WO2009043393A1

WO2009043393A1 - Fertility test

Info

Publication number: WO2009043393A1
Application number: PCT/EP2008/004170
Authority: WO
Inventors: Gunther Wennemuth; Helmut Stauber
Original assignee: Nanorepro Gmbh
Priority date: 2007-09-25
Filing date: 2008-05-26
Publication date: 2009-04-09
Also published as: DE102007045935B4; DE102007045935A1

Abstract

The invention relates to the variant of the surface antigen ABCG2 that is present on sperm and antibodies directed against a surface antigen on sperm, in other words, against an antigen located on the outer membrane or a membrane accessible from the exterior. A sperm membrane is accessible from the exterior only when the sperm does not lose the acrosome part due to damage, in other words, leaving the sperm undamaged to the largest extent possible. An additional component of the invention is a fertility test (1) and a method for the use thereof. The fertility test (1) comprises a reagent layer (2) having a test zone (10) of partial surfaces A, B, and C, which are adapted in size, and an immobilized agent provided in each of said surfaces. The agent is an antibody according to the invention or pores that have been reduced in size. In a further developed embodiment, the fertility test further comprises a carrier body (4); in another embodiment according to the invention, the fertility test comprises a filter layer (8) having a reduced pore size; in a further embodiment, said fertility test comprises at least two strips, one of which is the reagent layer (2). The method for activating the fertility test (1) comprises the incubation of a sample, application of an aliquot thereof to the fertility test (1), an additional incubation for a predeterminable time period, and finally reading the test.

Description

PATENT OFFICES ■ EUROPEAN PATENT, TRADEMARK AN D DESIGN ATTORN EYS AM WBNBERG 15 ■ D-35096 WEIMAR (LAHN) - TEL +49 (0) 6421 30993-0 • FAX +49 (0) 64217153

26.05.2008 0979WO1441

Applicant: NanoRepro GmbH, 35043 Marburg

fertility test

The invention relates to a sperm-specific surface antigen according to claim 1, a sperm-specific surface antigen antibody according to claim 4 and sperm-specific antibody-containing fertility test according to claim 10. Further, the invention relates to a method for detecting male fertility according to claim 45 with the fertility test and its use ,

In the course of advancing industrialization and the resulting increasing strain on people and the environment, especially due to stress and restricted movement, masculine infertility is increasing in the industrialized nations. Today, as many as 20 to 40% of men in these countries are infertile, ie they have too little sperm, ie oligozoospermia with a sperm concentration <10 ⁶ sperm per milliliter in the ejaculate. Some male individuals no longer produce sperm and suffer from azoospermia. Also in veterinary medicine tests are needed to determine the fertility of stallions, breeding bulls and other domestic and domestic animals. Thus, there is a continuing need for simple, inexpensive and reliable fertility tests for men and for male animals.

WO 96 13225 A2 describes an apparatus and a method for separating mobile and immobile sperm. An ejaculate sample is brought into contact with a liquid that is only passed through by mobile sperm. The latter one examines then on their ability, one Cause pregnancy. This is determined by the amount and thus activity of the enzyme sperm superoxide dismutase (SOD) with an antibody directed against this enzyme. Detecting 20 to 40 sperm / ml of sample fluid in this way will give an unclear result, while samples with more than 40 sperm per ml will most probably trigger pregnancy.

Other indicators of fertile sperm include: glutathione peroxidase, the amount of oxo-8-deoxyguanosine, the ratio between saturated and unsaturated fatty acids, creatine kinase activity, antibodies to sperm lipids, the amount of sperm phospholipids, and sperm protein α-tubulin Amounts of lactate dehydrogenase, protamine, acrosin, or proteins from the acrosome area and the sperm scourge. Further mentioned are a glycolytic enzyme, a nuclear protein, a mitochondrial protein, the ratio of PE / PC or saturated to unsaturated lipids.

However, all these indicators have in common that they are in the sperm and not on its surface. In addition, they are located in different places and therefore found in intact as damaged spermatozoa. That every sperm, whether integer or injured, fertile or infertile, is used for evidence. This is of no concern since motile and probably fertile sperm are separated from immobile before the actual indicator determination. However, this extra step makes the test described time-consuming and uncertain in its test statement.

It is therefore an object of the present invention to obviate the disadvantages of the prior art and to provide substances, an inexpensive device and a method for the simple, inexpensive, rapid and meaningful determination of male fertility. The aim is also to achieve high precision with low susceptibility due to external influences and to enable a user to obtain a semiquantitative statement about the amount of sperm present in the ejaculate. Finally, other compounds involved in the measurement or contained in the sample should leave the measurement unaffected.

Main features of the invention are in the characterizing part of claims 1, 4 and

10 indicated. Embodiments are the subject of claims 2 and 3, 5 to 9 and

11 to 53. A surface antigen according to the invention is the sperm variant of the protein ABCG2. This variant has not been known to date and it is particularly well suited as a marker for detecting the amount of intact sperm. It is a surface antigen present on or in the outer sperm membrane and will therefore be found with much less probability or not at all on injured and hence infertile sperm without or with a damaged surface membrane. However, it is present on intact fertile spermatozoa. Of particular relevance is the variant of ABCG2 present on human spermatozoa, because it can provide information on the fertility of male spermatozoa.

ABCG2, which carries a group that is colored or converts a substrate into an optically detectable substance, is also essential to the invention because it can be used in a competitive variant of the fertility test.

An essential component of the invention is also a sperm-specific antibody which is directed against a sperm-specific surface antigen in particular against sperm ABCG2. The term sperm-specific surface antigen includes all those antigens which are located on or in the plasma membrane of the sperm, ie on or in its outer membrane or on a membrane which is accessible from the outside when the sperm are intact.

The invention is particularly developed in that the antibody is directed against a surface antigen present in the acrosomal area of the sperm. The acrosomal area is the rather fragile head of the sperm and is due to mechanical or chemical influences caused quickly lost. When it is blown off, sperm are infertile because it is no longer possible to merge with the egg cell because the enzymes required for this purpose are located in the acrosome area, ie in the tip of the egg that first touches the nucleus of the sperm and then is missing. Surface antigens in the acrosome area indicate intact functional sperm. Therefore, antibodies directed against them are particularly valuable to the invention.

In one embodiment, the antibody is directed against the surface antigen ABCG2. This comes in the acrosomal area of the sperm at the surface in front. The ABCG2 variant found on human spermatoids is particularly suitable as a surface antigen.

The antibody is conjugated with gold particles for visual recognition with colored or coloring particles and in a direct perceptible to the eye preferred variant. These have a diameter of 40 to 60 nm and thereby combine good flow properties in the fertility test with a sufficiently good visual recognition.

Another essential component of the invention is a fertility test according to the flow-through principle or according to the lateral-flow principle, which contains at least one sperm-specific inventive antibody. This antibody is directed against a sperm-specific surface antigen and thus provides a fairly accurate picture of detected antigens present on intact sperm. This image is further improved if the antibody present in the fertility test is directed against a sperm-specific surface antigen in the acrosomal area, ie against the fragile cap of the sperm. Particularly good results can be achieved with an antibody which is intended in the fertility test and which is directed against the variant of the surface antigen ABCG2 present on sperm, wherein the human ABCG2 variant stands out.

The antibodies present in the fertility test are conjugated with colored or coloring particles for optical recognition, these particles having a diameter of 40 to 60 nm for better flow behavior, while maintaining good visual recognition.

Furthermore, the fertility test consists of at least one carrier body and at least one reagent layer of a flow-through material, wherein the reagent layer has at least one antibody and / or at least one surface antigen. These features make it possible to capture and detect a surface antigen present on a sperm.

The spreading layer provided in a continuation on the fertility test at the upstream end of the reagent layer or just below the upstream end of the reagent sheath permits a metered and uniformly fine distribution of the sample solution and contributes to a quick and accurate assay performance. The same applies to the or another in her provided antibody or for the (optically) labeled surface antigen. These particles bind to or compete with the surface antigen applied to the distribution layer with a measurement sample for a binding site downstream in the fertility test. Initial contact or mixing between the test sample and labeled surface antigen or antibody present in the distributor layer takes place in this embodiment of the invention already in the distributor layer and not only in the reagent layer, and means a more homogeneous and therefore more precise test procedure.

