Peptide that can serve as a standard for Proakap4 dosage - Dosage and related use kit

Abstract

The present invention relates to an isolated peptide that can serve as a standard for the assay of proAKAP4. Characteristically, said peptide is selected from peptides having an N-terminal end which comprises or is formed of a region which has 100% identity with a region of the sequence formed by the first 188 amino acids of the N-terminal end of the proAKAP4 protein represented by the sequence SEQ ID NO 4 and a C-terminal end which comprises or is formed of a region which has 100% identity with a region of the C-terminal sequence of said proAKAP4 protein formed by the last 100 amino acids of said proAKAP4 protein, said N-terminal end of said peptide comprising in particular at least 6 and in particular 11 amino acids, said C-terminal end of said peptide comprising at least 6 and in particular 10 amino acids and said peptide comprising from 16 to 50 amino acids, and in particular 41 amino acids.

Description

Title of the invention: Peptide that can serve as a standard for the dosage of Proakap4 - Dosing Kit and associated uses

[0001] The present invention relates to an isolated peptide that can be used as a standard or benchmark for the assay of the proAKAP4 protein.

[0002] The present invention also relates to a proAKAP4 detection / assay kit which contains the peptide of the invention.

[0003] The present invention also relates to the use of the peptide of the present invention for the detection or quantification, in particular by spectrometry, of the human proAKAP4 protein or that of another mammal.

[0004] The AKAP4 protein is a known protein present in most organisms, including mammals and reptiles. It is also called AKAP82, particularly in mice.

[0005] The AKAP4 protein has a precursor, called proAKAP4 or proAKAP82 protein. This precursor has a higher molecular mass than that of the protein.

[0006] In spermatozoa, particularly mammalian spermatozoa, the AKAP4 protein is present simultaneously with its precursor, the proAKAP4 protein. The two proteins have very similar structures and are therefore difficult to distinguish and thus to quantify when they are mixed. For example, the publication entitled "Regulation of protein tyrosine phosphorylation in human sperm by a calcium / calmodulin-dependent mechanism: identification of A kinase anchor proteins major substrates for tyrosine phosphorylation," published in 1996 in the journal "Developmental Biology," 180, pages 284 to 296, indicates that it is possible to detect the AKAP4 protein and its precursor (proAKAP4) in human sperm without distinguishing between the two. In the aforementioned document, detection is carried out using polyclonal antibodies directed against the AKAP82 protein, which has been previously separated by electrophoresis.This document specifies that in practice the antibodies bind to both the AKAP4 protein and its precursor and therefore do not allow the AKAP82 (or AKAP4) protein to be distinguished from its precursor (or proAKAP4).

[0007] There are also commercial kits available for measuring the AKAP4 protein. For example, CUSABIO® markets an ELISA kit for detecting the human AKAP4 protein. However, the kit's instructions indicate that it cannot distinguish FAKAP4 from its homologs, and therefore, in particular, from proAKAP4.

[0008] Mybiosource® also markets a Sandwich ELISA kit for the assay of porcine AKAP4. This kit uses antibodies that target conformational epitopes, not linear epitopes. The kit cannot be used to assay all AKAP4s and, in any case, cannot be used to measure AKAP4 alone or proAKAP4 alone.

[0009] Document WO 2015 / 032842 A1 describes a method for determining sperm quality that implements the measurement of AKAP4 protein expression levels in spermatozoa. This document describes a sandwich ELISA-type device that conventionally uses two antibodies, a polyclonal and a monoclonal. One is directed against the C-terminal portion (the polyclonal) of the AKAP4 protein, the other against its N-terminal portion (the monoclonal). This kit also uses recombinant human AKAP4 protein as a standard; it thus allows the quantification of FAKAP4 (full-length, native protein) contained in a mixture obtained from lysed spermatozoa.

[0010] Recombinant human AKAP4 protein is expensive. Furthermore, the kit described in document WO 2015 / 032842 A1 does not allow for the measurement of proAKAP4. However, proAKAP4 is useful to measure, particularly in mammalian and especially human spermatozoa. Thus, in humans, quantitative proAKAP4 assays by ELISA reveal at least two groups in normo-zoospermic patients, depending on the amount of proAKAP4: group A characterized by the presence of proAKAP4 and group B characterized by its presence at low concentration or absence. The concentration of proAKAP4 is correlated with progressive sperm motility in normo-zoospermic individuals. A statistical analysis was performed between the groups expressing proAKAP4 (groups A and B). In group A, the miscarriage rate is significantly reduced compared to group B.For in vitro fertilization techniques, IVF and ICSI combined, better embryo quality, an increased cryopreservation rate, an increase in the clinical pregnancy rate and live birth rate were observed in group A compared to group B.

[0011] There is therefore a real need to accurately measure proAKAP4, especially when it is in mixture with the AKAP4 protein.

[0012] One problem that the present invention aims to solve is to provide a kit that allows the proAKAP4 protein to be measured.

