A fluorescent labeling kit and method for evaluating sperm function and fertilization potential

Abstract

The present application relates to the field of biomedical detection reagent manufacturing and reproductive medicine technology, and particularly relates to a fluorescently labeled kit and method for evaluating sperm function and fertilization potential. The present application prepares a high-titer rabbit-derived monoclonal antibody combination capable of specifically recognizing and binding to key proteins such as DEFB126 highly related to the quality of embryo development in human sperm; the antibody is assembled with a fluorescently labeled secondary antibody, a nuclear dye, a concentrated washing solution, a mounting agent, a fixing agent, and a multi-well glass slide into a special detection kit. The kit can be used with a fluorescence microscope, an ordinary optical microscope, and image analysis software to achieve precise and quantitative analysis of the expression level and subcellular localization of key proteins such as DEFB126 and CATSPER2 in sperm, while being compatible with traditional sperm morphology and kinematic analysis, thereby providing an integrated molecular detection solution for sperm quality screening and embryo development potential prediction in assisted reproductive technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical detection reagent manufacturing and reproductive medicine technology, in particular to a fluorescently labeled kit and method for evaluating sperm function and fertilization potential. BACKGROUND

[0002] Male infertility affects about 15% of couples of childbearing age worldwide, and abnormal sperm quality is one of the main reasons. Traditional semen analysis (such as sperm concentration, motility and morphology) is the basis for evaluation, but it is difficult to fully reflect the functional status of sperm, especially the fertilization potential and embryo development support ability. About 30% of patients with unexplained infertility have normal routine semen parameters, highlighting the limitations of existing technologies in functional evaluation.

[0003] Sperm function involves multiple links such as occurrence, maturation, capacitation, acrosome reaction and egg binding, among which the own motility and egg binding capacity are particularly important. Although there are computer-assisted semen analysis (CASA) and in vitro functional experiments (such as zona pellucida binding test), there are still problems such as complex operation, low standardization, insufficient throughput and inability to realize simultaneous detection of multiple parameters.

[0004] With the progress of molecular biology, key proteins such as DEFB126 and CATSPER2 have been confirmed to be closely related to sperm function. There is an urgent need for an integrated kit that can integrate multiple key protein detection, simple operation, and quantitative results to improve the accuracy and efficiency of the diagnosis of male infertility and the prognosis evaluation of assisted reproduction. SUMMARY

[0005] The present application provides a fluorescently labeled kit and method for evaluating sperm function and fertilization potential to overcome the shortcomings of the prior art. The kit prepared by the method can accurately, quantitatively and multi-target analyze the expression levels of proteins such as DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1 and MIF, thereby comprehensively evaluating the sperm motility and egg binding capacity.

[0006] The present application is achieved by the following technical solutions:

[0007] In a first aspect, the present application provides a fluorescently labeled kit for evaluating sperm function and fertilization potential, comprising kit A and kit B, and kit A comprises the following components:

[0008] Antibody components: including specific antibodies of rabbit anti-human DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, MIF;

[0009] Labeling system: fluorescently labeled secondary antibody, nuclear fluorescent dye;

[0010] Solid support: sperm protein slide, the sperm protein slide is a multi-well glass slide, and the well is pre-coated with positive and negative control human sperm samples;

[0011] Kit B is the following components:

[0012] Concentrated washing solution, mounting medium, fixative.

[0013] Further, the specific sequences of the specific antibodies of rabbit anti-human DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, MIF are as follows:

[0014] (a) DEFB126 antibody

[0015] The sequence of the DEFB126 antibody light chain is shown as SEQ ID NO. 1, the variable region VL of the DEFB126 antibody light chain is shown as SEQ ID NO. 2, the complementarity determining region CDR-L1 of the DEFB126 antibody light chain is shown as SEQ ID NO. 3, the complementarity determining region CDR-L2 of the DEFB126 antibody light chain is shown as SEQ ID NO. 4, the complementarity determining region CDR-L3 of the DEFB126 antibody light chain is shown as SEQ ID NO. 5; the sequence of the DEFB126 antibody heavy chain is shown as SEQ ID NO. 6, the variable region VH of the DEFB126 antibody heavy chain is shown as SEQ ID NO. 7, the complementarity determining region CDR-H1 of the DEFB126 antibody heavy chain is shown as SEQ ID NO. 8, the complementarity determining region CDR-H2 of the DEFB126 antibody heavy chain is shown as SEQ ID NO. 9, and the complementarity determining region CDR-H3 of the DEFB126 antibody heavy chain is shown as SEQ ID NO. 10;

[0016] (b) CATSPER2 antibody

[0017] The CATSPER2 antibody light chain sequence is shown as SEQ ID NO. 11, the CATSPER2 antibody light chain variable region VL is shown as SEQ ID NO. 12, the CATSPER2 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 13, the CATSPER2 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 14, the CATSPER2 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 15; the CATSPER2 antibody heavy chain sequence is shown as SEQ ID NO. 16, the CATSPER2 antibody heavy chain variable region VH is shown as SEQ ID NO. 17, the CATSPER2 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 18, the CATSPER2 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 19, the CATSPER2 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 20;

[0018] (c) ACROSIN antibody

[0019] The ACROSIN antibody light chain sequence is shown as SEQ ID NO. 21, the ACROSIN antibody light chain variable region VL is shown as SEQ ID NO. 22, the ACROSIN antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 23, the ACROSIN antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 24, the ACROSIN antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 25; the ACROSIN antibody heavy chain sequence is shown as SEQ ID NO. 26, the ACROSIN antibody heavy chain variable region VH is shown as SEQ ID NO. 27, the ACROSIN antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 28, the ACROSIN antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 29, the ACROSIN antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 30;

[0020] (d) IZUMO1 antibody

[0021] The IZUMOl antibody light chain sequence is shown as SEQ ID NO. 31, the IZUMOl antibody light chain variable region VL is shown as SEQ ID NO. 32, the IZUMOl antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 33, the IZUMOl antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 34, the IZUMOl antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 35; the IZUMOl antibody heavy chain sequence is shown as SEQ ID NO. 36, the IZUMOl antibody heavy chain variable region VH is shown as SEQ ID NO. 37, the IZUMOl antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 38, the IZUMOl antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 39, the IZUMOl antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 40;

[0022] (e) SPACA6 antibody

[0023] The SPACA6 antibody light chain sequence is shown as SEQ ID NO. 41, the SPACA6 antibody light chain variable region VL is shown as SEQ ID NO. 42, the SPACA6 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 43, the SPACA6 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 44, the SPACA6 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 45; the SPACA6 antibody heavy chain sequence is shown as SEQ ID NO. 46, the SPACA6 antibody heavy chain variable region VH is shown as SEQ ID NO. 47, the SPACA6 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 48, the SPACA6 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 49, the SPACA6 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 50;