A significant extension of the fertility test is the provision of a filter layer on or above or below the reagent layer downstream and spaced from its upstream end, and preferably also downstream and spaced from the manifold layer, if the latter is present. By this measure, good results can be obtained even with large amounts of color-labeled antibody in addition to low surface antigen levels, because the excess of color-labeled antibody is taken up in parts of the filter layer. However, in order to prevent sperm also entering the filter layer and thus losing the measurement, the filter layer is provided in a logical continuation with pores smaller than those of the reagent layer. And they are so small that they are not permeable to sperm and antibodies labeled sperm.

Particularly important for a semiquantitative statement is that the fertility test on the reagent layer downstream of its upstream end, and preferably also downstream of the filter layer, if provided, has a test zone with means for indicating whether the surface antigen is present in at least one of the concentration ranges I, II or III is present in a test sample. Concentration range I comprises an amount of the surface antigen in the measurement sample corresponding to a cell count of less than 2 cells per nanoliter of sample liquid and concentration range II an amount corresponding to a cell count of 2 cells to 20 cells per nanoliter sample liquid, preferably from 2 cells to 10 cells per nanoliter sample liquid and more preferably from 2 to 5 cells per nanoliter sample liquid. Finally, concentration range III comprises an amount of the surface antigen in the test sample which corresponds to a cell count greater than 20 cells per nanoliter sample liquid, preferably greater than 10 cells per nanoliter of sample liquid, and more preferably greater than 5 cells per nanoliter of sample liquid. The means for indicating at least one of the concentration ranges 1, 11 or IM are arranged on at least one subarea A, B or C of the test zone of the fertility test such that each concentration range I, II, III or one of the subareas A, B, C is assigned , Only in this way can a semiquantitative statement about the amount of sperm in the test sample be obtained in a simple way in the manner of a line image with the fertility test.

To increase the performance and for more accurate measurement results, an embodiment of the fertility test envisages assigning the partial area A to the concentration area 1 and arranging it furthest upstream of all partial areas A, B, C. This also serves the measure of dimensioning the partial areas A, B, C of different widths and also the embodiment of making the partial area A narrower than that assigned to the concentration area II and / or the concentration area III. Because surface antigen is least likely to be detected in subarea A, it makes sense to concentrate the applied agents in as small a space as possible in order to obtain a visible color signal even with small amounts of sperm.

The means provided on the surface A are arranged therein in an amount sufficient to detect an amount of the surface antigen corresponding to a cell number of less than 2 cells per nanoliter of sample liquid. Those agents in sub-surface B are provided in an amount sufficient to detect an amount of surface antigen ranging from 2 cells to 18 cells per nanoliter sample liquid, preferably from 2 cells to 10 cells per nanoliter sample liquid and in a particularly preferred embodiment 2 cells to 5 cells per nanoliter of sample liquid. Finally, on the partial area C assigned to the concentration range III, means are arranged in an amount sufficient to detect an amount of the surface antigen having a cell count of more than 18 cells per nanoliter of sample liquid, preferably more than 10 cells per nanoliter of sample liquid, and more preferably more than 5 cells per nanoliter of sample liquid. It is only on the basis of this graduation of the measuring ranges in the three partial areas that a layman can quickly determine whether he is still able to produce or not.

In order to effect a marked line or signal formation in each of the subareas A, B or C, the means are immobilized on at least one of them. Especially good Results are obtained as long as the means are immovable in all partial areas, because this leads to a precise separation of the concentration ranges I to IM.

Usually, the agents consist of a sperm-specific surface antigen-binding substance, preferably of a sperm-specific surface antigen-binding substance present in the acrosomal region and very particularly preferably of a substance binding the surface antigen according to one of claims 1 to 3.

However, if this is expensive or cumbersome to obtain, costs can be saved if the means consist of size-reduced pores of the partial surface C of the reagent layer in an embodiment essential to the invention.

In another continuation of the invention, the fertility test consists of at least two strips which lie on one another in an overlap region, wherein at least one of the strips consists of the reagent layer or of a combination of reagent layer and carrier body or of a combination of reagent layer, carrier body and distributor layer or of a combination consists of reagent layer, carrier body, distributor layer and filter layer. By means of this construction variant of the fertility test, an extremely exact determination of surface antigen present in the test sample in all three subareas A, B and C is possible, because in each of these subareas of the test zone, only unmarked sperm with free surface antigen enter the sample solution. Interactions with mobile antibodies or labeled mobile antibodies did not occur before entry because the latter did not flow into the test zone from the second or another strip.

However, a prerequisite for this is that there is a flow connection between the strips, at least in an overlap region, in order to bring marked antibodies from strips two onto the test zone or into the respective partial surface of strip 1. Therefore, the strips in the overlapping area are so permeable that there flows the liquid sample from the first strip on the second strip and / or each other strip. If the overlapping area and partial area (s) cover, but at least partially, surface antigens ie intact sperm from the test sample are at least partially held in the respective partial area (s) and the test sample depleted of intact sperm flows into two strips. On the other hand, an influx of labeled antibodies from strips two into strips one is unimpeded possible. This effect is further enhanced if one of the strips in the overlapping area is freed from the carrier body.

In a further development of the invention, application zones are arranged on the upstream sides of the strips in such a way that, with simultaneous or almost simultaneous application of a liquid to each of the application zones, liquid flows are formed which meet in the overlapping area. If, for example, sample solution is applied to the strip to be regarded as a reagent layer and one liquid is applied to the or a further strip, then both liquids meet approximately in the overlap region of both strips and distortions can therefore not occur which would occur if the liquid consisted of strips 2 loaded with the antibody would flow through the overlap area before the antigen from the measurement solution is present in a partial area arranged directly below the overlap area.

In order to save expensive antibodies for capturing sperm even in the two-strip or multi-strip arrangement in subarea C and furthermore to lose sperm during the measurement, at least one of the strips has a different pore width, at least in the overlap region, than the other and each further strip. It is permeable to antibodies and to labeled antibodies but retains intact sperm and antibody-bound intact spermatozoa. Thus, antibodies and labeled antibodies can flow freely from one strip to the next, but sperm remain as a first strip on the reagent layer and do not pass into the second strip in the overlap area. They are therefore still available for the measurement in the test zone, so that a more accurate measurement result is obtained.

This also contributes to the fact that in the overlap region, a substance binding a sperm-specific surface antigen, preferably a sperm-specific surface antigen-binding substance present in the acrosomal region and very particularly preferably a substance binding the surface antigen according to one of claims 1 to 3 immobilized on at least one strip and im wet state is not movable.

For exact sample delivery, ie to increase the accuracy of the fertility test this has a dosing device in an extended design. Further, a fertility test is performed particularly quickly if it has a suction pad at the downstream end of the reagent layer or at the downstream end of each strip. In that case, the capillary effect present in the reagent layer anyway is supported by an additional suction effect. Such a suction pad is made of a material with a large surface area and large pores, ie a polymer foam or a large-pored non-woven fabric.

Another essential aspect of the invention relates to a method for detecting the fertility of a male with a fertility test, which according to the invention is characterized in that an ejaculate sample is incubated for a certain residence time, a part of this sample applied to the distribution layer or on the reagent layer of the fertility test incubating the fertility test for a prescribable time, and by counting the number of bands of color forming on the fertility test, the amount of sperm present in the ejaculate sample is determined. This will enable the user to determine very accurately whether or not he is capable of being produced and the whole without medical assistance.

He also likes to incubate his ejaculate sample at room temperature. This usually occurs during a residence time of 2 to 30 minutes, preferably 10 to 25 minutes, in a selected embodiment of 13 to 22 minutes and in a particularly preferred embodiment of 15 to 20 minutes. The time is advantageously chosen so that on the one hand comes to a sperm liquefaction, on the other hand, however, degradation processes on the sperm, in particular the blowing off of the acrosome cap not yet take place.

The part of the sample to be measured is 5 to 500 μl, preferably 25 to 250 μl and more preferably 50 to 100 μl. These levels cause the fertility test to run quickly, but without obtaining an unsafe test result due to low levels.

The fertility test is incubated for a predetermined time by adjustment in a flow agent or by application of a flow agent which is 2 to 15 minutes, preferably 4 to 12 minutes and more preferably 6 to 10 minutes. In this case, the flow agent is either the sample solution itself or another liquid that is able to flow through the fertility test. Anyhow needs It is the time taken to reach the sample through the fertility test.