[0013] Another problem that the present invention aims to solve is to provide a proAKAP4 assay kit that can be used for the assay of proAKAP4, even when the latter is mixed with the AKAP4 protein and in particular in a mixture from lysed spermatozoa.

[0014] Another problem that this invention proposes to solve is to provide a A kit that allows for the detection and / or quantification of the proAKAP4 protein, which is easy to manufacture and inexpensive.

[0015] Another problem that the present invention aims to solve is to provide a proAKAP4 assay kit which makes it possible to detect and / or measure human proAKAP4 but also that present in the spermatozoa of other animals, in particular mammals, such as for example, pigs (in particular the boar), sheep (in particular the ram), cattle (in particular the bull), goats (in particular the buck), equines (in particular the horse), canids (in particular the dog), rodents (in particular the mouse, the rat and the rabbit).

[0016] Another problem that the present invention aims to solve is to provide a standard allowing the assay of proAKAP4 and which is not a recombinant protein.

[0017] The present invention relates to a synthetic peptide which can in particular serve as a standard for the assay of proAKAP4.Characteristically, the synthetic peptide is selected from peptides having an N-terminal end which comprises or is formed from a region which has 100% identity with a region of the sequence formed by the first 188 amino acids of the N-terminal end of the proAKAP4 protein represented by the sequence SEQ ID NO 4 and a C-terminal end which comprises or is formed from a sequence which has 100% identity with a region of the C-terminal sequence comprising or consisting of the last 100 amino acids of said proAKAP4 protein, in that said sequence forming said N-terminal end of said peptide comprising in particular at least 6 and up to 12 amino acids, in that said sequence forming said C-terminal end of said peptide comprising at least 6 and up to 10 amino acids and said peptide comprising from 16 to 50 amino acids, and more precisely 41 amino acids.

[0018] According to another embodiment, the percentage of identity between the regions of the peptide and those of the aforementioned proAKAP4 protein is equal to 70% or between 70% and 90% or 95% or equal to 90% or 95%. The percentage of identity of one region may be different from another.

[0019] The proAKAP4 protein represented by the sequence SEQ ID NO 4 corresponds to the protein whose entry number is Q5JQC9 in the Uniprot database, entry version 111 (of February 28, 2018).

[0020] The first 188 amino acids of the proAKAP4 protein correspond to the amino acids located from position 1 to position 188 of the SEQ ID NO 4 sequence.

[0021] The last 100 amino acids correspond to the amino acids located from position 753 to position 853 on the SEQ ID NO 4 sequence.

[0022] All protein entry numbers cited in Table 1 are Uniprot database entry numbers, version 111 (from February 28, 2018). The Applicant observed that such a peptide could be used for the detection and quantification of proAKAP4; unexpectedly, a peptide like the one described above behaves like the proAKAP4 protein and can be recognized by anti-proAKAP4 monoclonal or polyclonal antibodies. It can be used for the detection or quantification of proAKAP4, particularly in a complex mixture, such as the mixture resulting from sperm lysis. Indeed, in such a mixture, there are numerous interactions between proteins; the close proximity of the proAKAP4 and AKAP proteins makes them difficult to distinguish from one another. The three-dimensional shape of the proAKAP4 protein is unknown and can change depending on the medium in which the protein is found. It was therefore not obvious to a person skilled in the art that a peptide like the one described above could behave and be recognized like the proAKAP4 protein itself.

[0023] According to a particular embodiment, the peptide comprises 42 amino acids and the N-terminal sequence comprises 12 amino acids, while the C-terminal sequence comprises 10 amino acids.

[0024] According to a particular embodiment, the peptide comprises 28 amino acids, the N-terminal sequence comprises 12 amino acids and the C-terminal sequence comprises 10 amino acids.

[0025] The Applicant also demonstrated that the peptide of the invention allows for the measurement of human proAKAP4 as well as that of other animals, particularly mammals. It thus allows, for example, the measurement of proAKAP4 in the animals listed in Table 1 below.

[0026] Regardless of the embodiment of the peptide of the invention, it may be contained in a composition, in particular a buffer solution, containing as a solvent PBS (a phosphate saline buffer conventionally containing NaCl, KCl, Na2HPO4, and KH2PO4), a mixture of PBS and casein, in particular a mixture containing PBS and 0.04% by mass of casein, Tris (2-amino-2-hydroxymethyl-1,3-propanediol), HEPES, or a mixture of at least two of these buffers. Similarly, the kit of the invention, regardless of its embodiment, may comprise a solution such as the one described above.

[0027] According to a first particular embodiment, the peptide is chosen from

[0028] - peptides comprising a sequence of general formula I:

[0029] X1X2X3 (I) in which

[0030] XI is a peptide represented by the sequence SEQ ID NO: 1 DLQKYALGFQHA;

[0031] X2 is a peptide containing 4 to 20 amino acids;

[0032] X3 is a peptide represented by the sequence SEQ ID NO2: EVMKFAKERQ; and

[0033] - peptides exhibiting at least 80% identity and up to at least 90% identity with the general formula I or with peptides XI and / or X2, the amine function linked to peptide XI and / or the carboxyl group associated with peptide X3 may be protected by a protecting group.