[0024] (f) SPACA6 antibody

[0025] The SPACA6 antibody light chain sequence is shown as SEQ ID NO. 51, the SPACA6 antibody light chain variable region VL is shown as SEQ ID NO. 52, the SPACA6 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 53, the SPACA6 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 54, the SPACA6 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 55; the SPACA6 antibody heavy chain sequence is shown as SEQ ID NO. 56, the SPACA6 antibody heavy chain variable region VH is shown as SEQ ID NO. 57, the SPACA6 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 58, the SPACA6 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 59, the SPACA6 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 60;

[0026] (g) GRIM-19 antibody

[0027] The GRIM-19 antibody light chain sequence is shown as SEQ ID NO. 61, the GRIM-19 antibody light chain variable region VL is shown as SEQ ID NO. 62, the GRIM-19 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 63, the GRIM-19 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 64, the GRIM-19 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 65; the GRIM-19 antibody heavy chain sequence is shown as SEQ ID NO. 66, the GRIM-19 antibody heavy chain variable region VH is shown as SEQ ID NO. 67, the GRIM-19 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 68, the GRIM-19 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 69, the GRIM-19 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 70;

[0028] (h) ODF2 antibody

[0029] The ODF2 antibody light chain sequence is shown as SEQ ID NO. 71, the ODF2 antibody light chain variable region VL is shown as SEQ ID NO. 72, the ODF2 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 73, the ODF2 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 74, the ODF2 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 75; the ODF2 antibody heavy chain sequence is shown as SEQ ID NO. 76, the ODF2 antibody heavy chain variable region VH is shown as SEQ ID NO. 77, the ODF2 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 78, the ODF2 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 79, the ODF2 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 80;

[0030] (i) PMCA4 antibody

[0031] The PMCA4 antibody light chain sequence is shown as SEQ ID NO. 81, the PMCA4 antibody light chain variable region VL is shown as SEQ ID NO. 82, the PMCA4 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 83, the PMCA4 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 84, the PMCA4 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 85; the PMCA4 antibody heavy chain sequence is shown as SEQ ID NO. 86, the PMCA4 antibody heavy chain variable region VH is shown as SEQ ID NO. 87, the PMCA4 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 88, the PMCA4 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 89, the PMCA4 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 90;

[0032] (j) GPX5 antibody

[0033] The GPX5 antibody light chain sequence is shown as SEQ ID NO. 91, the GPX5 antibody light chain variable region VL is shown as SEQ ID NO. 92, the GPX5 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 93, the GPX5 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 94, the GPX5 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 95; the GPX5 antibody heavy chain sequence is shown as SEQ ID NO. 96, the GPX5 antibody heavy chain variable region VH is shown as SEQ ID NO. 97, the GPX5 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 98, the GPX5 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 99, the GPX5 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 100;

[0034] (k) HPBP1 antibody

[0035] The HPBP1 antibody light chain sequence is shown as SEQ ID NO. 101, the HPBP1 antibody light chain variable region VL is shown as SEQ ID NO. 102, the HPBP1 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 103, the HPBP1 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 104, the HPBP1 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 105; the HPBP1 antibody heavy chain sequence is shown as SEQ ID NO. 106, the HPBP1 antibody heavy chain variable region VH is shown as SEQ ID NO. 107, the HPBP1 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 108, the HPBP1 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 109, the HPBP1 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 110;

[0036] (l) MIF antibody sequence

[0037] The MIF antibody light chain sequence is shown as SEQ ID NO. 111, the MIF antibody light chain variable region VL is shown as SEQ ID NO. 112, the MIF antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 113, the MIF antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 114, the MIF antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 115; the MIF heavy chain sequence is shown as SEQ ID NO. 116, the MIF antibody heavy chain variable region VH is shown as SEQ ID NO. 117, the MIF antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 118, the MIF antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 119, and the MIF antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 120.

[0038] Further, the antibody diluent is phosphate buffer, bovine serum albumin, sodium azide.

[0039] Further, the fluorescently labeled secondary antibody is a fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG antibody.

[0040] Further, the concentrated washing solution is 0.3M phosphate buffer at pH 7.4.

[0041] Further, the mounting agent is mounting glycerol.

[0042] Further, the nuclear dye is propidium iodide (PI) solution.

[0043] In a second aspect, the present application provides a method for using a fluorescently labeled kit for evaluating sperm function and fertilization potential, comprising the following steps:

[0044] (1) Sample pretreatment and smear preparation

[0045] The treated semen sample is applied to a multi-well glass slide, and an optical microscope is used for preliminary morphological detection;

[0046] (2) Incubation of primary antibody

[0047] After fixing the smear obtained in step (1), the smear is incubated with specific first antibodies against DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF.

[0048] (3) washing: the smear obtained after incubation of the first antibody in step (2) is taken out, washed with 1x washing solution once, then immersed in 3 portions of 1x washing solution, each for 5 minutes, finally immersed in deionized water for 30 seconds, and dried;

[0049] (4) incubating the smear after washing and drying in step (3) with the second antibody labeled with FITC;

[0050] (5) re-washing: the smear obtained after incubation of the second antibody in step (4) is re-washed and dried by the steps in step (3);

[0051] (6) after washing and drying in step (5), the smear is subjected to nuclear re-staining with propidium iodide PI solution, and incubated at room temperature in the dark;

[0052] (7) final washing: the smear after incubation of nuclear re-staining in step (6) is washed and dried by the steps in step (3);

[0053] (8) the smear obtained after sealing in step (7) is sealed, and images are collected by a confocal microscope after sealing;

[0054] (9) the qualitative expression of each target protein on sperm is determined by software.

[0055] Further, the 1x washing solution is a 0.3M phosphate buffer solution with a pH of 7.4.

[0056] Compared with the prior art, the present application has the following beneficial effects:

[0057] (1) marker innovation: DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1 and MIF are used as core markers and combined with other key proteins to apply to a kit, realizing direct molecular evaluation of sperm fertilization potential and solving the clinical pain point;

[0058] (2) the present application uses high-titer and high-specificity monoclonal antibodies, and combines with an internal control system to ensure high sensitivity, accuracy and comparability of the results of detection; morphological, kinematic and multiple protein molecular function analysis are simultaneously completed on one platform, providing more comprehensive diagnostic information and saving samples and time;

[0059] (3) the standardized kit preparation process and intelligent analysis software of the present application greatly reduce the operation complexity and dependence on personnel, and are easy to promote in clinic; the present application provides a powerful tool for etiological diagnosis of male infertility, prognosis, prediction and efficacy evaluation of assisted reproductive technology, has clear market demand, and has great clinical value and commercial potential. BRIEF DESCRIPTION OF DRAWINGS

[0060] The application will be further described below with reference to the accompanying drawings.