It is also essential to the invention to make the ejaculate sample of low viscosity, for example by adding dithiothreitol, if a very liquid sample is required.

Another aspect of the invention claims the use of a fertility test to diagnose the fertility of male animals, preferably male mammals, and more preferably males, or the use of diagnosing malformations in sperm and its components.

Further features, details and advantages of the invention will become apparent from the wording of the claims and from the following description of exemplary embodiments with reference to the drawing. It shows:

Fig. 1 is a plan view of a fertility test

A fertility test 1 according to the invention serves to determine the number of sperm in an ejaculate sample and thus to predict whether the fertilization of an egg cell is to be expected. In a first basic variant, it consists of a reagent layer 2 and a carrier body 4. The latter supports and stabilizes the reagent layer 2, by reinforcing it in its thickness or by surrounding it peripherally. At the upstream end of the reagent layer 2, antibodies directed against a sperm-specific surface antigen, preferably against the variant of the protein ABCG2 present on sperm, are applied to the reagent layer 2. These are fixed in the dry state, but movable during the application of liquid along the reagent layer 2. They are conjugated to particles that are colored or capable of producing a color signal. Downstream, the reagent layer 2 has a test zone 10 with faces A, B, and C. Each of these faces A, B, C contains a fixed site means for holding a complex of surface antigen and antibody within the face. The individual Teiifiächen A, B, C contain different amounts of the agent, so that in each sub-area only a certain amount of the complex of surface antigen and antibody is bound. This amount is the greater, the further the sub-surface is arranged downstream. To actuate the fertility test 1 according to the invention, a sufficiently liquid ejaculate sample is applied to the reagent layer 2 as sample liquid at the upstream end of the test 1. This (sample) flows in a sequence of capillary action or driven by the gravitational force into the layer 2, which is also referred to as the reaction layer, or along it. Sperms of a sample solution bind with their surface antigen to the antibodies provided with colored or coloring particles and form the complex. This flows further downstream into the partial area A. If at least so many sperm are present in the ejaculate sample that there are a cell number of less than 2 cells per nanoliter of sample liquid, a sandwich of complex and fixed-site agent is formed in the partial area A. This sandwich is optically recognizable in a basic design and indicates that the ejaculate sample contains less than two cells per ml.

If the sperm concentration in the sample of ejaculate to be measured is at or above 2 cells / nl, the agent immobilized in subarea A can no longer completely capture the complexes of surface antigen (preferably sperm-specific ABCG2) and antibody because the mean amount in subarea A is insufficient ,

The uncleared or excess amount of complex flows downstream on Reagent Layer 2 into Substrate B. This also contains undissolved agents, but in an amount that binds as much sperm-specific surface antigen as is present in an ejaculate sample, the 2 to 18 sperm cells contains per sample of sample liquid. In a more refined embodiment, the amount of agent corresponds to a concentration range of 2 to 10 cells per 1 ej ejaculate and in a more finely tuned variant of 2 to 5 cells per nanoliter sample. In subarea B, sperm remain in the basic form up to a concentration of 18 cells / nl and adhere to their cell surface, preferably bound to their surface antigen ABCG2 antibodies are thus concentrated in one place. This creates a second line on or in the fertility test 1, which is directly visible in a basic version.

Two lines on the fertility test indicate that the ejaculate sample contains at least as much surface specific antigen as contained in a sample with at least 2 sperm cells / ml and at most 20 cells per nanoliter. The antigen contained on less than two sperm cells / nl produces a signal in partial area A and the surface antigen present on up to 18 further spermatozoa per nanoliter can be recognized in partial area B.

If the sample contains more than 20 sperm per nanoliter, these can not be completely bound in the areas A and B. The excess portion flows on the reaction layer 2 farther downstream in sub-area C, in which an excess of means is present. These agents bind all excess complexes of sperm, sperm-specific surface antigen and antibodies. Because the latter is color-marked in a basic version, a colored line is also formed in sub-area C of the test zone.

A fertility test 1 with three lines thus indicates to the user that his examined ejaculate sample contains more than 20 sperm cells / nl sample. These sperm also have a sufficient ability to fertilize an egg, because they have sufficient mobility required for this. If the latter were not present, the sperm would not have actively moved to sub-area C of the fertility test and no or a weaker color line would have been detected.

A second basic variant of the fertility test 1 is almost identical to the first. However, on the reagent layer 2, instead of the antibody directed against a sperm-specific surface antigen, surface antigens according to the invention which carry a group which is colored or converts a substrate into an optically detectable substance are located upstream of the test zone 10.

When a liquid ejaculate sample is applied to such a fertility test 1, it moves downstream on the reagent layer 2 and, on its way towards the test zone 10, entrains the surface antigens, which are dried and can be moved in a moist state. These stained or suitable for staining, ie labeled surface antigens (preferably consisting of sperm-specific labeled ABCG2) mix with the spermatozoa. Thus, there is a mixture of surface antigen present on sperm so not labeled and free labeled surface antigen.

This mixture initially flows into the partial area A of the test zone 10. The agents fixed there and immobile in the wet state bind both free labeled surface antigen and the unlabelled surface antigen present on the spermatozoa. antigen. If the mark is a colored mark of the surface antigen as in the basic design described below, a color signal forming in face A of the test zone 10 indicates that the test sample contains an amount of surface antigen having a sperm concentration of less than two cells per nl sample liquid corresponds. For then all binding sites of the agent are exclusively occupied with color-marked surface antigen and sperm bind with unlabelled surface antigen in too small numbers to prevent this color impression in part A competitive.

If the test sample contains an amount of surface antigen corresponding to a concentration of 2 to 18 spermatozoa / ml of sample liquid, the surface antigens of the sperm cells compete with those of the labeled surface antigens dried on the reagent layer 2 by those on the means in the partial area A of the test zone 10 binding sites. However, unlabeled surface antigens of the spermatozoa produce no color impression in subarea A, so that absence of the signal in subarea A indicates to the user that his test sample contains spermatozoa in a concentration of 2 to 18 cells per ml.

If the sample to be measured contains more than 2 spermatozoa per nanoliter of sample liquid, surface antigens of the sperm with labeled surface antigens compete both in partial area A and in partial area B for the binding sites of the respective fixed means. If the sample to be measured contains more than 20 sperm per nanoliter of measuring fluid, the agents present in the areas A and B are completely saturated with the unlabelled surface antigen of these spermatozoa and no color signal occurs in both partial areas. Thus, the absence of a color signal in subareas A and B of test zone 10 of fertility test 1 indicates a sample whose sperm content is greater than or equal to 20 cells per nl.

Thus, three color lines on fertility test 1 indicate that the examined ejaculate sample contains less than two surface-antigened sperm per nl of sample solution, two lines indicate that the ejaculate sample has from 2 to less than 20 cells per nl of sample and 1 line in area C indicates that there are 20 spermatozoa and more in one nanoliter of sample fluid.

The above-mentioned test results are particularly well achieved when in a competitive reaction between labeled surface antigen and unlabelled Spermienoberflächenantigen the latter in each case competes against the labeled surface antigen. This is especially the case when the spermatozoa of the test sample reach the partial surfaces A ₁ B and C of the test zone 10 in front of the labeled surface antigen and already bind to the means provided there. Then remain for the binding of the labeled surface antigen only agents that are not already saturated with the present on the sperm cells surface antigen.

Therefore, an extension of the second basic variant of the fertility test 1, vorzuhalten the marked surface antigen as a suspension or solution in a vial or in a distribution layer 6 vorzushalten. However, this is located at the upstream end of the reagent layer 2 or downstream of the upstream end but upstream of the test zone 10. In one embodiment, the manifold layer 6 is a closed compartment which is not opened until it has a mandrel provided in the manifold layer 6 or mechanically applied externally Pressure with the reagent layer 2 comes into flow contact and releases the labeled surface antigen to the reagent layer 2.

In such an extension, the test sample is first applied to the reagent layer 2 and waited until it has flowed through it in the direction of the test zone 10. Next, a buffer solution is applied to the distributor layer 6 and elutes the marked surface antigens which have dried there and which flow in the direction of the test zone 10. Alternatively, surface antigen dissolved or suspended on the surface-antigen-free distributor layer 6, which likewise flows in the direction of the test zone 10, is added. In a further alternative, it is intended to mechanically perforate the sealed compartment of the distribution layer 6 provided with labeled surface antigen and thereby to transport said antigen into the test zone 10.