[0034] The protecting group is, by way of example, a phenylmethoxycarbonyl group, a 1,1-dimethylethoxycarbonyl group, an ethyl, methyl or benzyl group.

[0035] According to a particular embodiment of the first embodiment of the invention, peptide X2 has the following general formula II:

[0036] II (X4X5)nn being an integer from 3 to 10 inclusive, in which X4 is an amino acid chosen from D, E, K and R

[0037] X5 is an amino acid selected from A, G, I, V, L.

[0038] X4 and X5 can be chosen independently of each other.

[0039] Advantageously, X4 and X5 are identical. They can, for example, both be alanine.

[0040] The Applicant found that the peptide allowed for accurate dosing when the peptide X2 was not too long and in particular, when n was equal to or greater than 3 or equal to or less than 10 in the aforementioned formula II.

[0041] Advantageously, n=3 or 5 in the formula IL The Applicant has indeed observed that the peptide thus obtained behaved very similarly to proAKAP4, particularly when it is in a complex mixture and that it could therefore be used to quantify the latter, particularly in a complex mixture.

[0042] According to a particular embodiment, the peptide of the invention is represented by the sequence SEQ ID NO 3: DLQKYALGFQHDADADAEVMKFAKERQ. The Applicant has shown that such a peptide is a particularly effective standard.

[0043] The present invention also relates to a kit for the detection and / or quantification of the proAKAP4 protein. Characteristically, the kit of the invention comprises, in particular as a standard, the peptide according to the invention, optionally in solution, and in particular in a solution such as the one mentioned above. This kit may be a kit that allows the quantification of proAKAP4 by spectrometry, in particular by spectrophotometry using optical density measurement or by fluorescence spectrometry, for example.

[0044] The present invention also relates to any proAKAP4 detection kit (without assay) which contains the peptide according to the invention.

[0045] The kit of the invention is not limited to a specific type of kit. It may be, for example, an ELIS A kit, a sandwich ELIS A kit, or an immunochromatographic strip to which one or more antibodies are attached. Based on the sandwich ELISA principle, it may involve well-known state-of-the-art methods such as rapid detection tests like the lateral flow test, tests using gadolinium for antibody detection by NMR, tests allowing measurement at the molecular level such as the test marketed by Quanterix®, or the tests implementing flow cytometry variation (CyTOF).

[0046] Thus, according to a particular embodiment of the kit of the invention, it comprises first antibodies directed against the N-terminal portion of the proAKAP4 protein or directed against the C-terminal portion of the proAKAP4 protein and optionally second antibodies directed against the end of the proAKAP4 protein that is not detected by said first antibodies, and a detection marker linked to said first or second antibodies or capable of reacting with the first or second antibodies. When the kit comprises two types of antibodies as described above, it may be a sandwich ELISA kit or an immunochromatographic strip kit.

[0047] Thus, the kit may include an immunochromatographic strip which includes said first and / or second antibodies or a plate having a plurality of wells at the bottom of which said first or second antibodies are fixed or may be fixed.

[0048] The marker is not limited according to the invention. It can be chosen from radio-detectable markers, dyes, enzymes including peroxidases, biotin, colloids and fluorochromes, gadolinium, colored nanobeads or nanoparticles; the kit can also include, depending on the type of marker, a development solution suitable for reacting with said marker in order to allow visual detection of the latter, either in natural light or by fluorescence.

[0049] When the kit includes an immunochromatographic strip, the marker is advantageously chosen from colored nanobeads, fluorescent nanobeads, colloidal gold beads, polystyrene beads, latex beads and dyes, thus allowing visual detection, without revelation, of the presence of a certain given (and known) quantity of proAKAP4 in the sample being measured.

[0050] The antibodies used in the kit of the invention are not limited. According to a particular embodiment thereof, said first antibodies are capable of binding to the region of the proAKAP4 protein comprising amino acid at position 117 to amino acid at position 128 and said second antibodies are chosen from among the antibodies capable of binding to the region of said proAKAP4 protein comprising amino acid at position 824 to amino acid at position 833.

[0051] The combination of such antibodies with the peptide of the invention, used as a standard, allows the precise quantification of proAKAP4.

[0052] The antibodies may be, for example, the anti-AKAP4 (clone 7E10) antibodies and the anti-AKAP4 (clone 6F12) antibodies marketed by the company SPQI®.

