. This enables the identification of antibodies against HPV contained in body fluid. In particular, the labelled antibodies are the labelled antibodies described above in relation to the improved method.

[0052] In one embodiment, the improved test kit comprises a carrier medium on which a predetermined amount of the labelled antibodies is provided in a mobile manner and a predetermined amount of the antigens is present at a predetermined location, in particular is provided in an immobile manner, for example is fixed. This enables the improved method according to the first embodiment described above to be carried out. In particular, the labelled antibodies are arranged at a distance from the antigens. In particular, the labelled antibodies are arranged in the region of an application site for the body fluid. In particular, the labelled antibodies are designed in such a way that, for example in the case of binding, i.e. binding to the linear epitope of the antigens, they result in a change that is measurable and / or perceptible by a user if there is a spatial accumulation, i.e. a spatial accumulation of labelled antibodies.

[0053] The improved test kit of this embodiment can be reduced to the carrier medium as its sole component. The antigens are already stored on the carrier medium. This is possible because the antigens do not require a physiologically active liquid to maintain their reactivity. For example, the antigens are present in dried form, for example, dried on the carrier medium. In principle, the labeled antibodies can also be present in dried form, for example, dried on the carrier medium.

[0054] In another embodiment, the improved test kit comprises a predetermined amount of the antigens, for example in a container, for example dissolved in a buffer liquid, in particular buffer solution. Furthermore, the improved test kit comprises a carrier medium on which a predetermined amount of the labeled antibodies is provided in a mobile manner and a predetermined amount of capture molecules is present at a predetermined location, in particular is provided immobilely, for example is fixed. This enables implementation of the improved method according to the second embodiment described above.

[0055] In particular, the labelled antibodies are arranged at a distance from the capture molecules. In particular, the labelled antibodies are arranged in the region of an application site for the body fluid. In particular, the labelled antibodies are designed in such a way that, for example, in the event of binding, i.e. binding to the linear epitope of the antigens, they result in a measurable and / or user-perceptible change if there is a spatial clustering, i.e. a spatial clustering of labelled antibodies. If the antigens are multimeric, i.e. present as a multimer, the capture molecules can be designed in such a way that they can each bind specifically to the linear epitope of the antigens. In this case, implementation of the improved method according to the first embodiment of the second embodiment described above is possible.For example, the capture molecules are based on the same antibodies that also serve as the basis for the labeled antibodies. This saves development costs because only a single antibody needs to be developed.

[0056] Alternatively, the antigens can be monomeric. This results in cost advantages because the antigens used are relatively inexpensive as monomers. If the antigens are monomeric, i.e. present as monomers, the capture molecules can be designed such that they can each bind specifically to a different binding site on the antigens than the linear epitope. In this case, it is possible to carry out the improved method according to the second embodiment of the second embodiment described above. The other binding site can be an epitope. In this case, the capture molecules are designed, for example, such that they can each bind specifically to a different epitope on the antigens than the linear epitope. The other epitope can be a linear or a conformational epitope.

[0057] The aforementioned buffer liquid can be a saline-based solution. Alternatively, the buffer liquid can be a TRIS-BSA (tris(hydroxymethyl)aminomethane with bovine serum albumin) or a histidine buffer.

[0058] The carrier medium of the above embodiments of the improved test kit can be a test strip. For example, the test strip comprises or consists of a material that exerts a capillary force on a liquid applied thereto. For example, the test strip comprises or consists of an absorbent paper material. The test strip can be present in a test cassette.

[0059] In yet another embodiment, the improved test kit comprises a predetermined amount of the antigens, preferably presented on a carrier medium, in particular immobile on the carrier medium, for example fixed. The improved test kit further comprises a predetermined amount of the labelled antibodies. The labelled antibodies are in particular such that they cause a reaction that is measurable and / or perceptible by a user in the presence of a reactant. In addition, the improved test kit comprises a predetermined volume of a reactant that is such that the labelled antibodies cause the reaction that is measurable and / or perceptible by a user.

[0060] This embodiment enables implementation of the improved method according to the third embodiment described above. This embodiment allows the detection limit for identifying any antibodies against HPV contained in the body fluid to be modified, for example, reduced, by adjusting the incubation time and / or the incubation temperature.

[0061] In this case, the carrier medium can be a test tube, for example a microtiter plate. The antigens can be present therein, for example on the bottom and / or on the walls. The antigens are preferably fixed in the test tube, in particular on the bottom and / or on the walls. For example, the bottom and / or the walls of the test tube are coated with the antigens.

[0062] In a modification of the above embodiment, the improved test kit now comprises, instead of labeled antibodies that bind to the linear epitope of the antigens, labeled antibodies that can specifically bind to human antibodies, wherein these human antibodies are designed such that they bind or can specifically bind to the linear epitope of the antigens. The labeled antibodies used here are, in particular, designed such that, in the presence of a reactant, they cause a measurable and / or user-perceptible reaction.

[0063] This modification enables implementation of the improved method according to the fourth embodiment described above. In this respect, this modification enables any antibodies against HPV present in body fluid to be determined qualitatively and / or quantitatively and, moreover, to make a statement about the type of antibody present. The labeled antibodies mentioned above can be based on monoclonal antibodies, for example monoclonal mouse antibodies, which are labeled. These monoclonal antibodies can be obtained from a hybridoma cell line. This has the advantage that the antibodies obtained therefrom show no or at least no relevant variation from one another. The term “hybridoma cell line” is understood to mean, in particular, a cell line which is obtained using hybridoma technology.