In each of the cases described above, the labeled surface antigen passes into the test zone 10 only after the sperm, which helps to achieve a comparatively high degree of precision in determining a sperm-specific surface antigen and in particular the sperm-specific ABCG2 in the competitive operation of the fertility test 1 according to the invention. This is also the case if the sample to be tested contains 2 or 20 sperm per ml. The agents which are present or immobilized in the partial surfaces A, B and C are effector particles or, in the case of the partial surface C, reduced-size pores or narrow pores within the reagent layer 2.

By the term effector particles are meant all entities which are capable of binding to a sperm-specific surface antigen. Preferably, these include antibodies which attach to a surface antigen in the acrosomal region of the sperm. Because this area is a labile sperm region and is already damaged or blasted off many sperm that are no longer fertile, an antibody that accumulates in the acrosome area indicates intact and therefore highly fertile spermatozoa. This is to be assumed in any case if the antibody is directed against the surface antigen ABCG2 present on sperm, in particular human sperm, in the acrosomal region.

The term effector particles include both monoclonal and polyclonal antibodies as long as they have a surface antigen as defined previously as the target. Further, it is to be understood as referring to the surface antigen directed antibody fragments such as the F _c fragment or one or both F (ab) fragments. Finally, one or more of the light or heavy chains of an antibody directed against the surface antigen, for example the variable light chains or the variable heavy chains or single-chain antibodies, may also be included under the term effector particles.

The term effector particles also includes proteins, enzymes which do not cleave peptide bonds of surface antigens, ligands such as biotin and streptavidin, lectins, aptamers and polymers, provided that they are capable of binding specifically to a sperm-specific surface antigen preferably in the acrosome region and in particular to ABCG2 ,

Narrow pores are all those pores on the reagent layer 2 that retain sperm with surface antigen. Furthermore, they also trap the complex of sperm with surface antigen and conjugated antibody, that is, antibodies to which a colored or color-producing particle is bound, and prevent it from flowing further through the reagent layer 2. However, free conjugated antibodies do not retain the narrow pores. Narrow pores or size-reduced pores are located on the reagent layer 2 exclusively in partial area C.

If sperm with surface antigen are present in a test sample in an amount that corresponds to a sperm concentration of more than 20 cells per nl, they are only partly prevented from flowing on in the areas A and B of the fertility test 1. One part is either in free form or bound to conjugate antibody in sub-area C. This sub-area has narrow pores or immobilized sperm-selective, preferably against acrosomal surface antigens directed effector, which further movement of sperm or a complex of sperm and bound to its surface antigen color-labeled antibody prevention. Thus, all the complexes that have passed the partial area A and the partial area B concentrate in area C in order to produce a visually perceptible line there. Non-sperm bound conjugated antibodies continue to flow downstream and therefore can not falsify the measurement result in patch C.

Surface antigens according to the invention are all those antigens which are on the surface of a sperm, say, on or in its plasma membrane. They may also be located on or in the outer acrosomal membrane, as long as the plasma membrane of the spermatozoa is damaged but the acrosome area is not yet broken or destroyed.

Preferably, the surface antigen according to the invention is present only in the acrosomal region of the sperm. Intact fertile sperm still have an acrosome or acrosome cap that can easily be lost as a result of pH changes or lytic events. However, because the acrosome contains enzymes that allow the spermatozoon to penetrate into the egg cell, and in the absence of this organelle fertilization of the egg cell does not take place any more, the probability of detecting reproductive intact spermatozoa is quite high, if one exists only in the acrosomal area Surface antigen is used as a species to be detected.

In a particularly favorable embodiment, the surface antigen comprises the sperm variant of the protein ABCG2. It has a molecular weight of 51 KD after SDS-PAGE. This protein is located in the acrosomal area of the sperm and does not form until late in spermatogenesis. It is therefore a good pointer for a fully developed and functional sperm. The sperm-specific surface antigen ABCG2 includes not only the species present on human sperm but also those which are present on stallion sperm, on bull sperm, on boar sperm, on ovine and caprine sperm, on male sperm, on cat sperm and on rabbits, hares, mouse and rat sperm.

In the first basic variant, the antibodies dried on the reagent layer 2 are conjugated to colored or coloring particles. The labeled surface antigens used in the second basic variant also contain a group which is colored or converts a substrate into an optically detectable substance.

All particles or groups have in common that they have a size which is below the pore size of the reagent layer 2. Only in this way is it possible to achieve a sufficient flow through the reagent layer 2 with complexes of surface antigen and with particles conjugated to particles effector molecules, preferably antibodies. Effector molecules are all molecules that are freely movable on the one hand on the reagent layer 2 and on the other hand bind a surface antigen according to the invention. The labeled surface antigens, ie the surface antigens which carry a group which is colored or converts a substrate into an optically recognizable substance, also have a size which is smaller than that of the pores of the reagent layer 2.

The colored or colorant particles or the colored or colorant group are to be understood as the following compound or material families:

Metal sols, ie colloidal solutions of finely dispersed, preferably monodisperse, dissolved metal particles. These include gold as well as silver, platinum or copper particles, readily bind to antibodies and thus represent coloring particles or coloring groups. The same applies to carbon particles. All particles preferably have a size of 40 to 60 nm.

Color particles or particles of at least one of the following dyes; Coomassie Brilliant Blue, Congo Red, Malachite Green, Fuchsin, Crystal Violet, Benzaurin, Phenolphthalein, Fluorescein and other fluorescent dyes, such as Rhodamine, for example Rhodamine 123, Allophycocyanin, Phycoerythin, Erythrosine, Europian, Luminol, Luciferin and Cumarine, Phosphorescent Dyes, Eosin, Propidium Iodide, Methyl orange, alizarin yellow, hematoxylin, various toluidines, azure B, also known under the name methylenazur or NNN'-trimethylthionine, methyl green or Paris green also heptamethyl-p-rosanilin chloride, or ethyl green, Basic Blue 20, double green SF, Double Green SF, Hoffmann's Green, green powder, Vert Lumiere, Vert de Paris, Vert Etincelle, Methyl Green-zinc chloride salt, Foron Blue SRP, Resolin Blue BBLS, Anthraquinone, Palanil Violet 6R ₁ Palanil Yellow 3G, Palanil Yellow G, Palanil White G ₁ Terasil Brilliant Flavin 8GFF , Terasil Brilliant Pink 4BN, Cibacet Violet 2R, Foron Brilliant Flavin S8 GF, Resolin Brilliant Blue RRL, Procinyl Blue R, Samaron Brilliant Red H6GF, Samaron Brilliant Yellow MOGF, Samaron Brilliant Orange HFR, Samaron Violet HFRL, Tetrabromophenol, Thiazine Blue, and Acridine Orange Chlortetrazykline.

Enzymes according to the invention are also colorant particles or groups which convert a substrate into an optically detectable substance, usually into a dye, ie are capable of producing a color signal. They are either covalently linked to the effector particle, preferably the antibody, or adsorbed to a polymer which is chemically linked to the antibody. The enzymes include dehydrogenases, ureases, peroxidases, phosphatases or galactosidases. Examples are horseradish peroxidase, alkaline phosphatase or β-galactosidase. β-galactosidase hydrolyzes the substrate X-GaI (5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside) into galactose and a blue water-soluble indigo dye. Alkaline phosphatase also converts the substrate 5-bromo-4-chloro-3-indoxyl phosphate (BCIP, X-Phos) in the presence of nitroblue tetrazolium chloride (NBT) into a blue indigo dye. Another enzyme substrate is 2,2-azino-di (3-ethylbenzthiazolene sulphonic acid), ABTS.

Furthermore, color-coded protein A, color-labeled biotin, histidine or color-labeled streptavidin are included among the coloring particles.

All of these dyes or coloring particles either attach themselves directly to the antibodies as sol particles or they are adsorbed to carrier particles which form a sol and bind with the antibody.

Suitable carrier particles are all particles which are so small that they can easily pass through the pores of the reagent layer 2. Particularly preferred are latex particles of different particle size, which are dyed with one of the dyes. Furthermore find dispersible, preferably monodisperse particles such as Beads, liposomes and microparticles application. These include cellulose beads, silica particles, glass beads of defined particle size, magnetic or magnetizable Sepharose or Sephadex particles, starch and collagen aggregates or PVC biobeads. Finally, polyclonal antibodies aggregated around an antigen can also be used as carrier particles for dyes or coloring particles. These are on the one hand in contact with the antibody, on the other hand with the dye or coloring particles.