[0061] Figure 1 is a schematic diagram of the kit according to the application;

[0062] Figure 2 is a functional correlation diagram of the core protein marker combination according to the application;

[0063] Figure 3 is a process flow diagram for the preparation of monoclonal antibodies;

[0064] Figure 4 is an analysis schematic of the multi-modal image analysis software. DETAILED DESCRIPTION

[0065] In order to make the objects, technical solutions and advantages of the application clearer, the application will be further described below in detail with reference to specific embodiments. The following embodiments are only illustrative and are not intended to limit the protection scope of the application.

[0066] Example 1 Preparation of rabbit anti-human monoclonal antibody (taking DEFB126 as an example)

[0067] The process flow diagram for the preparation of monoclonal antibodies is shown in Figure 3 and is as follows:

[0068] 1. Preparation of immunogen: recombinantly express human DEFB126 full-length protein through an E. coli prokaryotic expression system, purify through a Ni column affinity and verify through SDS-PAGE to obtain a protein solution with a purity of >95%;

[0069] 2. Animal immunization: take 6-8 month old New Zealand white rabbits, emulsify 200 μg DEFB126 protein with Freund's complete adjuvant and perform multi-point subcutaneous injection on the back; perform booster immunization every 3 weeks for a total of 3 times;

[0070] 3. Cell fusion and screening: 7 days after the last immunization, take rabbit spleen cells and rabbit myeloma cells (such as 240E-W2) and perform electrofusion or PEG fusion according to the conventional method; culture with HAT selection medium and screen positive hybridoma cell wells through indirect ELISA using recombinant DEFB126 as the coating antigen;

[0071] 4. Subcloning and expansion: perform limited dilution subcloning on the positive well cells until a monoclonal cell strain stably secreting antibodies is obtained;

[0072] 5. Ascites preparation and purification: Hybridoma cells were injected into the abdominal cavity of pre-treated rabbits to prepare ascites, or the hybridoma cells were cultured in vitro in serum-free medium to obtain large amounts of supernatant, and the rabbit-derived monoclonal antibody was purified by Protein A or Protein G affinity chromatography column;

[0073] 6. Antibody identification: The titer of the purified rabbit anti-human DEFB126 monoclonal antibody was determined to be 1:100 (immunofluorescence method); Western Blot results showed a single specific band at the expected molecular weight. Immunofluorescence experiments showed that the signal was localized to the sperm head, and this signal could be completely blocked by DEFB126 recombinant protein, confirming its binding specificity.

[0074] Eleven other specific rabbit-derived monoclonal antibodies were successfully prepared and verified according to the same process and quality control standards. All antibodies met the requirements of immunofluorescence titer not less than 1:100, and no cross-reaction with other target proteins, providing high-quality core raw materials for the preparation of subsequent kits.

[0075] The protein sequences are as follows:

[0076] (a) DEFB126, the immunogenic protein sequence (SEQ ID NO. 121) is as follows: MKSLLFTLAVFMLLAQLVSGNWYVKKCLNDVGICKKKCKPEEMHVKNGWAMCGKQRDCCVPADRRANYPVFCVQTKTTRISTVTATTATTTLMMTTASMSSMAPTPVSPTG;

[0077] (b) CATSPER2, the immunogenic protein sequence (SEQ ID NO. 122) is as follows: 264-277: CSEYTRSPRQDLEYH-NH2; This short peptide is a specific epitope selected by bioinformatics analysis to stimulate high-specificity antibodies;

[0078] (c) ACROSIN, the immunogenic protein sequence of which (SEQ ID NO. 123) is as follows: MVEMLPTAILLVLAVSVVAKDNATCDGPCGLRFRQNPQGGVRIVGGKAAQHGAWPWMVSLQIFTYNSHRYHTCGGSLLNSRWVLTAAHCFVGKNNVHDWRLVFGAKEITYGNNKPVKAPLQERYVEKIIIHEKYNSATEGNDIALVEITPPISCGRFIGPGCLpHFKAGLPRGSQSCWVAGWGYIEEKAPRPSSILMEARVDLIDLDLCNSTQWYNGRVQPTNVCAGYPVGKIDTCQGDSGGPLMCKDSKESAYVVVGITSWGVGCARAKRPGIYTATWPYLNWIASKIGSNALRMIQSATPPPPTTRPPPIRPPFSHPISAHLPWYFQPPPRPLPPRPPAAQPRPPPSPPPPPPPPASPLPPPPPPPPPTPSSTTKLPQGLSFAKRLQQLIEVLKGKTYSDGKNHYDMETTELPELTSTS;

[0079] (d) IZUMOl, the immunogenic protein sequence of which (SEQ ID NO. 124) is as follows: MGpHFTLLCAALAGCLLPAEGCVICDPSVVLALKSLEKDYLPGHLDAKHHKAMMERVENAVKDFQELSLNEDAYMGVVDEATLQKGSWSLLKDLKRITDSDVKGDLFVKELFWMLHLQKETFATYVARFQKEAYCPNKCGVMLQTLIWCKNCKKEVHACRKSYDCGERNVEVPQMEDMILDCELNWHQASEGLTDYSFYRVWGNNTETLVSKGKEATLTKPMVGPEDAGSYRCELGSVNSSPATIINFHVTVLPKMIKEEKPSPNIVTPGEATTESSISLQPLQPEKMLASRLLGLLICGSLALITGLTFAIFRRRKVIDFIKSSLFGLGSGAAEQTQVPKEKATDSRQQ;

[0080] (e) SPACA6, the immunogenic protein sequence of which (SEQ ID NO. 125) is as follows: MALLALASAVPSALLALAVFRVPAWACLLCFTTYSERLRICQMFVGMRSPKLEECEEAFTAAFQGLSDTEINYDERSHLHDTFTQMTHALQELAAAQGSFEVAFPDAAEKMKKVITQLKEAQACIPPCGLQEFARRFLCSGCYSRVCDLPLDCPVQDVTVTRGDQAMFSCIVNFQLPKEEITYSWKFAGGGLRTQDLSYFRDMPRAEGYLARIRPAQLTHRGTFSCVIKQDQRPLARLYFFLNVTGPPPRAETELQASFREVLRWAPRDAELIEPWRPSLGELLARPEALTPSNLFLLAVLGALASASATVLAWMFFRWYCSGN;

[0081] (f) SPACA6, the immunogenic protein sequence of which (SEQ ID NO. 126) is as follows: [MEPPGRRECPFPSWRFPGLLLAAMVLLLYSFSDACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDVWVIAVIVIAIDIFKGGRRKGKQMVELNMPLTRLNQPLQQSREAE;

[0082] (g) GRIM-19, the immunogenic protein sequence of which (SEQ ID NO. 127) is as follows:

[0083] MAASKVKQDMPPPGGYGPIDYKRNLPRRGLSGYSMLAIGIGTLIYGHWSIMKWNRERRRLQIEDFEARIALLPLLQAETDRRTLQMLRENLEEEAIIMKDVPDWKVGESVFHTTRWVPPLIGELYGLRTTEEALHASHGFMWYT;

[0084] (h) ODF2, the immunogenic protein sequence of which (SEQ ID NO. 128) is as follows:

[0085] MSASSSGGSPRFPSCGKNGVTSLTQKKVLRAPCGAPSVTVTKSHKRGMKGDTVNVRRSVRVKTKVPWMPPGKSSARPVGCKWENPpHCLEITPPSSEKLVSVMRLSDLSTEDDDSGHCKMNRYDKKIDSLMNAVGCLKSEVKMQKGERQMAKRFLEERKEELEEVAHELAETEHENTVLRHNIERMKEEKDFTILQKKHLQQEKECLMSKLVEAEMDGAAAAKQVMALKDTIGKLKTEKQMTCTDINTLTRQKELLLQKLSTFEETNRTLRDLLREQHCKEDSERLMEQQGALLKRLAEADSEKARLLLLLQDKDKEVEELLQEIQCEKAQAKTASELSKSMESMRGHLQAQLRSKEAENSRLCMQIKNLERSGNQHKAEVEAIMEQLKELKQKGDRDKESLKKAIRAQKERAEKSEEYAEQLHVQLADKDLYVAEALSTLESWRSRYNQVVKEKGDLELEIIVLNDRVTDLVNQQQTLEEKMREDRDSLVERLHRQTAEYSAFKLENERLKASFAPMEDKLNQAHLEVQQLKASVKNYEGMIDNYKSQVMKTRLEADEVAAQLERCDKENKILKDEMNKEIEAARRQFQSQLADLQQLPDILKITEAKLAECQDQLQGYERKNIDLTAIISDLRSRIEHQGDKLEMAREKHQASQKENKQLSLKVDELERKLEATSAQNIEFLQVIAKREEAIHQSQLRLEEKTRECGTLARQLESAIEDARRQVEQTKEHALSKERAAQNKILDLETQLSRTKTELSQLRRSRDDADRRYQSRLQDLKDRLEQSESTNRSMQNYVQFLKSSYANVFGDGPYSTFLTSSPIRSRSPPA;

[0086] (i) PMCA4, the immunogenic protein sequence (SEQ ID NO. 129) of which is as follows:

[0087]

[0088] (j) GPX5, whose immunogenic protein sequence (SEQ ID NO.130) is shown below:

[0089] MTTQLRVVHLLPLLLACFVQTSPKQEKMKMDCHKDEKGTIYDYEAIALNKNEYVSFKQYVGKHILFVNVATYCGLTAQYPELNALQEELKPYGLVVLGFPCNQFGKQEPGD NKEILPGLKYVRPGGGFVPSFQLFEKGDVNGEKEQKVFSFLKHSCpHPSEILGTFKSISWDPVKVHDIRWNFEKFLVGPDGIPVMRWSHRATVSSVKTDILAYLKQFKTK;

[0090] (k) HPBP1, whose immunogenic protein sequence (SEQ ID NO.131) is shown below:

[0091] MSDEGSRGSRLPLALPPASQGCSSGGGGGGSSSAGGSGNSRPPRNLQGLLQMAITAGSEEPDPPPEPMSEERRQWLQEAMSAAFRGQREEVEQMKSCLRVLSQPMPPTAGEAEQAADQQEREGALELLADLCENMDNAADFCQLSGMHLLVGRYLEAGAAGLRWRAAQLIGTCSQNVAAIQ EQVLGLGALRKLLRLLDRDACDTVRVKALFAISCLVREQEAGLLQFLRLDGFSVLMRAMQQQVQKLKVKSAFLLQNLLVGHPEHKGTLCSMGMVQQLVALVRTEHSPFHEHVLGALCSLVTDFPQGVRECREPELGLEELLRHRCQLLQQHEEYQEELEFCEKLLQTCFSSPADDSMDR;

[0092] (l) MIF, whose immunogenic protein sequence (SEQ ID NO.132) is shown below:

[0093] MPMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVVPDQLMAFGGSSEPCALCSLHSIGKIGGAQNRSYSKLLCGLLAERLRISPDRVYINYYDMNAANVGWNNSTFA.

[0094] The antibody sequence is as follows:

[0095] (a)The DEFB126 antibody sequences (SEQ ID NO.1 - SEQ ID NO.10) are as follows:

[0096] Light chain:

[0097] DIVLTQSPASLAVSLGQRATISCKASQSVEFDGDSYMNWYQQKPGQPPKVLIYAISNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQTNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0098] Variable region of light chain (VL):

[0099] DIVLTQSPASLAVSLGQRATISCKASQSVEFDGDSYMNWYQQKPGQPPKVLIYAISNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQTNEDPWTFGGGTKLEIKR;

[0100] CDR regions:

[0101] CDR-L1: KASQSVEFDGDSYMN;

[0102] CDR-L2: AISNLES;

[0103] CDR-L3: QQTNEDPWT;

[0104] Heavy chain:

[0105] QIQLQQSGPEVVKPGASVKISCKASGYTFTDYWITWVKQKPGQGLEWIGWIYPGSANTRYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANSANYWFAYWGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0106] Heavy chain variable region (VH):

[0107] QIQLQQSGPEVVKPGASVKISCKASGYTFTDYWITWVKQKPGQGLEWIGWIYPGSANTRYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANSANYWFAYWGQGTQVTVSA;

[0108] CDR regions:

[0109] CDR-H1: GYTFTDYW;

[0110] CDR-H2: WIYPGSANTRYNEKFKG;

[0111] CDR-H3: ANSANYWFAY。

[0112] (b) The CATSPER2 antibody sequence (SEQ ID NO.11 - SEQ ID NO.20) is as follows:

[0113] Light chain:

[0114] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIRWYQQRTNGSPRLLIKYAGESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNGNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0115] Light chain variable region (VL):

[0116] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIRWYQQRTNGSPRLLIKYAGESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNGNWPTTFGAGTKLELKR;

[0117] Light chain CDRs:

[0118] CDR-L1: RASQSIGTNIR;

[0119] CDR-L2: YAGESIS;

[0120] CDR-L3: QQNGNWPT;

[0121] Heavy chain:

[0122] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTQYGFHWVRQSPGKGLEWLGIIWSGGNTEYNTWFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAVTYYDYEFAVWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0123] Heavy chain variable region (VH):

[0124] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTQYGFHWVRQSPGKGLEWLGIIWSGGNTEYNTWFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAVTYYDYEFAVWGQGTLVTVSA;

[0125] Heavy chain CDRs:

[0126] CDR-H1: GFSLTQYGFH;

[0127] CDR-H2: IIWSGGNTEYNTWFTS;

[0128] CDR-H3: AVTYYDYEFAV.