[0064] If the labelled antibodies are intended to produce a measurable and / or user-perceptible change when spatially clustered, these antibodies are labelled with colloidal gold and / or colloidal silver, for example.

[0065] If the labeled antibodies are intended to produce a measurable and / or user-detectable reaction in the presence of a reactant, these antibodies are labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The reactant used can be tetramethylbenzidine (TMB) or Fast-Red, so that the resulting reaction is a color change.

[0066] If the reaction or change caused is to be measured, the improved test kit may include a photometer or other photo-optical device.

[0067] According to one aspect, the use of an antigen in the improved method and / or the improved test kit is proposed. The antigen comprises a linear epitope, in particular at least one linear epitope or exclusively one linear epitope, which is such that several or many antibodies, each effective against a different HPV type, can alternatively bind specifically to it.

[0068] According to a further aspect, an antibody or labeled antibody is proposed for use in a diagnostic method, in particular in a method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid. The antibody or labeled antibody is designed such that it can specifically bind to a linear epitope of an antigen. The linear epitope of the antigen is designed such that several or many antibodies, each effective against a different type of HPV, can alternatively bind specifically to it.

[0069] In particular, the labelled antibody is such that, in the presence of a reactant, it causes a reaction that is measurable and / or perceptible by a user or, in the case of spatial accumulation, in particular spatial accumulation of bound labelled antibodies of this type, it causes a change that is measurable and / or perceptible by a user.

[0070] According to a further aspect, the use of a labeled antibody in the improved method and / or in at least one of the above-described embodiments of the improved test kit is proposed. The labeled antibody is designed such that it can specifically bind to a linear epitope of an antigen. The linear epitope of the antigen is designed such that several or many antibodies, each effective against a different HPV type, can alternatively specifically bind to it.

[0071] In particular, the labelled antibody is such that, in the presence of a reactant, it causes a reaction that is measurable and / or perceptible by a user or, in the case of spatial accumulation, in particular spatial accumulation of bound labelled antibodies of this type, it causes a change that is measurable and / or perceptible by a user.

[0072] According to a further aspect, the use of an antibody as a capture molecule is proposed in the improved method and / or in at least one of the above-described embodiments of the improved test kit. The antibody specifically binds to a linear epitope of an antigen, wherein the linear epitope is such that several or many antibodies, each effective against a different HPV type, can alternatively bind specifically to it.

[0073] According to a further aspect, the use of a molecule as a capture molecule is proposed in the improved method and / or in at least one of the above-described embodiments of the improved test kit. The molecule specifically binds to a binding site of an antigen other than a linear epitope, which is such that several or many antibodies, each effective against a different type of HPV, can alternatively bind specifically to it. In particular, the molecule is an antibody which is such that it can specifically bind to an epitope of the antigen other than the linear epitope.

[0074] Short description of the characters

[0075] Further details and features will become apparent from the following description of several embodiments based on the drawings.

[0076] Fig. 1 shows a first exemplary embodiment of a test kit for detecting antibodies against HPV in a schematic representation,

[0077] Fig. 2 shows a second exemplary embodiment of a

[0078] Test kits for the detection of antibodies against HPV in a schematic representation,

[0079] Fig . 3 a third exemplary embodiment of a

[0080] Test kits for the detection of antibodies against HPV in a schematic representation,

[0081] Fig. 4 shows a fourth exemplary embodiment of a

[0082] Test kits for the detection of antibodies against HPV in a schematic representation, and

[0083] Fig . 5 a fifth exemplary embodiment of a

[0084] Test kits for the detection of antibodies against HPV in a schematic representation.

[0085] Examples of implementation

[0086] Figure 1 shows a first exemplary embodiment of a test kit 1 for detecting antibodies against HPV. The exemplary test kit 1 comprises a carrier medium 2 on which a predetermined amount of antigens 10 and a predetermined amount of labeled antibodies 20 are present. Preferably, the predetermined amount of antigens 10 is arranged at a predetermined location 3 of the carrier medium 2. For example, the predetermined location 3 is designed as a test line. Preferably, the antigens 10 are immobilized, in particular fixed, at the predetermined location 3. Preferably, the labeled antibodies 20 are presented in a mobile manner and the predetermined amount of the labeled antibodies 20 is located at a spatial distance from the predetermined location 3. Preferably, the labeled antibodies 20 are arranged in the region of an application location 4. The application location 4 indicates a location on the carrier medium 2 which is intended to receive a liquid to be examined.

[0087] For example, the carrier medium 2 is a test strip. For example, the test strip is contained in a test cassette. For example, the test cassette has a test opening in the area of ​​the specified location 3. For example, the test cassette has an application opening in the area of ​​the application location 4, through which the liquid to be tested can be applied to the test strip.

[0088] For example, the carrier medium 2 comprises or consists of a material that exerts a capillary force on a liquid. A liquid applied to the carrier medium 2 in the area of ​​the application point 4 is thereby assisted in its flow toward the predetermined point 4. For example, the material is absorbent paper or the like.