The fertility test 1 consists of a reagent layer 2 and a carrier body 4. Its functionality is expanded or enlarged by the distributor layer 6, a filter layer 8 and a suction pad.

The reagent layer 2, also referred to as the reaction layer, is formed from a flow-through material on or through which the test sample applied to the fertility test 1 flows in the direction of the partial area A, the partial area B and then the partial area C. It has, at least to a large extent, a pore size which allows unrestricted flow through sperm cells and complexes of sperm cells and conjugated antibody. Similarly, surface antigens according to the invention which carry a group which is colored or converts a substrate into an optically detectable substance, ie labeled surface antigens, are able to flow through the reagent layer 2 unhindered and last but not least label-free surface antigens.

The layer 2 consists of a flow material, namely a sheet material or space material of nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), high density polyethylene (HDPE), polypropylene, glass fibers, micro glass fibers, polyvinyl chloride, a polyamide, such as nylon or other porous polymers, which satisfy the above-mentioned permeation conditions. Combination products such as Whatman's Fusion 5 ™ multifunctional paper have also proven their worth.

The reagent layer 2 is not only a sheet of low thickness, comparable to a sheet of paper, but also a space consisting of a three-dimensional network of a pore substance. This network-like three-dimensional pore substance, in addition to the compounds already mentioned above, which are not only flat but also in spherical or particulate form, also include layer materials such as Sephadex and Sepharose, Agarose, SM-Biobeads, reticulated polyacrylamide, various types of silica or pyrrolysed silica and aluminum hydroxide in different density and grain size. Furthermore, foams such as polystyrene foam, polyurethane foam, EIM foam, polyamide foam, polyacrylonitrile foam, polyethylene terephthalate and also polybutylene terephthalate foam and PVC foam can be included under this pore substance.

The reaction layer 2 can be subdivided into an area for sample application, into a flow area adjoining it downstream, into the test zone 10 with the partial areas A, B and C and into an outlet area. The partial surfaces A, B, C are spaced apart in the test zone 10.

Each partial area A, B, C contains immobilized agents with which a defined amount of surface antigen according to the invention is retained. Partial surface A contains immobilized agent in an amount that binds as much surface antigen as present on less than 2 sperm cells / μl of sample fluid. Subpart B contains as much agent as is necessary to detect surface antigen in a sample of 2 to 18 cells per ml of sample fluid. Subpart C finally contains so much agent that surface antigen is detected in an amount corresponding to more than 20 spermatozoa per ml of sample solution. Because the surface antigen of the invention is directly or indirectly bound to a colored or colorant particle, a line profile of one to three colored lines can quickly and fairly reliably tell whether a sperm sample is infertile, perhaps fertile or very likely capable To fertilize the egg.

If a test sample contains as much surface antigen (preferably ABCG2) as there are less than two sperm per nanoliter of sample solution, a line will be formed only in part A, provided that the fertility test is based on the sandwich principle in which the surface antigen is a first antibody with color marking or color indicator and bound by an effector particle. If the fertility test functions according to the competitive principle, surface antigens of the sperm compete with color-marked surface antigens dried on the reagent layer, and a color signal also occurs in sub-area A and additionally in sub-area B and sub-area C. In both cases, the color impression does not have to arise directly, but instead It can also be achieved by adding a solution with one of the above-mentioned substrates following the sample solution. This flows to the surface of the antigen-bound antibody conjugated to one of said enzymes and is converted by the latter into a dye.

At a concentration of fertile spermatozoa in the test sample, which corresponds to a cell number of 2 to 20 sperm per nl test sample, the sandwich method produces a colored line in subarea A and subarea B, but not in subarea C. According to the competitive principle, a Line in sub-area B and sub-area C, but not in sub-area A.

If the liquid or liquefied ejaculate sample contains as much surface-specific sperm antigen (which is preferably in the acrosome range and particularly preferably ABCG2) as the sample solution, that it corresponds to an amount of more than 20 sperm per nanoliter of sample liquid, three lines are detected on the fertility test the sandwich principle, ie, one in each of the partial surfaces A ₁ B and C. In the second variant of the fertility test 1, which operates on the competitive principle, surface antigens which are located on more than 20 sperm cells per nanoliter sample solution displace labeled surface antigens in the Partial areas A and B and partly also in C, so that missing lines in the areas A and B indicate a fertile sperm sample.

Immobilizing on the reagent layer three partial surfaces A, B, C with an adapted amount of means for detecting antigen is essential for a rapidly readable and meaningful fertility test according to the invention. Furthermore, it is essential that the farther a partial surface is located downstream on the reagent layer 2 of the fertility test 1, the amount of immobilized agent increases. Its precision is further increased if the partial area A, in which, according to the invention, the smallest amount of surface antigen is to be detected, is significantly narrower than the partial areas B and C arranged downstream of it.

If the effector particles to be immobilized in the subarea C are expensive, an inexpensive embodiment of the fertility test 1 is to provide regions of different pore size on the reagent layer 2. In this case, the reaction layer 2 is made either in one piece or else from at least two layer parts. In the test zone 10, the partial area C of the reagent layer 10 consists of one of the abovementioned materials with size-reduced pores, also called narrow pores. These narrow pores are a means of retaining sperm and antibody complexed sperm, non-sperm bound antibodies, or labeled ones However, antigens continue to flow downstream. Thus, without the use of effector particles, sperm and spermatozoa labeled with color-labeled antibodies are retained in patch C of test zone 10 and the latter form a colored line there.

In one extension of the invention, the narrow pores or size-reduced pores are sized to retain sperm provided with color-labeled antibodies. Furthermore, they retain those sperm bound to an antibody bearing particles capable of producing a color signal. Unlabeled sperm, i. However, sperm lacking the antibody-specific surface antigen, preferably a surface antigen from the acrosomal region, and most preferably the surface antigen ABCG2, pass through the narrow pores without being retained. Thus, this invention extension, in which most of the sperm detecting partial area C only allows to label, i.e., to allow. usually to detect intact sperm. This increases the precision and validity of the fertility test 1.

This is achieved by the coordinated dimensioning of size-reduced pores and carrier particles or color particles. The pores are so large that unmarked sperm pass through them, but sperm bound to carrier particles or color particles do not pass them. In other words, an uncomplexed sperm does not pass through the size-reduced pores of sub-area C, a complex of sperm and labeled antibody, that is, sperm and antibody provided with carrier particles or color particles. If the carrier particles or the color particles are significantly larger than sperm and antibodies, the size-reduced pores have a size smaller than that of the carrier particles or the color particles.

The metering device provided in a further embodiment of the fertility test 1 at the upstream end of the reagent layer 2 consists of a punctiform recorded surface onto which a predefined number of drops of test sample is to be applied.

In order to further increase the measurement comfort of the fertility test, the reagent layer 2 has in an extension a control zone, which is located downstream of the partial surface C. In a forwarding it is also located in the outlet region of the reaction layer 2. The control zone contains an immobilized substance, which indicates whether the liquid flow generated by the sample liquid reaches the control zone. This is a reacting to moisture with color change substance such as copper sulfate or silica gel. The same function is achieved by immobilized species antibodies that bind excess conjugated antibodies or labeled surface antigens.

Furthermore, a measurement can be accelerated with the fertility test according to the invention in a further embodiment by the reagent layer 2 has a suction pad at its downstream end. This pad consists of a sponge-like material, preferably of a non-woven or of a strongly adsorbing medium and promotes the downstream flow of the test sample.

A fast fertility determination is also achieved in a continuation of the invention in which a sperm-attractive reagent is fixed at the downstream end of the reagent layer 2 (for example in the control zone and / or in the absorbent pad). This is usually a flavor and, in a more advanced form, a flavor that also occurs in the female egg.

In a further continuation of the fertility test 1, the reagent layer 2 is hydrophobic, i.e., liquids applied to it only partially or not penetrate into it and instead move on its surface. They are mixed with alkylating reagents or silylation reagents. A liquid repellent hydrophobic character is also achieved when the reaction layer 2 is saturated with proteins and thus no longer provides free binding sites for polar species. In a particular embodiment of this continuation, the hydrophobic character decreases in the downstream direction on the reagent layer 2. This causes an even faster flow through the layer 2 in the direction of the test zone 10th

The manifold layer 6 as an invention extension is attached to the upstream end of the reagent layer 2 or not far from it adhering and undetachable. It consists of at least one of the materials that are used for the reagent layer 2, but is made thicker than the flat shape of the reaction layer 2. You have both filtering and suction properties. Therefore it is constructed in an extension of a fleece or a fibrous tissue. In a modification, the distributor layer 6 is arranged above the three-dimensional reagent layer 2 surrounded by the carrier body 4 in this case. The layer 6 consists of an adsorption material likewise selected from the substances provided for the reagent layer 2.