[0129] (c) The acrosin antibody sequences (SEQ ID NO.21 - SEQ ID NO.30) are shown below:

[0130] Light chain:

[0131] EIVLTQSPATLSLSPGERATLSCRASQSIGSYLAWYQQKPGQAPRLLIYDASQHATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNYWWALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0132] Variable light chain region (VL):

[0133] EIVLTQSPATLSLSPGERATLSCRASQSIGSYLAWYQQKPGQAPRLLIYDASQHATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNYWWALTFGGGTKVEIKR;

[0134] Light chain CDRs:

[0135] CDR-L1: RASQSIGSYLA;

[0136] CDR-L2: DASQHAT;

[0137] CDR-L3: QQRSNYWWALT;

[0138] Heavy chain:

[0139] QVQLQQWGAGLLKPSETLSLTCAVYGGSLSGWYWSWIRQSPEKGLEWIGEINHGSWFAYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGWSQPEWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0140] Heavy chain variable region (VH):

[0141] QVQLQQWGAGLLKPSETLSLTCAVYGGSLSGWYWSWIRQSPEKGLEWIGEINHGSWFAYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGWSQPEWYFDLWGRGTLVTVSS;

[0142] Heavy chain CDRs:

[0143] CDR-H1: AVYGGSLS;

[0144] CDR-H2: EINHGSWF;

[0145] CDR-H3: ARDYGWSQPEWYFDL.

[0146] (d) The IZUMO1 antibody sequence (SEQ ID NO.31-SEQ ID NO.40) is shown below:

[0147] Light chain:

[0148] EIVLTQSPATLSLSPGERATLSCRASQGLTAYLAWYQQKPGQAPRLLIYDAGQHTTGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSQYWWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0149] Variable light chain (VL):

[0150] EIVLTQSPATLSLSPGERATLSCRASQGLTAYLAWYQQKPGQAPRLLIYDAGQHTTGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSQYWWTFGQGTKVEIKR;

[0151] Light chain CDRs:

[0152] CDR-L1: RASQGLTAYLA;

[0153] CDR-L2: DAGQHTT;

[0154] CDR-L3: QQRSQYWWTFGQ;

[0155] Heavy chain:

[0156] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNGLTVSWVRQAPGKGLEWVSAISGGTAATDYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKFVYPAEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0157] Heavy chain variable region (VH):

[0158] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNGLTVSWVRQAPGKGLEWVSAISGGTAATDYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKFVYPAEPVFDYWGQGTLVTVSS;

[0159] Heavy chain CDRs:

[0160] CDR-H1: AVSGFTFN;

[0161] CDR-H2: SISGGTAATDYYADSVKG;

[0162] CDR-H3: AKDKFVYPAEPVFDY.

[0163] (e) The SPACA6 antibody sequences (SEQ ID NO.41 - SEQ ID NO.50) are as follows:

[0164] Light chain:

[0165] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSQSLAYLYWYLQKPGQSPQLLIYQFAQVVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCANQIDLPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0166] Variable region of light chain (VL):

[0167] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSQSLAYLYWYLQKPGQSPQLLIYQFAQVVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCANQIDLPYTFGGGTKVEIKR;

[0168] Light chain CDRs:

[0169] CDR-L1: RSSKSLLHSQSLAYLY;

[0170] CDR-L2: QFAQVVS;

[0171] CDR-L3: CANQIDLPYT;

[0172] Heavy chain:

[0173] QVQLVQSGAEVKKPGSSVKVSCKASGYAIAWGWINWVRQAPGQGLEWMGRIFPSESETDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVLESWWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0174] Heavy chain variable region (VH):

[0175] QVQLVQSGAEVKKPGSSVKVSCKASGYAIAWGWINWVRQAPGQGLEWMGRIFPSESETDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVLESWWLVYWGQGTLVTVSS;

[0176] Heavy chain CDRs:

[0177] CDR-H1: GYAIAW;

[0178] CDR-H2: RIFPSESETDYNGKFKG;

[0179] CDR-H3: NVLESWWLVY.

[0180] (f) The SPACA6 antibody sequence (SEQ ID NO.51-SEQ ID NO.60) is shown below:

[0181] Light chain:

[0182] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLWASQQKNYLAWYQQKPGQSPKLLIYWAGAHDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQWWGPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0183] Light chain variable region (VL):

[0184] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLWASQQKNYLAWYQQKPGQSPKLLIYWAGAHDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQWWGPRTFGGGTKLEIK;

[0185] Complementary determining regions (CDRs):

[0186] CDR-L1: KSSQSLLWASQQKNYLA;

[0187] CDR-L2: WAGAHDS;

[0188] CDR-L3: QQWWGPRT;

[0189] Heavy chain:

[0190] QVQLQQSGPEVVKPGASVKMSCKASGYTFERTYIHWVRQKPGQGLDWIGYINPYQESYDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKEQWSTGAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0191] Heavy chain variable region (VH):

[0192] QVQLQQSGPEVVKPGASVKMSCKASGYTFERTYIHWVRQKPGQGLDWIGYINPYQESYDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKEQWSTGAWFAYWGQGTLVTVSS;

[0193] Complementary determining regions (CDRs):

[0194] CDR-H1: GYTFERTYIH;

[0195] CDR-H2: YINPYQESYDYDEKFKG;

[0196] CDR-H3: AREKEQWSTGAWFAY.

[0197] (g) The GRIM-19 antibody sequences (SEQ ID NO.61 - SEQ ID NO.70) are shown below:

[0198] Light chain:

[0199] DIVMTQSPLSLPVTPGEPASISCRSSQSIVWGQSNTYLGWYLQKPGQSPQLLIYKVGQHLSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQSGRLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0200] Light chain variable region (VL):

[0201] DIVMTQSPLSLPVTPGEPASISCRSSQSIVWGQSNTYLGWYLQKPGQSPQLLIYKVGQHLSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQSGRLPYTFGQGTKLEIKR;

[0202] Light chain CDRs:

[0203] CDR-L1: RSSQSIVWGQSNTYLG;

[0204] CDR-L2: KVGQHLS;

[0205] CDR-L3: CFQSGRLPYT;

[0206] Heavy chain:

[0207] QVQLVQSGAEVKKPGASVKVSCKASGYTFTQWQLHWVRQAPGQRLEWMGTIYPGNEEAGYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGSWHTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0208] Heavy chain variable region (VH):

[0209] QVQLVQSGAEVKKPGASVKVSCKASGYTFTQWQLHWVRQAPGQRLEWMGTIYPGNEEAGYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGSWHTMDYWGQGTLVTVSS;

[0210] Heavy chain CDRs:

[0211] CDR-H1: GYTFTQW;

[0212] CDR-H2: TIYPGNEEAGYNQKFKD;

[0213] CDR-H3: GSWHTMDY.