[0089] In the exemplary test kit 1, the antigens 10 have a linear epitope. This linear epitope is reactive and such that several or many antibodies, each effective against a different HPV type, can bind specifically to it. The antigens 10 can be present as a monomer or multimer. Preferably, the antigens 10 are present on the carrier medium 2 in dry form and / or the antigens 10 are presented in dried form on the carrier medium 2.

[0090] Corresponding to the antigens 10, the labeled antibodies 20 are designed in such a way that they can specifically bind to the linear epitope of the antigens 10. The labeled antibodies 20 also have a label, specifically such that a spatial accumulation of the labeled antibodies 20 results in a measurable and / or user-perceptible change. For example, the labeled antibodies 20 are monoclonal mouse antibodies provided with such a label. For example, the labeled antibodies are labeled with colloidal gold and / or colloidal silver.

[0091] Preferably, the predetermined amount of antigens 10 is smaller than the predetermined amount of labeled antibodies 20. In principle, the predetermined amount of antigens 10 can also correspond to the predetermined amount of labeled antibodies 20 or be greater than the predetermined amount of labeled antibodies 20. Furthermore, the predetermined amount of antigens 10 and the predetermined amount of labeled antibodies 20 are preferably dimensioned in relation to the volume of a liquid sample to be examined such that, when the amount of antibodies contained in the liquid sample and specifically binding to the linear epitope of the antigens 10 is above a predetermined minimum amount, the labeled antibodies 20, when spatially concentrated, result in a measurable and / or user-perceptible change.

[0092] The exemplary test kit 1 enables the qualitative and / or quantitative determination of HPV antibodies present in body fluids using serological methods. The antigens 10 used, with their specific linear epitope, allow a single serological test to determine the presence of precancerous lesions and carcinomas caused by an entire group of HPV types.

[0093] To carry out the serological test, a sample of body fluid from a person must be provided. The body fluid is preferably blood, such as whole blood, or serum and / or plasma from blood. The blood can be obtained by venipuncture or whole blood from a fingertip. This body fluid is applied to the carrier medium 2 via the application site 4. There, the body fluid comes into contact with the labelled antibodies 20 and, together with at least some of the labelled antibodies 20, optionally by utilising a capillary force determined by the material of the carrier medium 2, runs via the carrier medium 2 in the direction of the predetermined site 3 and comes into contact with the antigens 10.Due to the contact, interactions occur between the antigens 10 on the one hand and the labelled antibodies 20 and any antibodies contained in the body fluid on the other hand, whereby the labelled antibodies 20 and the antibodies contained in the body fluid compete with each other for binding to the linear epitope of the antigens 10.

[0094] The more of the labelled antibodies 20 bind to the antigens 10, the more pronounced a measurable and / or user-perceptible change occurs on the carrier medium 2. This change is caused by a local accumulation of the labelled antibodies 20, which results from their binding to the antigens 10 and are thus held by the antigens 10 at the location of the accumulation. The location of the accumulation can be the area of ​​the predetermined location 3. For example, the accumulation has a linear extension, so that the change occurs along a line, as is indicated by way of example in Figure 1 using the predetermined location 3, which is shown as a dashed line.

[0095] Conversely, with fewer labelled antibodies 20 binding to antigens 10, the change on carrier medium 2 is weaker. This effect is used to make a statement about any antibodies against HPV contained in the body fluid. This is because a weaker change on carrier medium 2 means that the body fluid contains more antibodies that can bind to the linear epitope of antigens 10, and these antibodies are now bound to antigens 10 instead of the labelled antibodies 20.

[0096] Preferably, the exemplary test kit 1 is adjusted or calibrated by measuring the amount of antigens 10 applied to the carrier medium 2 and / or by measuring the amount of labeled antibodies 20 applied to the carrier medium 2 such that the measurable and / or user-perceivable change has or exceeds a preferably predetermined maximum strength when the body fluid contains an amount of antibodies that specifically bind to the linear epitope of the antigens 10 that is below the minimum amount described above. Thus, the exemplary test kit 1 enables a qualitative determination of antibodies against HPV present in the body fluid, i.e., whether such antibodies are present in the body fluid or not.

[0097] Given that with increasing amounts of antibodies contained in the body fluid that specifically bind to the specific epitope of antigen 10, the measurable and / or user-perceptible change from the maximum strength decreases, the exemplary test kit 1 also enables a quantitative determination of antibodies against HPV contained in the body fluid. In this case, the degree of attenuation of the change compared to the maximum strength can be used as a measure of the amount of antibodies against HPV contained in the body fluid.

[0098] The occurring change, particularly its severity, can be perceived by a user, who then, based on their perception, performs a qualitative and / or quantitative identification for antibodies against HPV. Additionally or alternatively, a photometer or other auxiliary device that detects the severity of the change can be used to perform a qualitative and / or quantitative identification for antibodies against HPV.

[0099] Figure 2 shows a second exemplary embodiment of a test kit 1.1 for detecting antibodies against HPV. Components or functional sections of the exemplary test kit 1.1 that are identical or functionally equivalent to components or functional sections of the exemplary test kit 1 of Figure 1 are provided with the same reference numerals; in this respect, reference is made to the description of the exemplary test kit 1 of Figure 1.

[0100] In this exemplary test kit 1 . 1 , a carrier medium 2 ' is provided, for example in the manner of the carrier medium 2 of the exemplary test kit 1 of Figure 1 , and in this carrier medium 2 ' the labeled antibodies 20 are also presented in a mobile manner, for example in the area of ​​the application site 4 .

