WO2009043393A1 - Test de fertilité - Google Patents

Test de fertilité Download PDF

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Publication number
WO2009043393A1
WO2009043393A1 PCT/EP2008/004170 EP2008004170W WO2009043393A1 WO 2009043393 A1 WO2009043393 A1 WO 2009043393A1 EP 2008004170 W EP2008004170 W EP 2008004170W WO 2009043393 A1 WO2009043393 A1 WO 2009043393A1
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WO
WIPO (PCT)
Prior art keywords
sperm
surface antigen
fertility test
fertility
sample
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Application number
PCT/EP2008/004170
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German (de)
English (en)
Inventor
Gunther Wennemuth
Helmut Stauber
Original Assignee
Nanorepro Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanorepro Gmbh filed Critical Nanorepro Gmbh
Publication of WO2009043393A1 publication Critical patent/WO2009043393A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • 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 ,
  • 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.
  • SOD superoxide dismutase
  • 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 ⁇ -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.
  • 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.
  • other compounds involved in the measurement or contained in the sample should leave the measurement unaffected.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l, preferably 25 to 250 ⁇ l and more preferably 50 to 100 ⁇ l. 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.
  • 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.
  • 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.
  • Fig. 1 is a plan view of a fertility test
  • a fertility test 1 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.
  • 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.
  • 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.
  • the reagent layer 2 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 Teiifiumbleen 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.
  • a sufficiently liquid ejaculate sample is applied to the reagent layer 2 as sample liquid at the upstream end of the test 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a liquid ejaculate sample 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.
  • labeled surface antigens preferably consisting of sperm-specific labeled ABCG2
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • test results are particularly well achieved when in a competitive reaction between labeled surface antigen and unlabelled Spermienoberfestantigen 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • effector particles all entities which are capable of binding to a sperm-specific surface antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, Basic Blue 20, double green SF, Double Green SF, Hoffmann's Green, green powder, Vert Lumiere, Vert de Paris, Vert Etincelle, Methyl green, Basic Blue 20, double green SF, Double Green SF,
  • 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 ⁇ -galactosidase.
  • ⁇ -galactosidase hydrolyzes the substrate X-GaI (5-bromo-4-chloro-3-indoxyl- ⁇ -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.
  • color-coded protein A color-labeled biotin, histidine or color-labeled streptavidin are included among the coloring particles.
  • 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.
  • 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.
  • 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.
  • 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.
  • PVDF polyvinylidene fluoride
  • HDPE high density polyethylene
  • polypropylene polypropylene
  • glass fibers glass fibers
  • micro glass fibers polyvinyl chloride
  • a polyamide such as nylon or other porous polymers
  • 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.
  • 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 / ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • surface antigen preferably ABCG2
  • the sandwich method produces a colored line in subarea A and subarea B, but not in subarea C.
  • 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.
  • 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.
  • an inexpensive embodiment of the fertility test 1 is to provide regions of different pore size on the reagent layer 2.
  • the reaction layer 2 is made either in one piece or else from at least two layer parts.
  • 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.
  • 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.
  • 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.
  • size-reduced pores and carrier particles or color particles 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reagent layer 2 is surrounded or enveloped by the carrier body 4 almost over the whole area.
  • 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.
  • 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.
  • 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.
  • a cylindrical tube is introduced, which is open on one side.
  • a cylindrical rotary plug with a small opening is fitted.
  • space A and space B 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.
  • sample samples can be precisely metered.
  • the preferably diluted test sample in room A of the cup so that it rests on the intermediate bottom.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fertility test 1 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 alsdrrocknete 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the carrier body 4 of the strip which rests on the strip acting as reagent layer 2, is permeable to labeled or conjugated antibodies.
  • the strip resting on the strip formed as reagent layer 2 has no carrier body 4 in the overlapping area.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • dithiothreitol is also suitable for liquefaction of an ejaculate sample in the previously described manner of use.
  • liquefaction also contributes to the fact that the reagent layer 2 is hydrophobic at the sample application zone, for example by silylation or alkylation.
  • 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.
  • the incubated sample moves horizontally due to capillary action through the reagent layer 2.
  • sample liquid flows vertically through the reagent layer 2.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne la variante de l'antigène superficiel ABCG2 présent sur des spermatozoïdes, ainsi que des anticorps qui neutralisent un antigène de surface sur des spermatozoïdes, c'est-à-dire un antigène qui se trouve sur la membrane extérieure du spermatozoïde, ou sur une membrane du spermatozoïde qui est accessible de l'extérieur. Une membrane du spermatozoïde n'est accessible de l'extérieur que si le spermatozoïde ne perd pas à cet effet sa part d'acrosome par dégradation, donc si le spermatozoïde reste essentiellement intact. L'invention concerne également un test de fertilité (1) et son procédé d'utilisation. Le test de fertilité (1) est constitué d'une couche de réactif (2) avec une zone de test (10) constituée de surfaces partielles A, B et C de tailles adaptées, et un agent immobilisé dans une quantité prédéfinie dans chacune de ses surfaces. Cet agent est un anticorps selon l'invention ou des pores de taille réduite. De plus, le test de fertilité comprend selon une forme structurelle développée un corps support (4), et selon une autre configuration de l'invention, une couche filtrante (8) à taille de pores réduite; selon un développement supplémentaire, il est constitué d'au moins deux bandes dont l'une est la couche de réactif (2). Le procédé de mise en action du test de fertilité (1) comprend : l'incubation d'un échantillon; l'application d'une partie aliquote de cet échantillon sur le test de fertilité (1); une incubation supplémentaire pendant une durée prescriptible; et finalement une lecture.
PCT/EP2008/004170 2007-09-25 2008-05-26 Test de fertilité WO2009043393A1 (fr)