[0053] To perform the proAKAP4 assay using a sandwich ELISA kit, initial antibodies are first fixed to the bottom of the wells of a plate. Known concentrations of the peptide of the invention are added to the wells that will serve as standards. The mixture to be assayed is added to the other wells, if applicable. The peptide is diluted (the dilution ratio being known). It binds to the first antibody, as does the proAKAP4 protein. The second antibody is then added, binding to the other end of the peptide or to the proAKAP4 protein. The optical density of the mixtures in the wells is then measured, and using the calibration curve obtained for the wells containing the peptide, the concentration of proAKAP4 in the other wells containing the mixture to be assayed is deduced. If the second antibody contains a fluorochrome, the fluorescence emitted by the wells is measured either with a densitometer equipped with a UV system or with a spectrofluorometer. If the marker contains a radioisotope, the intensity of the emitted radiation is measured.If the marker is an enzyme (for example, peroxidase or alkaline phosphatase), the labeled antibody is detected by adding the enzyme's substrate; this yields a colored product whose color is measured by spectrophotometry. If the antibody is labeled with biotin, it is detected by adding avidin or streptavidin, itself labeled with several enzymes or several fluorochromes.

[0054] The present invention also relates to the use of the peptide according to the invention for the spectrometric determination of proAKAP4, in particular in a mixture obtained by lysis of spermatozoa, in particular mammalian spermatozoa and in particular human spermatozoa.

[0055] The present invention also relates to a first method for quantifying human or other proAKAP4 contained in a sample, according to which:

[0056] - solutions are prepared to serve as a standard, each containing a quantity known of the peptide according to the invention, said solutions each containing a different quantity of said peptide;

[0057] - a given quantity of a first antibody is deposited on the bottom of several wells as mentioned above;

[0058] - a given quantity of each of the said is added into separate wells, a given quantity of the said standard solutions and a given quantity of a second labeled antibody as above is added;

[0059] - rinsing is optional to remove said secondary supernumerary antibodies which are not related to the peptide contained in the said standard solutions.

[0060] - the optical density of each of the solutions contained in the wells is measured and From this we deduce a standard curve which correlates the optical density of each of the solutions with the logarithm of their molar or mass concentration in proAKAP4 or, in the case of the assay of a non-human proAKAP4, we determine from the following mass ratio: molecular mass of human proAKAP4 / Molecular mass of proAKAP4 contained in the sample, the optical density as a function of the logarithm of the mass concentration of proAKAP4 contained in the sample.

[0061] According to this method, the concentration of proAKAP4 in the sample is determined either simultaneously with the preparation of the standard curve or separately, in the following manner:

[0062] - a well is added to the bottom of which the same given quantity has been fixed said first antibody, for the preparation of the standard curve, a given quantity of said sample, possibly diluted;

[0063] - a given quantity of said second labeled antibody is added and the product is rinsed evenly temporarily to eliminate excess antibodies;

[0064] - the optical density of the solution contained in the well(s) is measured and then... deduced using the standard curve, the concentration of proAKAP4 of said sample.

[0065] According to the invention, according to a second method, it is also possible to deposit onto a substrate, for example, an immunochromatographic strip, a given quantity of said first antibody and the same quantity of said second labeled antibody. The antibodies are as defined with reference to the first method. The same quantity of the peptide according to the invention is also deposited on said support, at a distance from said antibodies. In both methods, the term quantity refers not to the mass of substance deposited but to the number of moles deposited. One end of the strip is then placed with a given quantity of said sample, possibly diluted in an elution solution.The appearance of a more or less colored band on the substrate indicates, in a cruder way, the concentration of proAKAP4, by comparison with a standard that shows colored bands and associates concentration ranges with these bands, or even a numerical index or other indicator of sperm quality.

[0066] The two aforementioned methods can be applied, in particular, to a sample of human or other spermatozoa whose spermatozoa are lysed before application of the assay method.

[0067] The term proAKAP4 refers to the human protein named AKAP4, whose entry number is Q5JQC9 in the Uniprot database, entry version 111 (of February 28, 2018). This is human proAKAP4. When reference is made to a proAKAP4 protein of non-human origin, the organism of origin is specified.

[0068] The term “percentage of identity” refers to the percentage of identical amino acid residues between the sequence of the invention and the sequence to be compared, across a comparison window, said percentage being obtained after best alignment. This alignment can be global (the entire sequence being compared is aligned with the peptide sequence) or local (only the C- and N-terminal parts of the peptide being compared are aligned with the SEQ ID NO. 1 and SEQ ID NO. 2 sequences of the peptide of the invention). Peptides having a % of identity with the peptide of the invention, as defined in Formula I, may thus include insertions or deletions of one or more amino acids (for example "gaps") with respect to the sequence of the peptide of the invention (which therefore does not include these insertions or deletions).

[0069] The terms "assay" and "quantification" refer, unless otherwise specified, to either measuring the amount of proAKAP4 in a sample or determining that the concentration of proAKAP4 in the sample is at least equal to a given and known value, which corresponds, in particular, to the amount of antibody used for quantification. Thus, in the case of a kit containing an immunochromatographic strip, it is ensured that the amount (concentration) of proAKAP4 contained in the tested sample is equal to or greater than a given value.