[0214] (h) The ODF2 antibody sequence (SEQ ID NO.71 - SEQ ID NO.80) is shown as follows:

[0215] Light chain:

[0216] ESALTQPASVSGSPGQSITISCTGTSSDVGSWQGVSWYQQHPGKAPKLMIYEVGQHYSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCNSYAGAGMVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0217] Light chain variable region (VL):

[0218] ESALTQPASVSGSPGQSITISCTGTSSDVGSWQGVSWYQQHPGKAPKLMIYEVGQHYSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCNSYAGAGMVFGGGTKLTVLG;

[0219] Light chain CDRs:

[0220] CDR-L1: TGTSSDVGSWQGV;

[0221] CDR-L2: EVGQHYS;

[0222] CDR-L3: NSYAGAGMV;

[0223] Heavy chain:

[0224] EVQLVQSGAEVKKPGASVKVSCKASGYTLAGWSISWVRQAPGQGLEWMGWVSFYNSQAQYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARSWSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0225] Heavy chain variable region (VH):

[0226] EVQLVQSGAEVKKPGASVKVSCKASGYTLAGWSISWVRQAPGQGLEWMGWVSFYNSQAQYAQKLQGRGTMTTDPSTSTAYMELRSLRSDDTAVYYCARSWSMDVWGQGTTVTVSS;

[0227] Heavy chain CDRs:

[0228] CDR-H1: GYTLAGWS;

[0229] CDR-H2: WVSFYNSQAQYAQKLQG;

[0230] CDR-H3: SWSMDV.

[0231] (i) The PMCA4 antibody sequences (SEQ ID NO.81 - SEQ ID NO.90) are shown below:

[0232] Light chain:

[0233] DIVMTQSPDSLAVSLGERATINCKSSQSVLYAGNNHQWFAWYQQKPGQPPKLLINQTGAKASGVPDRFSGSGSGTEFTLIISSLQAEDVAIYYCQQYWAYYLAFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0234] Variable light chain region (VL):

[0235] DIVMTQSPDSLAVSLGERATINCKSSQSVLYAGNNHQWFAWYQQKPGQPPKLLINQTGAKASGVPDRFSGSGSGTEFTLIISSLQAEDVAIYYCQQYWAYYLAFGGGTKLEIKR;

[0236] Light chain CDRs:

[0237] CDR-L1: KSSQSVLYAGNNHQ;

[0238] CDR-L2: QTGAKAS;

[0239] CDR-L3: QQYWAYYLA;

[0240] Heavy chain:

[0241] EVQLVESGGGLVKPGGSLRLSCAASGLTFEHTYLNWVRQAPGKGLEWVGRIKRKTESDATDYAAPVKGRFSISRDDSKNTLYLQMNSLKTEDTAVYYCAGGQKTFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0242] Heavy chain variable region (VH):

[0243] EVQLVESGGGLVKPGGSLRLSCAASGLTFEHTYLNWVRQAPGKGLEWVGRIKRKTESDATDYAAPVKGRFSISRDDSKNTLYLQMNSLKTEDTAVYYCAGGQKTFDIWGQGTMVTVSS;

[0244] Heavy chain CDRs:

[0245] CDR-H1: GLTFEH;

[0246] CDR-H2: RIKRKTESDATDYAAPVKG;

[0247] CDR-H3: GGQKTFDI.

[0248] (j) The GPX5 antibody sequences (SEQ ID NO.91 - SEQ ID NO.100) are shown as follows:

[0249] Light chain:

[0250] DILLTQSPAILSVSPGERVSFSCRASQFVSGGLHWYQQRTNGSPRLLIKYTGDGMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQARAYPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0251] Variable light chain region (VL):

[0252] DILLTQSPAILSVSPGERVSFSCRASQFVSGGLHWYQQRTNGSPRLLIKYTGDGMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQARAYPFTFGSGTNLEVKR;

[0253] Analysis of light chain CDR

[0254] CDR-L1: RASQFVSGGLH;

[0255] CDR-L2: YTGDGMS;

[0256] CDR-L3: QQARAYPFT;

[0257] Heavy chain:

[0258] EVKLEESGGGLVQPGGSMKLSCVASGFIFSQRYVNWVRQSPEKGLEWVAEIRSKSIQTGSHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYWATSYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0259] Heavy chain variable region (VH):

[0260] EVKLEESGGGLVQPGGSMKLSCVASGFIFSQRYVNWVRQSPEKGLEWVAEIRSKSIQTGSHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYWATSYDYWGQGTTLTVSS;

[0261] Heavy chain CDR analysis

[0262] CDR-H1: GFIFSQRYV;

[0263] CDR-H2: EIRSKSIQTGSHYAES;

[0264] CDR-H3: SRNYWATSYDY.

[0265] (k) The HPBP1 antibody sequences (SEQ ID NO.101 - SEQ ID NO.110) are shown below:

[0266] Light chain:

[0267] EIVLTQSPGTLSLSPGERATLSCRASQSVSTTWIAWYQQKPGQAPRLLIYGATTKGTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYATTWCTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-TR;

[0268] Variable region of light chain (VL):

[0269] EIVLTQSPGTLSLSPGERATLSCRASQSVSTTWIAWYQQKPGQAPRLLIYGATTKGTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYATTWCTFGQGTRLEIKR;

[0270] Analysis of light chain CDR

[0271] CDR-L1: RASQSVSTTWIA;

[0272] CDR-L2: GATTKGT;

[0273] CDR-L3: QQYATTWCTF;

[0274] Heavy chain:

[0275] EVQLVESGGGLVQPGGSLRLSCAASGFTFSQWYLNWVRQAPGKGLEWVAAINQDGSDHPPVGSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDILSEWPIHYWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0276] Heavy chain variable region (VH):

[0277] EVQLVESGGGLVQPGGSLRLSCAASGFTFSQWYLNWVRQAPGKGLEWVAAINQDGSDHPPVGSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDILSEWPIHYWYFDLWGRGTLVTVSS;

[0278] Heavy chain CDR analysis

[0279] CDR-H1: GFTFSQWY;

[0280] CDR-H2: AINQDGSDHPPVGSVKGR;

[0281] CDR-H3: VRDYYDILSEWPIHYWYFDL.