In both variants, the distribution layer 6 has a pore size that allows sperm without problems to pass through it. Larger particles, such as, for example, clumped sperm or other cell or protein aggregates, however, prevent the distribution layer 6 from passing through and to this extent take on the function of a filter which allows only species smaller than a test-specific size to flow through to the reagent layer 2.

For easier and faster application of a test sample to the reagent layer 2 or to the distributor layer 6, at least one of them has a sample application zone which is excellent at the upstream end. This consists of a color-coded field or of an exposed, i.e. uncovered portion of the reagent layer 2 or the manifold layer 4.

The fertility test 1 has certain embodiments, a support body 4 as an important component. It is attached on the back or on the circumference of the reagent layer 2 and supports and stabilizes the reagent layer 2.

A surface-extending reaction layer 2 wins by the also flat-shaped carrier body 4, which is mounted in one embodiment on the underside of the reagent layer 2, considerably in stability. The carrier body 4 is formed in addition to the materials already mentioned above for the reagent layer 2 also from a polyamide such as nylon, nitrocellulose, cellulose acetate, glass fibers or other porous polymers. They also include membranes of polyethylene, polypropylene, polyvinylidene difluoride and glass fiber and glass cellulose blended papers or plates.

In a further embodiment of the fertility test 1, the reagent layer 2 is surrounded or enveloped by the carrier body 4 almost over the whole area. For then the latter has the form of a tube, stick or dipstick, a hollow cuboid or a hollow prism, in which the reagent layer 2 is set or filled. All forms of the support body 4 according to this embodiment are open at the end and consist of a transparent material such as glass or a transparent Plastic. These include glass and plastic capillaries, straws or pipettes. In order to prevent sperm of the sample solution provided with surface antigen or antibodies or antibodies provided with colorant particles from being adsorbed on the carrier body 4, in a further development of the invention it is coated with at least one substance which gives the carrier body 4 a hydrophobic surface. This substance comprises a compound which has arisen from a reagent selected from the group of the alkylating reagents, silylating reagents, alkyl fluorinating reagents or the at least partially hydrophobic peptides or proteins.

The carrier body 4 has, if it is made of a non-transparent and the reagent layer 2 enveloping or enclosing in its length plastic, a plurality of openings or transparent window, through which the test zone 10 and the control zone are visible. The sample application zone of the reagent layer 2 is likewise visible and accessible through an opening in the carrier body 4 which completely surrounds the reagent layer 2.

In a further development of the invention, the carrier body 4 is an airtight sealable housing in the manner of a cup with a lid. This cup has a bottom and an intermediate floor, so it is divided into the rooms A and B. Between floor and intermediate floor in room B, a cylindrical tube is introduced, which is open on one side. In this cylindrical tube, a cylindrical rotary plug with a small opening is fitted. In a selected position of the plug there is a flow connection between space A and space B, in all other positions of the plug both spaces are not in flow contact with each other. The area between the bottom and the intermediate bottom, ie space B, is in fluid communication with the reagent layer 2. The latter gets up on the floor of room B.

With such a housing, sample samples can be precisely metered. For this purpose, the preferably diluted test sample in room A of the cup, so that it rests on the intermediate bottom. Then close the lid of the housing airtight and rotate on the stopper until a flow connection between space A and space B is formed. It flows as long as measuring sample from room A to room B, until in the airtight space A through the liquid drain creates a negative pressure. Consequently, with this device, a defined amount of sample solution reaches room B and thus to the upstream part of the reagent layer. A fertility test 1 with such a carrier body 4 is a fertility test with a dosing device.

In an embodiment of the invention which further enhances the precision of the fertility test 1, the reagent layer 2 is provided with a filter layer 8. This is located in a first variant on the areal reagent layer 2 between the upstream end and the test zone 10. The layer 8 consists of one of the above for the reagent layer 2 mentioned materials and has a relation to the reagent layer 2 different. smaller pore size. Through the pores of this layer 8, free antibodies as well as antibodies provided with a colorant or colored particle can flow through unhindered, but not spermatozoa or spermatozoa with bound antibody. Layer 8, in a variant, also has binding substances which are capable of binding both mobile and mobile antibodies conjugated to a colored or coloring particle, as long as these antibodies are not bound to a sperm-present surface antigen according to the invention.

In a fertility test 1 operating on the sandwich principle, sample solution flows downstream from the sample application zone and entrains mobile antibody provided with a dye or a coloring particle and dried on the reagent layer 2. These are applied in a two- to ten-fold excess on the reagent layer 2, in order to guarantee a binding to all existing spermatozoa in any case, even with large amounts of sperm with surface antigen. In the case of small amounts of motile sperm, accordingly, there is a great deal of excess mobile antibody which can influence a measurement result, especially in partial area A. On the way to the test zone 10 all particles flow past the filter layer 8. Excess antibody not bound to spermatozoa either flows further downstream on the reagent layer 2 into the test zone 10 or into the filter layer 8. This dilutes the amount of free mobile antibody which flows into the test zone 10 and the measurement result obtained in the partial area A is due this dilution even more accurate. Antibodies bound to sperm are too large for the pores of the filter layer 8 and therefore can not be absorbed by it.

In a second variant, the filter layer 8 is part of a tubular fertility test 1. It consists of one of the spatial materials already given for the reaction layer 2 above. It is for example in a serving as a carrier body 4 Tube above, ie upstream of the reagent layer 2 and downstream of the sample application zone arranged or introduced as a layer. The filter layer 8 in this embodiment has pores passable for free or dye-labeled or colorant-labeled antibodies as well as for sperm and antibody-containing sperm, but retains larger particles.

Sample applied to the sample application zone of this form of fertility test 1 flows into the filter layer 8. Any particles of the sample that are larger than sperm labeled with color-labeled antibodies will retain the filter layer 8 and thus function as a particle screen for impure samples. Smaller particles, i.a. Sperm, antibodies, etc. continue to flow unhindered into the test zone 10, as already described.

In one embodiment of the second variant, the carrier body 4 of the fertility test 1 has the shape of a T-piece with a long tube part and a portion attached to the side of the long tube part in fluid communication. The long tube part contains the reagent layer 2 and is from top to bottom, i.e. in the direction of the gravitational force of the sample liquid can be flowed through. The section consists of one of the materials intended for the carrier body 4 and is connected to the filter layer 8, i. completely filled with one of the space materials used for the reagent layer 2. These materials have a pore size that does not allow the passage of sperm and antibody-labeled sperm, but allows smaller particles to freely enter the portion. Filter layer 8 and reagent layer 2 directly adjoin one another. In a material-saving continuation of the invention, the portion consists only of the filter layer 8, which is inserted through an opening in the carrier body 4 in the reagent layer 2.

If one applies to the fertility test 1 with laterally arranged filter layer 8 - ie to the fertility test 1 in the form of a T-piece - sample liquid, it flows in the direction of the test zone 10 and ruptures on its way aufdrrocknete color-labeled antibodies with it. These antibodies, which are usually present in a two- to ten-fold excess, can influence the measurement result, in particular in partial area A of the test zone 10, owing to their high concentration with very low sperm quantities in the ejaculate. If they are not bound to a sperm, a part of them flows into the laterally arranged filter layer 8 and no longer penetrates into the test zone 10, that is also not in partial area A. This laterally arranged to the flow direction of the sample Filter layer 8 causes a dilution of the color-labeled antibodies and thus can be obtained even at low sperm concentrations in the presence of much color-labeled antibodies unadulterated precise results.

In a further embodiment of the invention, the fertility test 1 comprises at least two strips, one of the two strips corresponding to the reagent layer 2 with its partial surfaces A, B, C. The strips overlap in a common area, so that the fertility test 1 has the shape of a cross, a T or a circular or semi-circular fan-cut arrangement. Each strip not corresponding to the reagent layer 2 has pores at least in the region of overlap which are smaller than those of the reagent layer 2 and do not permit the passage of sperm. It also has color-labeled surface antigens or antibodies with conjugated coloring or colored particles. Both antigens and antibodies are dried downstream of the overlap region on the strip (s) resting on the strip acting as reaction layer 2.