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DE202011104802U1 (de) * 2011-08-20 2012-01-26 Christian Kober Strohhalm zur Messung schädlicher Stoffe bzw. von Partydrogen in Getränken
DE202012005997U1 (de) 2012-06-20 2012-07-18 Claus Linder Gefäß für Getränke und Flüssigkeiten jeglicher Art zur Messung schädlicher Stoffe bzw. Rauschgift und Partydrogen in Getränken
CN104459152A (zh) * 2014-12-03 2015-03-25 青岛拓新生物科技有限公司 一种基于胶体金免疫层析技术的精子浓度快速检测方法
US10417481B2 (en) 2015-06-22 2019-09-17 The Brigham And Women's Hospital, Inc. Home evaluation of the quality of semen samples

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ES2900645T3 (es) * 2019-05-14 2022-03-17 Hilgenberg GmbH Pajitas para beber de uso múltiple y su producción

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WO2000050639A2 (fr) * 1999-02-22 2000-08-31 Variagenics, Inc. Variations de sequences geniques presentant une utilite pour la selection du traitement d'une maladie
WO2001060968A1 (fr) * 2000-02-16 2001-08-23 Genosis Limited Dispositifs pour separation de spermatozoides motiles
EP1743649A1 (fr) * 1997-03-06 2007-01-17 The University of Adelaide Traitement ou diagnostic d'une stérilité par le TGF-beta ou l'activine
WO2007025166A2 (fr) * 2005-08-25 2007-03-01 Repair Technologies, Inc. Dispositifs, compositions et methodes de protection et de reparation de cellules et de tissus

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AU4214799A (en) * 1998-05-28 1999-12-13 St. Jude Children's Research Hospital Expansion of hematopoietic stem cells transduced with mdr-1 and methods of use thereof
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US5665556A (en) * 1990-03-02 1997-09-09 Brigham And Women's Hospital Complement components and binding ligands in fertility
EP1743649A1 (fr) * 1997-03-06 2007-01-17 The University of Adelaide Traitement ou diagnostic d'une stérilité par le TGF-beta ou l'activine
WO2000050639A2 (fr) * 1999-02-22 2000-08-31 Variagenics, Inc. Variations de sequences geniques presentant une utilite pour la selection du traitement d'une maladie
WO2001060968A1 (fr) * 2000-02-16 2001-08-23 Genosis Limited Dispositifs pour separation de spermatozoides motiles
WO2007025166A2 (fr) * 2005-08-25 2007-03-01 Repair Technologies, Inc. Dispositifs, compositions et methodes de protection et de reparation de cellules et de tissus

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE202011104802U1 (de) * 2011-08-20 2012-01-26 Christian Kober Strohhalm zur Messung schädlicher Stoffe bzw. von Partydrogen in Getränken
DE202012005997U1 (de) 2012-06-20 2012-07-18 Claus Linder Gefäß für Getränke und Flüssigkeiten jeglicher Art zur Messung schädlicher Stoffe bzw. Rauschgift und Partydrogen in Getränken
CN104459152A (zh) * 2014-12-03 2015-03-25 青岛拓新生物科技有限公司 一种基于胶体金免疫层析技术的精子浓度快速检测方法
US10417481B2 (en) 2015-06-22 2019-09-17 The Brigham And Women's Hospital, Inc. Home evaluation of the quality of semen samples
US10977477B2 (en) 2015-06-22 2021-04-13 The Brigham And Women's Hosptial, Inc. Home evaluation of the quality of semen samples

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