[0101] In contrast to the exemplary test kit 1 in Figure 1, in the exemplary test kit 1.1 in Figure 2, a predetermined amount of capture molecules 30 is provided on the carrier medium 2'. Preferably, the predetermined amount of capture molecules 30 is arranged at the predetermined location 3 of the carrier medium 2'. Preferably, the capture molecules 30 are immobilized, in particular fixed, at the predetermined location 3. Preferably, the capture molecules 30 are designed such that they can specifically bind to the linear epitope of the antigens 10'. For example, the capture molecules 30 are based on the same antibody that also forms the basis of the labeled antibodies 20.

[0102] In contrast to the exemplary test kit 1 in Figure 1, the exemplary test kit 1.1 in Figure 2 provides a predetermined amount of antigens 10' which are present separately from the carrier medium 2'. The antigens 10' have, like the antigens 10 of the exemplary test kit 1 in Figure 1, the said linear epitope which is designed in such a way that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it. The exemplary test kit 1.1 in Figure 2 is designed so that the antigens 10' are present as a multimer. The said linear epitope is therefore present at least twice in the respective antigen 10'.

[0103] For example, the antigens 10' are dissolved in a preferably predetermined volume of a buffer liquid 40. The buffer liquid 40 serves to keep the antigens 10' in solution and to be able to utilize any capillary property of the carrier medium 2'. The buffer liquid 40 does not need to have a physiological effect on the antigens 10', since the reactivity of the linear epitope of the antigens 10' is retained even with a non-physiologically acting buffer liquid. For example, the buffer liquid is a saline-, Tris-, or histidine-based solution. For example, the buffer liquid 40 with the antigens 10' is accommodated in a preferably separate container 5. For example, the container 5 has an opening for filling and / or emptying.

[0104] The exemplary test kit 1 . 1 can be used as follows: Body fluid, in particular a predetermined volume of body fluid, is brought together with antigens 10 ', in particular the buffer liquid 40 with the antigens 10 ' dissolved therein. For example, the body fluid is introduced into the container 5 for this purpose. For example, the body fluid is the body fluid described above for the exemplary test kit 1 in Figure 1.

[0105] Subsequently, preferably after a predetermined exposure time of the body fluid with the antigens 10', the mixture with the body fluid and the antigens 10', in particular the mixture with the body fluid, the buffer solution 40 and the antigens 10', or at least a volume part of the mixture is applied to the carrier medium 2' via the application point 4. There, the mixture, in particular the body fluid, comes into contact with the labelled antibodies 20 and runs together with at least a portion of the labelled antibodies 20, optionally by utilising a capillary force caused by the material of the carrier medium 2', via the carrier medium 2' in the direction of the predetermined point 3 and comes into contact with the capture molecules 30.Due to the contact, the capture bodies 30 bind to those of the linear epitopes of the antigens 10 ' which are still free, in particular not yet occupied by one of the labelled antibodies 20 or an antibody contained in the body fluid.

[0106] In principle, the antigens 10 ' , in particular the buffer liquid with the antigens 10 ' dissolved therein, and separately the body fluid , can be applied, for example, simultaneously to the carrier medium 2 ', i.e. brought together with the labeled antibodies 20 . However, if the antigens 10 ' are brought together first with the body fluid and later with the labeled antibodies, the interactions between the antigens 10 ' and any antibodies contained in the body fluid that bind to the linear epitope of the antigens 10 ' are shifted forward in time . These interactions therefore have a temporal head start over any interactions between the antigens 10 ' and the labeled antibodies 20 .

[0107] Preferably, the exemplary test kit 1 . 1 is adjusted or calibrated by measuring the amount of labeled antibodies 20 placed on the carrier medium 2 ' and / or measuring the amount of capture molecules 30 placed on the carrier medium 2 ' such that after bringing the antigens 10 ' together with the labeled antibodies 20 without the assistance of the body fluid, but with the assistance of a liquid, for example the buffer liquid, and after this mixture has been brought together and flows towards the capture bodies 30, on the one hand the labeled antibodies 20 bind specifically to the antigens 10 ' and on the other hand there are still sufficient antigens present from these bound antigens which still have at least one of the linear epitopes free, so that the capture molecules bind specifically to it.Only by binding to the capture molecules 30 are the labelled antibodies 20 retained on the carrier medium 2' and a local accumulation of the labelled antibodies 20 occurs and thus a measurable and / or user-perceptible change on the carrier medium 2'.

[0108] If the antigens 10 ' and the body fluid with the labeled antibodies 20 are now brought together, at least one of the linear epitopes of at least some of the antigens 10 ' is occupied by any antibodies contained in the body fluid and bindable to the linear epitope of the antigens 10 ', so that a smaller number of the antigens 10 ' have both a binding with the labeled antibodies 20 and also a binding with the capture molecules 30 and consequently the measurable and / or user-perceivable change is weaker.

[0109] Preferably, the exemplary test kit 1 . 1 is adjusted or measured by measuring the amount of antigens 10 ' compared to the amount of labeled antibodies 20 and / or the amount of capture molecules such that the measurable and / or user-perceptible change has or exceeds a preferably predetermined maximum strength when the body fluid contains an amount of antibodies that specifically bind to the linear epitope of the antigens 10 ' below the minimum amount described above. The exemplary test kit 1 . 1 thus enables a qualitative and / or quantitative determination of antibodies against HPV contained in the body fluid in the same way as the exemplary test kit 1 in Figure 1. In this respect, with regard to the analysis of the change occurring due to the accumulation of labeled antibodies, reference is made to the description of the exemplary test kit 1 in Figure 1.