[0070] The present invention, its features and the various advantages it provides will become clearer upon reading the following examples, which are given by way of non-limiting example and which refer to the accompanying drawings in which:

[0071] Fig. 1 represents a standard curve (optical density as a function of the logarithm of mass concentration) obtained from the peptide of the present invention for the assay of horse proAKAP4 in previously lysed horse spermatozoa;

[0072] Fig. 2 represents a standard curve (optical density as a function of the logarithm of mass concentration) obtained from the peptide of the present invention for the assay of bull proAKAP4 in bull spermatozoa, previously lysed;

[0073] Figure 3 represents a standard curve (optical density as a function of the logarithm of mass concentration) obtained from the peptide of the present invention for the assay of porcine proAKAP4 in previously lysed porcine spermatozoa.

[0074] Fig. 4 represents a standard curve (optical density as a function of the logarithm of mass concentration) obtained from the peptide of the present invention for the assay of human proAKAP4 in previously lysed human spermatozoa;

[0075] [EXAMPLES]

[0076] Peptide synthesis

[0077] A peptide of the invention, corresponding to the sequence SEQ ID NO°3, was synthesized in solid phase using conventional methods. The peptide synthesis was carried out using amino acids whose amine groups were protected (Fmoc). The synthesis begins with the last amino acid in the carboxy-terminal position moving towards the first amino acid at the amino-terminal end. In each synthesis cycle, the N-terminal protecting group on the C-terminal amino acid of the peptide is first The amino acid is unprotected. After the removal of the protecting group, the added amino acid is activated at its carboxyl group by a coupling agent such as DCC (dicyclohexyl carbodiimide) or DIC (diisopropyl carbodiimide), thus facilitating the formation of a peptide bond between the unprotected amino-terminal portion of the first amino acid and the activated carboxyl group. The newly synthesized amino-terminal group is again unprotected and coupled to the next amino acid. This coupling cycle is repeated until the peptide sequence described above is obtained.

[0078] Example of a protocol for the assay of proAKAP4 in a mixture obtained from sperm lysis using a sandwich ELISA kit

[0079] The following solutions are prepared and then stored at 4°C. The water is always water conforming to ISO 3696.

[0080] Carbonate buffer (coating buffer)

[0081] A 0.1 M NaHCO3 solution is prepared: (NaHCO3 PM=84.01), 4.2 g per 500 mL of H2O according to ISO 3696. Anhydrous sodium carbonate (2.645 g) is added to 250 mL of water according to ISO 3696 (Na2CO3 PM=105.99) to form a 0.1 M solution. The aforementioned solution is gradually added to the NaHCO3 solution until a pH of 9.6 is obtained.

[0082] Citrate / phosphate buffer (development buffer)

[0083] 10.5 g of citric acid is dissolved in 100 mL of water (C6H8O7, H2O PM=210.14). 14.91 g of Na2PO4 is dissolved in 210 mL of water. 207.5 mL of the Na2PO4 solution is mixed with 97 mL of citric acid solution. The volume is then made up to the required volume with water, and the pH is adjusted to 5 with citric acid.

[0084] Buffer pbs Ix:

[0085] One sachet of PBS buffer preparation (marketed by Sigma - Ref: P3813-10PAK) is diluted in IL of water.

[0086] Casein 0.4% (saturation buffer)

[0087] Prepare the day before and shake for several hours to homogenize

[0088] Weigh out 0.4g of casein, add PBS Ix buffer to obtain 100mL. Shake for half a day - Store at 4°C with shaking

[0089] PBS / Tween 0.05% tampon (washing tampon)

[0090] One sachet of PBS Ix is diluted in IL of water. 500 pL of Tween20 is added.

[0091] 0.04% PBS / casein buffer (dilution buffer)

[0092] The 0.4% PBS / Casein buffers are diluted one-tenth in PBS Ix.

[0093] sulfuric acid (reaction stop solution)

[0094] Mix 10.32 mL H2SO4 in 89.78 mL of H2O.

[0095] Plate coating

[0096] Coating solutions are produced. Anti-AKAP4 antibody clone 7E10 (ref. 4BDX-1702) diluted to 1.5 pg / mL in carbonate buffer (coating buffer). Coating in NUNC F8 Maxisorp® plates. 100 pL / well of anti-AKAP4 (previously diluted in coating buffer) is loaded. Incubate overnight at 4°C with shaking.

[0097] Saturation: The coating solution is removed by washing 3 times with 300pL / well of PBS buffer (washing buffer)

[0098] Sample incubation

[0099] Samples are prepared diluted in 0.04% PBS / Casein (dilution buffer):

[0100] A standard curve is prepared with the peptide of the invention (sequence SEQ ID NO° 3). The following solutions are prepared containing respectively: 300, 150, 75, 37, 5, 17.25, 8.6125, 4.362 ng / well.

[0101] Sperm treatment

[0102] The spermatozoa are lysed in a known manner. 100pL / well of lysed semen is deposited and incubated for 2 hours at room temperature.