In an embodiment essential to the invention each subarea A, B, C of the test zone 10 of the first strip overlaps with another strip so that as a whole four strips form the fertility test 1. This makes it possible to spend in each subarea A, B, C quantitatively and qualitatively different amounts of labeled or provided with coloring particles antibodies and thus to influence the intensity of the color measurement result. In addition, with this arrangement, it is possible to apply to each strip not corresponding to the reagent layer 2 a different antibody or a differently colored antibody for the sperm-specific surface antigen. This enhances the reading accuracy of the fertility test 1. Finally, labeled antibodies directed to different sperm-specific surface antigens, preferably derived from the acrosome region, can be introduced in each strip, which also increases the accuracy of the fertility test.

In the overlap area, means are provided and immobilized on the reagent layer 2, which bind or hold a surface antigen according to the invention. These are either effector molecules directed against a sperm-specific surface antigen or a reduced pore size in subarea C. At least one of the strips is applied to the support body 4 or surrounded by it. In the overlapping area of the strips, the carrier body 4 of the strip, which rests on the strip acting as reagent layer 2, is permeable to labeled or conjugated antibodies. In a further embodiment, the strip resting on the strip formed as reagent layer 2 has no carrier body 4 in the overlapping area.

Incidentally, the individual strips of the fertility tests have, in different combinations, the parts already mentioned for the reaction layer 2, or the parts surrounding it or connected to it.

In order to operate a fertility test 1 consisting of at least two strips with a test zone 10 consisting of 3 partial surfaces A, B, C, sample liquid is first added to the sample application zone of the reaction layer 2. If it contains sperm with sperm-specific surface antigen as described above, sperm are retained in subarea A, in subareas A and B or in subareas A, B and C by the agents immobilized there. Next, a liquid, buffer or pure water is added to the strip resting on the reagent layer 2. This liquid phase entrains the labeled antigens on the strip or with antibodies provided with colorant or color particles and transports them into the overlap area. Here, the antigens penetrate into the test zone 10 of the reagent layer 2 and bind in subregions A, B and C to immobilized effector particles not yet saturated by sperm-specific surface antigens. If previously mentioned antibodies are dried on the strips resting on the reagent layer instead of color-labeled antigens, these also flow into the overlap area and from there into the test zone 10 in order to bind sperm already immobilized there.

Sample liquid and conjugated antibodies or antigens bound to color particles or coloring particles on separate flow paths and preferably to be passed successively into the test zone 10 have the advantage that there is no interaction between surface antigen and mobile antibodies or antigens and binding in the test zone difficult or delayed. Furthermore, in most cases, sperm are already bound and aligned in the test zone 10 and are therefore exposed to conjugated antibodies or to color-coded antigens, respectively. In an extension consisting of a total of four strips to proceed with the measurement sample as previously mentioned. Next, liquid is applied to the strips overlapping the faces A ₁ B, C upstream. This solves the on each strip upstream of the respective overlap region the dried against the or one of the surface antigens antibody directed from the individual strips, and conveys it over the respective overlap regions in the partial areas A, B ₁ C of the test zone 10 of acting as a reagent layer 2 strip. There they connect with the already bound to the effector and thus immobilized sperm. Depending on the number of sperm present in the test sample, one, two or three detection lines are produced, which are different in color depending on the selected conjugate antibody dried on the individual strips. Color-different and thus more distinguishable test results can be obtained as it were, when applied in a competitive fertility test 1 antigens provided with different colorant or colored particles on the not acting as a reagent layer 2 strips of the fertility test 1.

Ejaculate is viscous and can not readily flow through a fertility test 1 according to the invention. It must first be brought into a low viscous form. For this purpose, according to the invention, an incubation is preferably provided between 15 and 20 minutes.

Alternatively, liquefaction by enzymes can be carried out as long as these enzymes do not damage or digest the outer membrane of the sperm or even its acrosome area. Suitable enzymes are chemotrypsin, pronase, trypsin, or amylase added to the sample or, in a particular embodiment, dried on the sample application zone of the reagent layer 2. In addition to the enzymes mentioned, dithiothreitol is also suitable for liquefaction of an ejaculate sample in the previously described manner of use.

Finally, liquefaction also contributes to the fact that the reagent layer 2 is hydrophobic at the sample application zone, for example by silylation or alkylation.

In an expanded version, which is used in particular in the hermetically sealable housing as a support body 4, the ejaculate sample is through Addition of buffer solution not only liquefied but diluted and thus converted into a flowable form.

Suitable incubation tubes are sample tubes with lids, but also condoms without spermicidal activity.

Fertility tests according to the invention can be operated according to the flow-through principle and the lateral flow principle. In the latter case, the incubated sample moves horizontally due to capillary action through the reagent layer 2. According to the flow-through principle, sample liquid flows vertically through the reagent layer 2. In both methods, a prefiltration of the sample by means of the filter layer 8 or the distributor layer 6 according to the invention can be carried out.

As immuno-detection method according to the invention in the competitive and the sandwich principle (s. O.) Fluorescent antibody-bound particles in a fluorescence immunoassay and above-mentioned bound to antibody enzymes representative of an enzyme immunoassay in particular an ELISA ((enzyme linked immunosorbent assay)) is used.

The invention is not limited to one of the above-described embodiments, but can be modified in many ways. Thus, instead of 3, 4 or more partial surfaces can also be provided, although this makes the handling more difficult.

It can be seen that the invention relates to the variant of surface antigen ABCG2 present on spermatozoa. It also relates to antibodies directed against a surface antigen on sperm, ie an antigen located on the outer membrane or an externally accessible membrane of the sperm. A sperm membrane is accessible from the outside only if the sperm does not lose its acrosome part due to damage, so the sperm remains intact for the most part. Another component of the invention is a fertility test 1 and a method for its use. The fertility test 1 consists of a reagent layer 2 with a test zone 10 of adapted in size faces A, B, and C, as well as immobilized in each of these areas in a predetermined amount means. It is an antibody according to the invention or reduced size pores. Furthermore, the fertility test in a redirected design comprises a carrier body 4, in a further inventive Embodiment a filter layer 8 with reduced pore size and in an additional forwarding it consists of at least two strips, one of which is the reagent layer 2. The procedure for the activation of the fertility test 1 comprises the incubation of a test sample, the application of an aliquot thereof to the fertility test 1, a further incubation for a predeterminable time and subsequent reading.

All of the claims, the description and the drawings resulting features and advantages, including design details, spatial arrangements and method steps may be essential to the invention both in itself and in various combinations.

LIST OF REFERENCE NUMBERS

1 fertility test

2 reagent layer, reaction layer

4 carrier body

6 distribution layer

8 filter layer

10 test zone

A partial area A

B partial area B

C partial area C

Claims

claims

1. surface antigen, characterized in that it is present on sperm variant of the protein ABCG2.

2. surface antigen according to claim 1, characterized in that it is present on human sperm.

3. surface antigen according to claim 1 or 2, characterized in that it carries a group which is colored or converts a substrate into an optically detectable substance.

4. Antibody, characterized in that it is sperm-specific and directed against a sperm-specific surface antigen in particular according to one of claims 1 to 3.

5. An antibody according to claim 4, characterized in that it is directed against an existing in the acrosomal area of the sperm surface antigen.

6. Antibody according to one of claims 4 or 5, characterized in that it is directed against the surface antigen ABCG2.

7. Antibody according to one of claims 4 to 6, characterized in that it is directed against the human surface antigen ABCG2.

8. An antibody according to any one of claims 4 to 7, characterized in that it is conjugated with colored or coloring particles and in a preferred embodiment with gold particles.

9. An antibody according to claim 8, characterized in that the colored or coloring particles have a diameter of 40 to 60 nm.

10. Fertitlitätstest after the flow-through principle or the lateral-flow principle, characterized in that it contains at least one sperm-specific antibody according to any one of claims 4 to 9.

11. fertility test according to claim 10, characterized Porsche Style nzeichnet that the antibody is directed against a sperm-specific surface antigen.

12. Fertility test according to claim 10 or 11, characterized in that the antibody is directed against a sperm-specific surface antigen in the acrosomal area of the sperm.