[0110] Figure 3 shows a third exemplary embodiment of a test kit 1.2 for detecting antibodies against HPV. Components or functional sections of the exemplary test kit 1.2 which are identical or functionally equivalent to components or functional sections of the exemplary test kit 1 of Figure 1 or the test kit 1.1 of Figure 2 are provided with the same reference numerals; in this respect, reference is made to the description of the exemplary test kit 1 of Figure 1 or the exemplary test kit 1.1 of Figure 2. The exemplary test kit 1.2 differs from the exemplary test kit 1.1 of Figure 2 in that a predetermined amount of antigens 10" is provided, which are present as monomers and therefore each have the said linear epitope only once. The antigens 10'' can be used instead of the antigens 10' of the exemplary test kit 1.1 of Figure 2, dissolved in the buffer liquid 40 and / or contained in the container 5.

[0111] The exemplary test kit 1.2 differs from the exemplary test kit 1.1 of Figure 2 further in that a carrier medium 2'' is used, on which a predetermined amount of capture molecules 50 in the form of antibodies are provided, which are designed such that they each specifically bind to a different epitope of the antigens 10'' than the said linear epitope. The capture molecules 50 are preferably arranged in the region of the predetermined location 3 of the carrier medium 2'''. The capture molecules 50 are preferably arranged at the predetermined location 3 of the carrier medium 2'''. The capture molecules 50 are preferably immobilized, in particular fixed, at the predetermined location 3.

[0112] A serological test can be carried out with the exemplary test kit 1.2 in the same way as with the exemplary test kit 1.1 of Figure 2. For example, a preferably predetermined volume of the body fluid is first brought together with the antigens 10'', in particular the antigens 10'' dissolved in the buffer solution 40, and in particular incubated. Thereafter, the body fluid and the antigens 10'', in particular the body fluid, the antigens 10'', and the buffer liquid 40, are brought together with the labeled antibodies 20 located on the carrier medium 2'', and this mixture then flows on the carrier medium 2'' toward the predetermined location 3 and comes into contact with the capture molecules 50.

[0113] Those of the antigens 10" that have formed a binding complex with one of the labeled antibodies 20 will be captured by at least one of the capture molecules 50, provided that the epitope required for binding on the antigens of the binding complex is still unoccupied and the at least one capture molecule specifically binds thereto. The subsequent analysis of the carrier medium 2" for a measurable and / or user-perceptible change caused by the accumulation of the captured labeled antibodies 20 can be carried out according to the procedure described above for the exemplary test kit 1 or the exemplary test kit 1.1.

[0114] Figure 4 shows a fourth exemplary embodiment of a test kit 1 . 3 for detecting antibodies against HPV . The exemplary test kit 1 . 3 comprises a carrier medium 2 ' ' ' on which a predetermined amount of the antigens 10 already described above is present. The carrier medium 2 ' ' ' is preferably a reagent vessel. For example, the reagent vessel is a component of a microtiter plate. The antigens 10 are preferably fixed to the bottom and / or to the walls of the reagent vessel. For example, the bottom and / or the walls of the reagent vessel are coated with the antigens 10 .

[0115] Furthermore, the exemplary test kit 1 . 3 comprises a predetermined amount of labeled antibodies 60 and a predetermined volume of a reactant 70 . The labeled antibodies 60 are preferably present separately from the carrier medium 2 ' ' '. The reactant 70 is preferably present separately from the carrier medium 2 ' ' ' and the labeled antibodies 60 .

[0116] The labelled antibodies 60 are based on antibodies such as those already used as the basis for the labelled antibodies 20 of the exemplary test kits 1, 1.1 and 1.2, which are therefore designed such that they can specifically bind to the linear epitope of the antigens 10. With regard to the labelling, the labelled antibodies 60 are designed such that, in the presence of a reactant, they cause a reaction that is measurable and / or perceptible to a user. The provided reactant 70 is designed such that it causes this reaction. For example, the labelled antibodies 60 are labelled with an enzyme such as horseradish peroxidase (HRP). For example, the reactant is tetramethylbenzidine (TMB).

[0117] A serological test can be carried out using the exemplary test kit 1 . 3 as follows: A preferably predetermined volume of body fluid is brought together with the antigens 10 of the carrier medium 2 ' ' ' and, in particular, incubated. For example, the body fluid is poured into the test tube for this purpose. The body fluid can be the body fluid described above.

[0118] The carrier medium 2 ' ' ' is then washed. Any antibodies contained in the body fluid that bind to the linear epitope of the antigens 10 remain on the carrier medium 2 ' ' ', particularly in the test tube, provided that specific binding to the antigens 10 has taken place and these antibodies are thereby retained.

[0119] After washing, the labeled antibodies 70 are applied to the carrier medium 2 '' '' , for example, filled into the reagent vessel, and in particular incubated. More or less interactions occur between the antigens 10 and the labeled antibodies 70, depending on how many of the antigens 10 still allow specific binding because the said linear epitope or at least one of the said linear epitopes is not yet occupied by an antibody contained in the body fluid.