[0103] Incubation Antibody detection

[0104] The plate is washed 3 times with 300 pL / well using PBS / Tween 0.05% / Proclin 0.035% buffer (wash buffer). The detection antibody is prepared in PBS / casein 0.04% (dilution buffer). The Anti-AKAP4-Nter:6F12 conjugated antibody, commercially available from SPQI (ref: 4BDX-1701), is used – mouse monoclonal – 0.5 pg / mL in PBS / casein 0.04% (dilution buffer). 100 pL / well of the detection antibody is added and the plate is incubated for 1 hour at room temperature with shaking.

[0105] Development (Day 2)

[0106] The plate is washed 5 times with 300 pL / well of PBS / Tween 0.05% / Proclin 0.035% buffer (washing buffer). The detection solution is prepared (at the last minute and protected from light). For 1 plate, 10 mL of Citrate / Phosphate buffer (detection buffer) and 1 TMB pellet are added. Once the pellet has dissolved, 2 pL of 30% H₂O₂ is added, or alternatively, ready-to-use TMB is used. 100 pL / well of the solution is added. The plate is incubated for 30 min at room temperature (in the dark). The reaction is stopped with 50 pL / well of 30% sulfuric acid (H₂SO₄). The reading is taken with a spectrophotometer at 450 nm.

[0107] The aforementioned protocol was used to obtain the standard curves in Figures 1 to 4 for the assay of proAKAP4 in boar, horse, human, and bull spermatozoa. In these examples, it is the proAKAP4 protein of the mammal in question that is assayed. The peptide of the invention makes it possible to measure the proAKAP4 proteins of various animals, particularly mammals, due to the presence of the N-terminal and C-terminal sequences of the peptide of the invention in the proAKAP4 proteins of these animals. Thus, the peptide corresponding to the SEQ ID NO° 3 sequence makes it possible, in particular, to detect or measure the proAKAP4 proteins of

[0108] Animals grouped in Table 1 below. This list is not exhaustive. [Tables 1] Protein Reference NBCEprotein Species Select seq ref IXP_013831926.ll Capra hircus Select seq ref IXP_014982904.ll Macaca mulatta Select seq reflXP_009195875. Il Papio anubis Select sequence reflXP_007989913.ll Chlorocebus sabaeus Select sequence reflXP_005593638.ll Macaca fascicularis Select sequence reflXP_011760281.11 Macaca nemestrina Select sequence reflXP_007989914.ll Chlorocebus sabaeus Select seq gblELK11371.ll Pteropus alecto Select seq refIXP_010604603.Il Fukomys damarensis Select seq reflXP_006743070.ll Leptonychotes weddellii Select seq reflXP_011962095.Il Ovis aries Select seq refIXP_017899481.11 Capra hircus Select sequence emblCAA75494.ll Homo sapiens Select sequence reflXP_001363577.ll Monodelphis domestica Select sequence reflXP_010604604.Il Fukomys damarensis Select sequence reflXP_001509793.ll Ornithorhynchus anatinus Select sequence reflXP_003779687.ll Pongo abelii Select sequence reflXP_004064208.2l Gorilla gorilla gorilla Select sequence reflXP_016799679.ll Pan troglodytes Select sequence reflXP_007989915.ll Chlorocebus sabaeus Select seq reflXP_005593639.ll Macaca fascicularis Select seq reflXP_003917767.Il Papio anubis Select seq refIXP_003806003.Il Pan paniscus Select seq reflXP_002808575.2l Macaca mulatta Select seq reflXP_003276909.ll Nomascus leucogenys Select seq dbjIBAF85152.il Homo sapiens Select seq reflNP_003877.2l Homo sapiens Select seq reflXP_011846734.Il Mandrillus leucophaeus Select seq gblKFO22638.ll Fukomys damarensis . Select seq reflXP_012408905.Il Sarcophilus harrisii Select seq reflXP_004670116.Il Jaculus jaculus Select seq gbIABL74506.ll Notamacropus eugenii Select seq reflXP_021009702. Il Mus caroli Select seq reflXP_014584335.ll Equus caballus Select seq reflNP_033781.2l Mus musculus Select seq gblELR62277.ll Bos mutus Select seq reflXP_021044175.ll Mus pahari Select seq reflXP_019812053.Il Bos indicus Select seq reflXP_016073998.ll Miniopterus natalensis Select seq reflXP_014682650.ll Equus asinus Select seq reflXP_012666755.Il Otolemur gamettii Select seq reflXP_008507887.ll Equus przewalskii Select seq gbIEFB29220.Il Ailuropoda melanoleuca Select seq reflNP_776660.ll Bos taurus Select seq reflXP_006059540.ll Bubalus bubalis Select seq reflXP_005977085.ll Pantholops hodgsonii Select seq reflXP_005887684.ll Bos mutus Select seq reflXP_005700789.ll Capra hircus Select seq reflXP_005657864.ll Sus scrofa Select seq reflXP_004022127.ll Ovis aries Select seq reflXP_003505846.ll Cricetulus griseus Select seq tpglDAA12829.ll Bos taurus Select seq reflXP_022363746.ll Enhydra lutris kenyoni Select seq reflXP_021534407.Il Neomonachus schauinslandi Select seq reflXP_020834628.ll Phascolarctos cinereus Select seq reflXP_020007990.ll Castor canadensis Select seq reflXP_019273047.ll Panthera pardus Select seq gblEDL83863.ll Rattus norvegicus Select seq reflXP_851545.ll Canis lupus familiaris Select seq reflXP_014928063.Il Acinonyx jubatus . Select sequel reflXP_014682651.ll Equus asinus Select sequel reflXP_006877688.ll Chrysochloris asiatica Select sequel reflXP_006902969.ll Elephantulus edwardii Select sequel reflXP_006743071.ll Eeptonychotes weddellii Select seq reflXP_008507888.ll Equus przewalskii Select seq reflNP_077378.ll Norwegian Rattus Select seq reflXP_007090150.ll Panthera tigris altaica Select seq reflXP_006097270.ll Myotis lucifugus Select seq reflXP_005864603.ll Myotis brandtii Select seq reflXP_004755176.ll Mustela putorius furo Select seq reflXP_004713195.ll Echinops telfairi Select seq reflXP_004606517.ll Sorex araneus Select seq reflXP_004376884.ll Trichechus manatus latirostris Select seq reflXP_002917787. Il Ailuropoda melanoleuca Select seq reflXP_019653617.ll Ailuropoda melanoleuca Select seq reflXP_012612405.Il Microcebus murinus Select seq reflXP_012500292.ll Propithecus coquereli Select seq reflXP_010604605.Il Fukomys damarensis Select seq reflXP_008053507.ll Carlito syrichta Select seq reflXP_007956069.ll Orycteropus afer afer Select seq reflXP_005085139.ll Mesocricetus auratus Select seq reflXP_004064207.2l Gorilla gorilla gorilla Select seq reflXP_016799680.ll Pan troglodytes Select seq reflXP_014982905.ll Macaca mulatta Select seq reflXP_012612406.Il Microcebus murinus Select seq reflXP_012500293.ll Propithecus coquereli Select seq reflXP_011885330.Il Cercocebus atys Select seq reflXP_011846735.Il Mandrillus leucophaeus Select seq reflXP_008156477.ll Eptesicus fuscus Select seq reflXP_007989916.ll Chlorocebus sabaeus Select seq reflXP_006995469.ll Peromyscus maniculatus bairdii . Select seq reflXP_005593640.ll Macaca fascicularis Select seq reflXP_003917766.ll Papio anubis Select seq reflXP_003806002. Il Pan paniscus Select seq reflXP_003276908.ll Nomascus leucogenys Select seq reflNP_647450.ll Homo sapiens Select seq reflXP_008053509.ll Carlito syrichta Select seq reflXP_007533976.ll Erinaceus europaeus Select seq reflXP_006217896.ll Vicugna pacos Select seq reflXP_006758966.ll Myotis davidii