[0282] (l) The MIF antibody sequence (SEQ ID NO.111 - SEQ ID NO.120) is shown as follows:

[0283] Light chain:

[0284] DIQMTQSPSSLSASVGDRVTITCKASRDVGLGFAWYQQKPGKVPKLLIYWAGSKRTGVPSRFSGSGSRTDFTLTISSLQPEDVADYFCHQWTTWPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0285] Variable light chain (VL):

[0286] DIQMTQSPSSLSASVGDRVTITCKASRDVGLGFAWYQQKPGKVPKLLIYWAGSKRTGVPSRFSGSGSRTDFTLTISSLQPEDVADYFCHQWTTWPFTFGSGTKLEIKR;

[0287] Light chain CDRs:

[0288] CDR-L1: KASRDVGLGFA;

[0289] CDR-L2: WAGSKRT;

[0290] CDR-L3: CHQWTTWPFT;

[0291] Heavy chain:

[0292] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTENSLHWMRQAPGQGLEWIGYIYPREETYKYNENFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCAIPDHTAWAWFIYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0293] Heavy chain variable region (VH):

[0294] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTENSLHWMRQAPGQGLEWIGYIYPREETYKYNENFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCAIPDHTAWAWFIYWGQGTLVTVSS;

[0295] Heavy chain CDRs:

[0296] CDR-H1: GYTFTENSLH;

[0297] CDR-H2: YIYPREETYKYNENFKG;

[0298] CDR-H3: AIPDHTAWAWFIY。

[0299] After successfully preparing rabbit-derived monoclonal antibodies targeting all twelve key proteins (DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF), systematic and standardized performance validation was performed to ensure they meet the requirements for high sensitivity, high specificity, and high stability in kit production. The functional association diagram of the core protein biomarker combination is shown below. Figure 2 As shown in the table below, all verification data are presented.

[0300] Table 1. Overview of rabbit-derived monoclonal antibody preparation and performance validation data for twelve key proteins.

[0301] .

[0302] High titer and high affinity: As shown in Table 1, all twelve antibodies exhibited extremely high titers (all not lower than 1:256,000) and nanomolar (10 -9 M) to Pimoll (10 -11 The high affinity at the M level ensures that even with highly diluted working solution concentrations, a strong and specific signal is generated in subsequent immunofluorescence assays, guaranteeing the high sensitivity of the kit.

[0303] These twelve key proteins form a precisely coordinated network in sperm function: the CATSPER family, together with PMCA4, regulates calcium homeostasis, driving sperm motility and capacitation; IZUMO1, SPACA6, and ACROSIN mediate the acrosome reaction, zona pellucida penetration, and sperm-egg membrane fusion, respectively; SPACA6, DEFB126, and MIF provide triple immune protection, ensuring sperm survival in the female reproductive tract; GPX5 resists oxidative damage, protecting sperm integrity; and ODF2 maintains tail structure, while GRIM-19 and HPBP1 regulate energy metabolism, providing the driving force and structural basis for the entire process. They cover the entire functional chain from motility, capacitation, immune escape to egg fusion; the absence of any protein will cause a break in the functional assessment chain, leading to missed detections or misdiagnosis.

[0304] Excellent specificity: Western blotting results showed that all antibodies recognized only one band at the expected molecular weight in the complex human sperm protein lysate, with no extraneous bands. Cross-reactivity assays further confirmed that each antibody bound only to its corresponding target protein, showing no cross-reactivity with the other eleven proteins. This demonstrates the high specificity of the antibody combination, which is the foundation for achieving precise parallel detection of multiple targets.

[0305] Precise subcellular localization: Immunofluorescence assays confirmed that all antibodies accurately labeled the native locations of their target proteins on sperm, with results fully consistent with existing literature. This demonstrates that antibodies can not only bind to target proteins but also specifically recognize them in near-native cellular structures, which is crucial for functional assessment.

[0306] Batch consistency: The above key indicators were verified for three consecutive batches of production samples for each antibody. The results showed that the batch-to-batch difference was less than 15%, which proved the stability of the production process and the controllability of product quality.

[0307] The above systematic verification data fully demonstrate that the twelve rabbit-derived monoclonal antibody combinations successfully prepared by this invention have reached top-level performance in terms of titer, affinity, specificity, and localization accuracy, fully meeting the core raw material requirements for constructing a high-performance, highly reliable integrated sperm function detection kit.

[0308] Example 2 Assembly of the detection kit

[0309] The assembly diagram of the test kit is shown below. Figure 1 As shown, assemble according to the specification of 5 servings / box (see Table 2):

[0310] 1. Antibody aliquoting: Dilute the purified DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF antibodies to the working concentration (0.05 mg / mL) with PBS containing 1% BSA, and aliquot 150 μL each into brown light-proof tubes.

[0311] 2. Labeling reagent aliquoting: Dilute FITC-goat anti-rabbit IgG secondary antibody and propidium iodide (PI) solution to working concentration and aliquot each into 750μL / vial.

[0312] 3. Slide preparation: Known positive and negative samples of fertile sperm (DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF) were washed with PBS, fixed with methanol, and then adjusted to a concentration of 50 × 10⁻⁶. 6 / mL. Using a spotting instrument, 1μL of sample was spotted into the A1 (positive control) and B1 (negative control) wells of a 12-well glass slide, dried at room temperature, and then sealed for storage. Three slides were randomly selected from each batch for immunofluorescence staining and analysis.

[0313] 4. Auxiliary reagent aliquots: aliquot 30×PBS concentrated wash buffer into 50mL / bottle; aliquot glycerol for mounting into 3mL / vial; aliquot methanol for fixative into 50mL / bottle.

[0314] 5. Final assembly: Place all components into a custom foam tray, cover with an aluminum foil bag and heat-seal, then affix a label printed with the batch number, expiration date, and storage conditions (-20℃, protected from light).

[0315] Table 2. Components of the kit

[0316] ;

[0317] .

[0318] Example 3: Detection using a kit

[0319] The specific detection process of the kit is as follows:

[0320] 1. Sample processing: Take 1 mL of liquefied semen, add 6 mL of 1× washing buffer, centrifuge at 1500 rpm for 5 min, discard the supernatant, and repeat the washing twice. Finally, resuspend in 1× washing buffer and adjust the concentration to 40-50×10⁻⁶. 6 / mL.

[0321] 2. Kinematic Analysis: A 10 μL sample of fresh semen was placed in a counting chamber, and a 30-second motion video was immediately captured using an optical microscope. The software automatically analyzed the kinematic parameters, including VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF.

[0322] 3. Smear and morphological analysis: Apply 1 μL of sample to the blank well of a glass slide and dry at 37°C for 30 min. Acquire bright-field images using a 40x objective lens and analyze morphological parameters.

[0323] 4. Immunofluorescence staining: Fix glass slides with methanol for 5-10 min, dry, and select 1-3 primary antibodies for the target proteins according to the detection requirements. Add 10 μL of the corresponding primary antibody to each well and incubate overnight in a humidified chamber at 2-8℃. Wash three times with 1× washing buffer, 5 min each time. Add 10 μL of the corresponding fluorescently labeled secondary antibody to each well and incubate at 37℃ in the dark for 30 min. After washing, add 10 μL of propidium iodide (PI) solution and incubate in the dark for 10 min. Finally, wash and dry.

[0324] 5. Fluorescence Imaging and Analysis: Add mounting medium and cover with a coverslip. Acquire images using a fluorescence microscope in the corresponding channels, photographing at least 200 sperm cells per sample.