[0120] The carrier medium 2' ' ' is then washed again. Those of the labelled antibodies 70 which were able to specifically bind to the antigens 10 remain on the carrier medium 2' ' ', in particular in the reagent vessel. Finally, the reactant 70 is applied to the carrier medium 2' ' ', in particular poured into the reagent vessel. As a result of the reactant 70, those of the labelled antibodies 60 which have remained on the carrier medium 2' ' ', in particular in the reagent vessel, will now show a reaction which can be measured and / or perceived by a user, such as a colour reaction.

[0121] The more of the labelled antibodies 60 that are able to bind to the antigens 10, the stronger the reaction that can be measured and / or perceived by a user. Conversely, with fewer labelled antibodies 60 bound to the antigens 10, the weaker the reaction. This effect is used to make a statement about any antibodies against HPV contained in the body fluid. A weaker reaction means that there are more antibodies in the body fluid that were able to bind to the linear epitope of the antigens 10, and that these antibodies are bound to the antigens 10 instead of the labelled antibodies.The subsequent analysis of the reaction with a view to a qualitative and / or quantitative determination of any antibodies against HPV present in the body fluid can in principle be carried out in the same way as in the serological tests described above, which are based on a measurable and / or user-perceptible change caused by the accumulation of the labelled antibodies 20 .

[0122] Figure 5 shows a fifth exemplary embodiment of a test kit 1.4 for detecting antibodies against HPV. The exemplary test kit 1.4 differs from the exemplary test kit 1.3 of Figure 4 in that a predetermined amount of labeled antibodies 80 is provided, which are such that they can specifically bind to human antibodies. For example, the labeled antibodies 80 do not have the ability to specifically bind to the linear epitope of the antigens 10. The labeled antibodies 80 can be based on a monoclonal mouse antibody. The labeled antibodies 80 can be labeled in the same way as the labeled antibodies 70 of the exemplary test kit 1.3, so that in the presence of a reactant they cause a reaction that is measurable and / or perceptible by a user.

[0123] A serological test can be carried out with the exemplary test kit 1 . 4 in the same way as with the exemplary test kit 1 . 3 of Figure 4; in this respect, reference is made to the above description. Due to the labeled antibodies 80, the exemplary test kit 1 . 4 is based on a sandwich approach. This has the result that the labeled antibodies 80 specifically bind to antibodies contained in the body fluid, which are themselves specifically bound to the said linear epitope of the antigens 10 and are thus retained on the carrier medium 2 '' '' , in particular in the reagent vessel.

[0124] The more of the labelled antibodies 80 bind to antibodies originating from the body fluid, the stronger the reaction that can be measured and / or perceived by a user. This means that the stronger the reaction, the more antibodies that specifically bind to the linear epitope of the antigens are contained in the body fluid. This effect is used here to make a statement about any antibodies against HPV contained in the body fluid. The analysis of the reaction with a view to a qualitative and / or quantitative determination of any antibodies against HPV contained in the body fluid can basically be carried out in the same way as the serological tests described above, which are based on a measurable and / or user-perceptible change brought about by an accumulation of the labelled antibodies 20.

[0125] Investigation results

[0126] The performance of the serological test described above is described below using the example of an examination of the body fluid of 138 women who were diagnosed with HPV-induced precancerous lesions or carcinomas. A sample of whole blood was used as the body fluid. The serological tests were carried out with test kits corresponding to the exemplary test kit 1 in Figure 2. The linear epitope of the antigens used was such that the several or many antibodies against HPV that can alternatively bind to it are each effective against HPV of a different one of the types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82. This covered the entire high-risk group.

[0127] The results of the serological tests are summarized in Table 1. Under the column labeled "Diagnosis," a distinction is made between cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CxCa). Cervical intraepithelial neoplasia is classified into three grades of severity: grade 1 ("CIN 1") indicates only mild changes in the affected body cells, grade 2 ("CIN 2") indicates moderate changes in the affected body cells, and grade 3 ("CIN 3") indicates severe changes in the affected body cells.

[0128] The number of women diagnosed with grade 1 cervical intraepithelial neoplasia, grade 2 cervical intraepithelial neoplasia, grade 3 cervical intraepithelial neoplasia, or cervical carcinoma is shown in the "Number" column. Table 1

[0129] A qualitative determination was made during the serological tests. The threshold for a positive result was set at 750 ng / ml for HPV antibodies in the body fluid, to which the test kit was adjusted. Serological test results above 750 ng / ml were considered positive, and results below or equal to 750 ng / ml were considered negative.

[0130] The number of test results rated as positive is given under the "positive" column and the number of test results rated as negative is given under the "negative" column. The detection rates are given under the "sensitivity" column. As can be seen from Table 1, women diagnosed with "CIN 1" were detected 100% of the time. Women diagnosed with "CIN 2" were also detected 100% of the time. A slightly lower detection rate was found for women diagnosed with "CIN 3" which was 86.6%. For diagnosed cervical carcinomas a detection rate of 82.4% was achieved. The overall detection rate was 89.9%.