[0109] Example of a standard curve for the determination of equine proAKAP4 contained in a mixture obtained from the lysis of equine spermatozoa

[0110] Fig. 1 represents the standard curve which represents the optical density as a function of the logarithm of the mass concentration of equine proAKAP4. Knowing the peptide mass concentration of each of the solutions used for the standard curve, the logarithm of the mass concentration of equine proAKAP4 can be deduced as a function of the ratio of the molecular mass 1 of the peptide / mass of equine proAKAP4 (which is known) (see Table 1).

[0111] Example of a standard curve for the assay of bull proAKAP4, which is contained in a mixture obtained from the lysis of bull spermatozoa

[0112] Fig. 2 represents the standard curve which represents the optical density as a function of the logarithm of the mass concentration of bovine proAKAP4. Knowing the peptide mass concentration of each of the solutions used for the standard curve, the logarithm of the mass concentration of bovine proAKAP4 (which is known) can be deduced as a function of the ratio of peptide mass to molecular mass of bovine proAKAP4 (which is known) (see Table 1).

[0113] Example of a standard curve for the assay of boar proAKAP4, which is contained in a mixture obtained from the lysis of boar spermatozoa

[0114] Fig. 3 shows the standard curve which represents the optical density as a function of the logarithm of the mass concentration of porcine proAKAP4. Knowing the peptide mass concentration of each of the solutions used for the standard curve, the logarithm of the mass concentration of porcine proAKAP4 can be deduced as a function of the ratio of the peptide mass to the molecular mass of porcine proAKAP4 (which is known) (see Table 1).

[0115] Example of a standard curve for the assay of human proAKAP4, which is contained in a mixture obtained from the lysis of human spermatozoa

[0116] Fig. 4 represents the standard curve which represents the optical density as a function of the logarithm of the mass concentration of proAKAP4. Knowing the peptide mass concentration of each of the solutions used for the standard curve, the logarithm of the mass concentration of human proAKAP4 can be deduced as a function of the optical density.