13. Fertility test according to one of claims 10 to 12, dadu rch in that the antibody is directed against the present on sperm variant of the surface antigen ABCG2.

14. Fertility test according to one of claims 10 to 13, characterized in that the antibody is directed against the present on human sperm variant of the surface antigen ABCG2.

15. fertility test according to any one of claims 10 to 14, dadu rch geken nzeich net that the antibody is conjugated with colored or coloring particles.

16. fertility test according to claim 15, characterized in that the particles have a diameter of 40 to 60 nm.

17. fertility test according to one of claims 10 to 16, dadu rch gekennzeich net, that it consists of at least one carrier body (4) and at least one reagent layer (2) consists of a flow-through material.

18. Fertility test according to claim 17, characterized in that the reagent layer (2) has at least one antibody according to one of claims 4 to 16 and / or at least one surface antigen according to claim 3.

19. Fertility test according to claim 17 or 18, characterized Porsche Style nzeichnet that at the upstream end of the reagent layer (2) or immediately below the upstream end of the Reagenzgeninschicht (2) a distributor layer (6) is provided.

20. The fertility test according to claim 19, characterized in that the distributor layer (6) has the or one further antibody according to one of claims 4 to 16 or the surface antigen according to claim 3.

21. Fertility test according to one of claims 17 to 20, characterized Porsche Style nke net that on or above or below the reagent layer (2) downstream and spaced from its upstream end and preferably also downstream and spaced from the manifold layer (6) if the latter exists is, a filter layer (8) is provided.

22. Fertility test according to claim 21, characterized Porsche Style nzeichnet that the filter layer (8) has pores which are smaller than those of the reagent layer (2).

23. Fertility test according to one of claims 17 to 22, characterized in that it on or in the reagent layer (2) downstream of its upstream end and preferably also downstream of the filter layer (8), if the latter is provided, a test zone (10) Having means which indicate whether the surface antigen is present in at least one of the concentration ranges I, II or III in a measurement sample.

24. fertility test according to claim 23, characterized Porsche Style nzeichnet that the concentration range I comprises an amount of the surface antigen in the measurement sample, which corresponds to a cell number of less than 2 cells per nanoliter sample liquid.

25. The fertility test according to claim 23 or 24, characterized in that the concentration range II comprises an amount of the surface antigen in the test sample which corresponds to a number of cells from 2 cells to 20 cells per nanoliter of sample liquid, preferably from 2 cells to 10 cells per nanoliter Sample liquid and more preferably from 2 to 5 cells per nanoliter sample liquid.

26. Fertility test according to one of claims 23 to 25, characterized in that the concentration range III comprises an amount of the surface antigen in the test sample, which corresponds to a cell count of more than 20 cells per nanoliter sample liquid, preferably of more than 10 cells per nanoliter of sample liquid and more preferably of more than 5 cells per nanoliter of sample liquid.

27. fertility test of any of claims 23 to 26, characterized in that the means for displaying at least one of the concentration ranges I ₁ II or IM on at least one part area A, B or C of the test zone (10) of the fertility tests (1) so are arranged, that each concentration range I, II, III respectively one of the partial surfaces A, B, C is assigned.

28. fertility test according to claim 27, dadu rch geken nzeich net, that the partial area A is assigned to the concentration area 1 and of all partial areas A, B, C is arranged furthest upstream.

29. fertility test according to claim 27 or 28, dadu rch gekennzeich net, that the partial surfaces A, B, C are different in width.

30. fertility test according to one of claims 27 to 29, characterized in that the partial area A is made narrower than that associated with the concentration range Il and / or the concentration range IM.

31. fertility test according to one of claims 27 to 30, dadu rch geken nzeich net that on the surface area A means are arranged in an amount sufficient to detect an amount of the surface antigen corresponding to a cell count of less than 2 cells per nanoliter sample liquid.

32. fertility test according to one of claims 27 to 31, dadu rch in that on the concentration range II associated patch B means are arranged in an amount sufficient to detect an amount of the surface antigen, a cell number of 2 cells to 18 cells per Nanoliter sample liquid, preferably from 2 cells to 10 cells per nanoliter sample liquid and in a particularly preferred design of 2 cells to 5 cells per nanoliter sample liquid corresponds.

33. fertility test according to one of claims 27 to 32, dadu rch in that on the concentration range III associated patch C means are arranged in an amount sufficient to detect an amount of the surface antigen having a cell count of more than 18 cells per nanoliter Sample liquid, preferably of more than 10 cells per nanoliter of sample liquid, and more preferably of more than 5 cells per nanoliter of sample liquid corresponds.

34. fertility test according to one of claims 23 to 33, characterized in that the means are immobilized on at least one of the partial surfaces A, B or C.

35. Fertility test according to one of claims 23 to 34, dadu rch in that the agent from a spermienespecific surface antigen binding substance, preferably from an existing in the acrosomal area, sperm-specific surface antigen binding substance and most preferably from a surface antigen after a consist of the claims 1 to 3 binding substance.

36. Fertility test according to one of claims 23 to 35, dadu rch geken Nzeichnet that the means consist of reduced size pores of the partial surface C of the reagent layer.

37. fertility test according to one of claims 21 to 36, characterized in that it consists of at least two strips which lie on one another in an overlap region, wherein at least one of the strips from the reagent layer (2) or from a combination of reagent layer (2) and carrier body (4) or a combination of reagent layer (2), carrier body (4) and distributor layer (6) or a combination of reagent layer (2), carrier body (4), distributor layer (6) and filter layer (8).

38. Fertility test according to claim 37, characterized Porsche Style nzeichnet that the strips are so permeable in the overlapping area that there flows the liquid sample from the first strip on the second strip and / or each other strip.

39. fertility test according to claim 37 or 38, characterized in that one of the strips in the overlapping region of the carrier body (4) is released.

40. fertility test according to one of claims 37 to 39, dadu rch geken net nets that are arranged on the upstream sides of the strips order zones such that form at the same time or almost simultaneous application of a liquid to each of the application zones liquid flows, which are in the overlap region to meet.

41. Fertility test according to one of claims 37 to 40, characterized Porsche Style nzeichnet that at least one of the strips has at least in the overlapping region with respect to the other and each further strip different pore width.

42. fertility test according to one of claims 37 to 41, characterized Porsche Style nke net that in the overlap area a sperm-specific surface antigen binding substance, preferably a present in the acrosomal area, sperm-specific surface antigen binding substance and most preferably a surface antigen according to any one of claims 1 to 3 binding substance immobilized on at least one strip and is not mobile in the wet state.

43. fertility test according to one of claims 10 to 42, dadu rch characterized in that it comprises a metering device.

44. Fertility test according to one of claims 10 to 43, characterized in that it comprises a suction pad at the downstream end of the reagent layer (2) or at the downstream end of each strip.

45. A method for detecting the fertility of a male living with a fertility test (1) according to any one of claims 10 to 44, characterized in that

- an ejaculate sample is incubated for a certain residence time,

a part of this sample is applied to the distribution layer (6) or to the reagent layer (2) of the fertility test (1),

- The fertility test (1) is incubated for a predeterminable time

and, by counting the number of bands of color forming on the fertility test (1), the amount of sperm present in the ejaculate sample is determined.

46. The method according to claim 45, characterized in that the ejaculate sample is incubated at room temperature.

47. The method according to claim 45 or 46, characterized in that the residence time is 2 to 30 minutes, preferably 10 to 25 minutes, in a selected embodiment 13 to 22 minutes and in a particularly preferred embodiment 15 to 20 minutes.

48. The method according to any one of claims 45 to 47, dadu rch geken Nzeichnet that the portion of the sample is 5 to 500 ul, preferably 25 to 250 ul and more preferably 50 to 100 ul.

49. The method according to any one of claims 45 to 48, characterized in that the fertility test (1) is incubated for a predetermined time by adjusting in a Füeßmittei or by applying a flow agent.

50. The method according to any one of claims 45 to 49, characterized in that the predeterminable time is 2 to 15 minutes, preferably 4 to 12 minutes and more preferably 6 to 10 minutes.

51. The method according to any one of claims 45 to 50, characterized in that the Ejakulatprobe is made low viscosity.

52. Use of a fertility test according to any one of claims 10 to 44 for the diagnosis of fertility of male animals, preferably male mammals and more preferably of men.

53. Use according to claim 52 for the diagnosis of malformations in sperm and their components.