[0131] List of reference symbols

[0132] 1 test kit

[0133] 1.1 Test kit

[0134] 1.2 Test kit

[0135] 1.3 Test kit

[0136] 1.4 Test kit

[0137] 2 Carrier medium

[0138] 2 ' carrier medium

[0139] 2 ' ' carrier medium

[0140] 2 ' ' ' carrier medium

[0141] 3 specified position

[0142] 4 Order office

[0143] 5 containers

[0144] 10 antigens

[0145] 10' antigens, multimeric

[0146] 10' ' antigens, monomeric

[0147] 20 labeled antibodies

[0148] 30 capture molecules

[0149] 40 buffer fluid

[0150] 50 capture molecules

[0151] 60 labeled antibodies

[0152] 70 reactant

[0153] 80 labeled antibodies

Claims

Patent claims 1. Serological method for the detection of antibodies against HPV, which uses antigens with a linear epitope that is reactive and such that several or many antibodies, each effective against a different type of HPV, can bind to it alternatively.

2. Serological method according to claim 1, in which labelled antibodies are used which are such that they can specifically bind to the linear epitope of the antigens and, in the presence of a reactant, cause a measurable and / or user-perceptible reaction or, in the case of spatial accumulation, cause a measurable and / or user-perceptible change.

3. A serological method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid, comprising or consisting of the steps of: i) providing a carrier medium (2) on which a predetermined amount of labelled antibodies (20) is mobile and a predetermined amount of antigens (10) is present at a predetermined location (3), wherein the antigens (10) have a linear epitope which is reactive and is such that several or many antibodies, each effective against a different type of HPV, can alternatively bind thereto specifically, and wherein the labelled antibodies (20) are such that they can specifically bind to the linear epitope of the antigens (10) and, when spatially concentrated, bring about a measurable and / or user-perceptible change; ii) bringing a preferably predetermined volume of body fluid together with the labelled antibodies (20);iii ) carrying out a qualitative and / or quantitative identification for antibodies against HPV by analyzing the carrier medium (2) for a change in the region of the predetermined site (3); 4. Serological method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid, comprising the steps or consisting of the steps: 36 i) Providing a predetermined amount of antigens (10'), wherein the antigens (10') are multimeric and have a linear epitope which is reactive and is such that several or many antibodies, each effective against a different type of HPV, can bind specifically thereto; ii) Providing a carrier medium (2') on which a predetermined amount of labelled antibodies (20) is mobile and a predetermined amount of capture molecules (30) is present at a predetermined location (3), wherein the labelled antibodies (20) and the capture molecules (30) are such that they can each bind specifically to the linear epitope of the antigens (10'), and the labelled antibodies (20) bring about a measurable and / or user-perceptible change when spatially clustered; iii) Bringing together a preferably predetermined volume of body fluid with the antigens (10');iv) bringing the body fluid and the antigens (10') together with the labelled antibodies (20); v) carrying out a qualitative and / or quantitative identification for antibodies against HPV by analyzing the carrier medium (2') for a change in the region of the predetermined site (3); 5. Serological method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid, comprising the steps or consisting of the steps: i) providing a predetermined amount of antigens (10"), wherein the antigens (10") are monomeric and have a linear epitope which is reactive and is such that several or many antibodies, each effective against a different type of HPV, can bind specifically thereto; ii) providing a carrier medium (1") on which a predetermined amount of labeled antibodies (20) is mobile and a predetermined amount of capture molecules (50) is present at a predetermined location (3), wherein the labeled antibodies (20) are such that they can each bind specifically to the linear epitope of the antigens (10") and the labeled antibodies (20) 37 when spatially clustered, cause a change that is measurable and / or perceptible by a user, wherein the capture molecules (50) are designed such that they can each specifically bind to a different binding site of the antigens (10'') than the linear epitope; iii) bringing a preferably predetermined volume of body fluid together with the antigens (10''); iv) bringing the body fluid and the antigens (10'') together with the labelled antibodies (20); v) carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis of the carrier medium (2'') for a change in the region of the predetermined site (3).

6. Serological method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid, comprising the steps or consisting of the steps: i) providing a predetermined amount of antigens (10) on a carrier medium (2' ' '), wherein the antigens (10) have a linear epitope which is reactive and is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind thereto specifically; ii) bringing a preferably predetermined volume of body fluid together with the antigens (10);iii) bringing into contact a predetermined amount of labelled antibodies (60) with the body fluid and the antigens (10), wherein the labelled antibodies (60) are such that they can specifically bind to the linear epitope of the antigens (10) and, in the presence of a reactant (70), bring about a reaction that is measurable and / or perceptible by a user; iv) washing the carrier medium (2 '' '); v) applying a predetermined volume of a reactant (70) to the carrier medium (2 '' '), wherein the reactant (70) is such that the labelled antibodies (60) bring about the reaction that is measurable and / or perceptible by a user; vi) carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis of the carrier medium (2 '' ') for a reaction.

7. Serological method for the qualitative and / or quantitative determination of antibodies against HPV contained in body fluid, comprising the steps or consisting of the steps: i) providing a predetermined amount of antigens (10) on a carrier medium (2'''), wherein the antigens (10) have a linear epitope which is reactive and is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind thereto specifically; ii) bringing a preferably predetermined volume of body fluid together with the antigens (10); iii) washing the carrier medium (2''');iv) applying a predetermined amount of labelled antibodies (80) to the carrier medium (2 '' '), wherein the labelled antibodies (80) are such that they can specifically bind to human antibodies which can specifically bind to the linear epitope of the antigens (10) and, in the presence of a reactant (70), bring about a reaction which is measurable and / or perceptible by a user; v) washing the carrier medium (2 '' '); vi) applying a predetermined volume of a reactant (70) to the carrier medium (2 '' '), wherein the reactant (70) is such that the labelled antibodies (80) bring about the reaction which is measurable and / or perceptible by a user; vii) carrying out a qualitative and / or quantitative identification for antibodies against HPV based on an analysis of the carrier medium (2 '' ') for a reaction.