Claims

Demands

1. Synthetic peptide which can in particular serve as a standard for the assay of the proAKAP4 protein, characterized in that said peptide is selected from peptides comprising a sequence of general formula I: X1X2X3 (I) in which XI is a peptide represented by the sequence SEQ ID NO: 1 DLQKYALGFQHA; X2 is a peptide containing 4 to 20 amino acids; X3 is a peptide represented by the sequence SEQ ID NO: 2 EVMKFAKERQ; the amine function linked to peptide XI and / or the carboxyl group associated with peptide X3 being optionally protected by a protecting group.

2. Peptide according to claim 1, characterized in that peptide X2 has the following general formula II: (II) (X4X5)nn being an integer from 3 to 10 inclusive, in which X4 is an amino acid selected from D, E, K and R; X5 is an amino acid selected from A, G, I, V, L.

3.

4. Peptide according to claim 2, characterized in that n=3 or 5. Peptide according to any one of the preceding claims, characterized in that it is represented by the sequence SEQ ID NO 3: DLQ-KYALGFQHADADADAEVMKFAKERQ

5. Kit enabling the detection and / or quantification, in particular of the proAKAP4 protein, characterized in that it comprises, in particular as a standard, the peptide according to any one of claims 1 to 4.

6. Kit according to claim 5, characterized in that it comprises first antibodies directed against the N-terminal part of the proAKAP4 protein or directed against the C-terminal part of the proAKAP4 protein and optionally second antibodies directed against the end of the proAKAP4 protein which is not detected by said first antibodies and a detection marker linked to said first or second antibodies or capable of reacting with the first or second antibodies and in that said first antibodies are in particular selected from antibodies capable of binding to the region of the proAKAP4 protein comprising amino acid at position 117 to amino acid at position 128 and said second antibodies are selected from among antibodies capable of binding to the region of said proAKAP4 protein comprising amino acid at position 824 to amino acid at position 833.

7. Kit according to any one of claims 5 and 6, characterized in that it comprises an immunochromatographic strip which includes said first and / or second antibodies or a plate comprising a plurality of wells at the bottom of which said first or second antibodies are fixed or may be fixed.

8. Kit according to any one of claims 6 to 7, characterized in that said marker is selected from radio-detectable markers, enzymes including peroxidases, biotin, gadolinium and fluorochromes, colored nanobeads and nanoparticles and in that it optionally comprises a development solution capable of reacting with said marker in order to allow visual detection of the latter, either in natural light or by fluorescence.

9. Use of the peptide according to any one of claims 1 to 4 for the detection and / or assay, in particular by spectrometry, of proAKAP4 of a mammal selected from humans, horses (equus caballus), pigs (Sus scrofa), Capra hircus, Macaca mulatta, Papio anubis, Chlorocebus sabaeus, Macaca fascicularis, Macaca nemestrina, Chlorocebus sabaeus, Pteropus alecto, Fukomys damarensis, Eeptonychotes weddellii, Ovis aries, Capra hircus, Homo sapiens, Monodelphis domestica, Fukomys damarensis, Ornithorhynchus anatinus, Pongo abelii, Gorilla gorilla gorilla, Pan troglodytes, Chlorocebus sabaeus, Macaca fascicularis, Papio anubis, Pan paniscus, Macaca mulatta, Nomascus leucogenys, Mandrillus leucophaeus, Sarcophilus harrisii, Jaculus jaculus, Notamacropus eugenii, Mus caroli, Equus caballus, Mus musculus, Bos mutus, Mus pahari, Bos indicus, Mi-niopterus natalensis, Equus asinus, Otolemur garnettii, Equus pr-zewalskii, Ailuropoda melanoleuca, Bos taurus,Bubalus bubalis, Pantholops hodgsonii, Bos mutus, Capra hircus, Sus scrofa, Ovis aries, Cricetulus griseus, Enhydra lutris kenyoni, Neomonachus schauinslandi , Phascolarctos cinereus, Castor canadensis, Panthera pardus, Rattus norvegiens, Canis lupus familiaris, Acinonyx jubatus, Equus asinus, Chrysochloris asiatica, Elephantulus edwardii, Eeptonychotes weddellii, Equus przewalskii, Panthera tigris altaica, Myotis lucifugus, Myotis brandtii, Mustela putorius furo, Echinops telfairi, Sorex araneus, Trichechus manatus latirostris, Ailuropoda melanoleuca, Microcebus murinus, Propithecus coquereli, Carlito syrichta, Orycteropus afer afer, Mesocricetus auratus, Pan troglodytes, Macaca mulatta, Microcebus murinus, Propithecus coquereli, Cercocebus atys, Mandrillus leucophaeus, Eptesicus fuscus, Chlorocebus sabaeus, Peromyscus maniculatus bairdii, Macaca fascicularis, Papio anubis, Pan paniscus, Nomascus leucogenys, Carlito syrichta, Erinaceus europaeus, Vicugna pacos, Myotis davidii.

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