8. Serological method according to one of claims 3 to 7, wherein the predetermined amount of antigens is less than the predetermined amount of labeled antibodies or corresponding to the predetermined amount of labeled antibodies.

9. Serological method according to one of claims 3 to 8, wherein the predetermined amount of antigens and the predetermined amount of labelled antibodies are measured relative to the volume of the body fluid such that, when the amount of antibodies contained in the body fluid and specifically binding to the linear epitope of the antigens is above a predetermined minimum amount, the labelled antibodies, in the presence of a reactant, cause a measurable and / or perceptible reaction by a user or, if spatially concentrated, result in a measurable and / or perceptible change by a user.

10. Serological method according to one of the preceding claims, wherein the linear epitope of the antigens is such that the plurality or plurality of antibodies which can be alternatively bound thereto are each effective against HPV of a different type from a class comprising at least two of the types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82.

11. Serological method according to one of the preceding claims, wherein the linear epitope of the antigens is such that the several or many antibodies alternatively bindable thereto are effective against the Ll protein of the HPV.

12. Test kit (1; 1.1; 1.2; 1.3; 1.4) for detecting antibodies against HPV, in particular for carrying out a method according to one of the preceding claims, comprising antigens (10; 10'; 10'') with a linear epitope which is reactive and is such that several or many antibodies, each of which is effective against HPV of a different type, can alternatively bind specifically to it.

13. Test kit according to claim 12, in particular for carrying out a method according to one of claims 1 to 6, comprising labelled antibodies (20; 60) which are such that they can specifically bind to the linear epitope of the antigens (10; 10'; 10'') and, in the presence of a reactant (70), cause a reaction which is measurable and / or perceptible by a user or, in the case of spatial accumulation, result in a change which is measurable and / or perceptible by a user.

14. Test kit according to claim 13, in particular for carrying out a method according to claim 3, comprising a carrier medium (2) on which a predetermined amount of the labelled antibodies (20) is mobile and a predetermined amount of the antigens (10) is present at a predetermined location (3).

15. Test kit according to claim 13, in particular for carrying out a method according to claim 4, comprising a predetermined amount of the antigens (10') and a carrier medium (2') on which a predetermined Amount of labelled antibodies (20) is provided in a mobile manner and a predetermined amount of capture molecules (30) is present at a predetermined location (3), wherein the antigens (10') are multimeric and the capture molecules (30) are such that they can each bind specifically to the linear epitope of the antigens (10').

16. Test kit according to claim 13, in particular for carrying out a method according to claim 5, comprising a predetermined amount of the antigens (10'') and a carrier medium (2'') on which a predetermined amount of the labelled antibodies (20) is provided in a mobile manner and a predetermined amount of capture molecules (50) is present at a predetermined location (3), wherein the antigens (10'') are monomeric and the capture molecules (50) are such that they can each bind specifically to a different binding site of the antigens (10'') than the linear epitope.

17. Test kit according to claim 13, in particular for carrying out a method according to claim 6, comprising a predetermined amount of the antigens (10), preferably on a carrier medium (2'' '), a predetermined amount of the labelled antibodies (60) and a predetermined volume of a reactant (70) which is such that the labelled antibodies (60) cause a reaction which is measurable and / or perceptible by a user.

18. Test kit according to claim 12, in particular for carrying out a method according to claim 7, comprising a predetermined amount of the antigens (10), preferably on a carrier medium (2'''), a predetermined amount of labelled antibodies (80) which are such that they can specifically bind to human antibodies which can specifically bind to the linear epitope of the antigens (10) and, in the presence of a reactant (70), bring about a reaction which is measurable and / or perceptible by a user, and comprising a predetermined volume of a reactant (70) which is such that the reaction takes place as a result of it.

19. Use of an antigen in a method according to any one of claims 1 to 11 and / or in a test kit according to any one of claims 12 to 18, the antigen comprising a linear epitope which is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it. 41 20 . Antibody or labelled antibody which is such that it can specifically bind to a linear epitope of an antigen, wherein the linear epitope is such that several or many antibodies, each effective against HPV of a different type, can alternatively specifically bind to it.

21. Use of a labelled antibody in a method according to any one of claims 2 to 6 and / or in a test kit according to any one of claims 12 to 17, the labelled antibody specifically binding to a linear epitope of an antigen, wherein the linear epitope is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it.

22. Use of an antibody as a capture molecule in a method according to claim 4 and / or in a test kit according to claim 15, the antibody specifically binding to a linear epitope of an antigen, wherein the linear epitope is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it.

23. Use of a molecule as a capture molecule in a method according to claim 5 and / or in a test kit according to claim 16, the molecule specifically binding to a binding site of an antigen other than a linear epitope, which is such that several or many antibodies, each effective against HPV of a different type, can alternatively bind specifically to it. 24 . Use according to claim 23 , wherein the molecule is an antibody which is such that it can specifically bind to an epitope of the antigen other than the linear epitope . 42

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