[0325] 6. Report Generation: Import the images into the analysis software. The software automatically identifies sperm and calculates the average fluorescence intensity, integrated optical density, and localization rate of the target protein. Combined with kinematic and morphological data, a comprehensive evaluation report is generated.

[0326] The kit exhibits excellent stability: it demonstrates outstanding accuracy, with intra-assay and inter-assay precision (CV%) consistently ranging from 4% to 10%, significantly better than the acceptable standard of 15%, ensuring excellent reproducibility of test results; in terms of accuracy, it shows a high correlation with the gold standard Western blot method (r=0.96), and its prediction accuracy for clinical outcomes reaches over 92%, demonstrating excellent clinical applicability.

[0327] Example 4: Analysis System Integration and Testing Applications

[0328] This embodiment details the hardware integration, software configuration, and complete process of using the multimodal analysis system for detection. A schematic diagram of the multimodal image analysis software is shown below. Figure 4 As shown.

[0329] 1. Hardware system integration: Fluorescence microscope and ordinary optical microscope, used for acquiring fluorescence images, bright field images and motion videos.

[0330] 2. Software Configuration: Includes "Human Sperm Localization Protein Quantitative Analysis Software" or a similar multimodal image analysis system, supporting:

[0331] Automatically identify sperm and analyze the fluorescence intensity, integrated optical density, and localization rate of DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF proteins; analyze sperm morphological parameters in bright-field images; analyze sperm morphological parameters in bright-field images of processed sperm smears acquired by ordinary optical microscopes and sperm motility parameters in motility videos; generate a comprehensive report and compare it with database reference values.

[0332] 3. System testing applications:

[0333] (1) Sample pretreatment and smear preparation

[0334] Take a fresh semen sample after liquefaction, add 6 mL of 1× washing buffer (diluted with deionized water at a ratio of 1:29 using 30× concentrated washing buffer), and mix gently. Place the mixture in a horizontal centrifuge and centrifuge at 1500 rpm for 5 minutes, discarding the supernatant. Repeat the washing process twice: each time, add 6 mL of 1× washing buffer to resuspend the sperm precipitate, centrifuge again, and discard the supernatant. Finally, resuspend the sperm in an appropriate amount of 1× washing buffer and adjust the sperm concentration to 40–50 × 10⁻⁶. 6 / mL (ensure 40–50 sperm are visible under a 40x microscope). Take 1 μL of the adjusted semen sample and spread it evenly in the blank wells of a multi-well slide. Incubate the smear in a 37°C constant temperature drying incubator for 30 minutes to allow it to dry completely.

[0335] (2) Examination by ordinary optical microscope: morphological and kinematic analysis

[0336] The dried smear was directly observed under a regular optical microscope. 5 μL of physiological saline or a special sperm diluent was placed over the well of the smear, and a coverslip was placed on top to prevent air bubbles. Bright-field images and motion videos were acquired using a 40x objective lens (recording at least 30 seconds is recommended). The images and videos were then imported into the "Human Sperm Localization Protein Quantitative Analysis Software" for the following analysis:

[0337] a) Morphological parameters: Automatically identify sperm heads and calculate head area, ellipticity, vacuolation ratio, etc.

[0338] b) Kinematic parameters: Track sperm motility trajectory and quantitatively calculate curvilinear velocity (VCL), linear velocity (VSL), average path velocity (VAP), linearity (LIN), forward momentum (STR), wobble (WOB), lateral swing amplitude (ALH), and whiplash frequency (BCF).

[0339] c) Generate a preliminary morphological and kinematic analysis report.

[0340] (3) Sample fixation and immunofluorescence staining

[0341] a) After completing the bright-field detection, immerse the smear in methanol for fixation for 5–10 minutes, remove it and allow it to air dry at room temperature for 5–10 minutes, followed by immunofluorescence staining:

[0342] b) Primary antibody incubation: Based on the target protein (DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, MIF), add 10 μL of the corresponding primary antibody working solution (including control wells A and B) to each well. Place the slide in a light-protected, humidified chamber and incubate at 2–8°C for at least 8 hours (or 37°C for 1 hour).

[0343] c) Washing: Remove the glass slide, gently rinse it with 1× washing solution, then immerse it in 3 portions of fresh 1× washing solution for 5 minutes each (you can shake it slightly), and finally rinse it with deionized water for 30 seconds and dry it.

[0344] d) Secondary antibody incubation: Add 10 μL of FITC-labeled goat anti-rabbit IgG secondary antibody to each well and incubate at 37°C for 30 minutes in a light-protected humidified chamber;

[0345] e) Wash again: Wash and dry;

[0346] f) Nuclear counterstaining: Add 10 μL of propidium iodide (PI) solution to each well and incubate at room temperature in the dark for 15 minutes;

[0347] g) Final wash: Wash and dry as described above;

[0348] h) Sealing: Add one drop of sealing glycerin to each well and cover with a coverslip to avoid air bubbles.

[0349] (4) Fluorescence microscopy detection

[0350] Multichannel fluorescence images were acquired using a fluorescence microscope (such as a Zeiss LSM800 or Nikon C2) under a 40x objective lens: the FITC channel (Ex / Em: 488 nm) was used to detect target proteins, and the PI channel (Ex / Em: 561 nm) was used for nuclear localization. Images of at least 200 sperm cells were acquired from each sample. First, the fluorescence signals of the A and B control wells were checked to ensure they were within the batch-specific reference range. After confirming the system was functioning correctly, full-slide images were acquired.

[0351] (5) Image analysis and report generation

[0352] Import the fluorescence images into the analysis software and perform the following processing:

[0353] a) Automatically identify the sperm head region and calculate the average fluorescence intensity, integrated optical density, and localization rate of DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF.

[0354] b) Combine the results of the previous bright-field analysis to generate a comprehensive report, including morphological, kinematic, and protein expression data;

[0355] c) Compare the results with the reference interval in the database and output the final evaluation report.

[0356] Note: All washing steps must use freshly prepared 1× washing solution and cannot be reused. Strictly avoid light during operation to prevent fluorescence quenching.

[0357] 4. Formula calculation:

[0358] (1) Sperm head detection algorithm

[0359] PI positive sperm identification formula:

[0360] I_PI(x,y) ≥ T_PI = μ_background + 3σ_background;

[0361] in:

[0362] I_PI(x,y): Pixel intensity of the PI channel;

[0363] T_PI: PI positive threshold;

[0364] μ_background: Average intensity of the background area;

[0365] σ_background: Standard deviation of background area intensity;

[0366] Head shape screening criteria:

[0367] A_min ≤ A_head ≤ A_max;

[0368] 0.7 ≤ E_head = (4πA_head) / (P_head²) ≤ 1.0;

[0369] in:

[0370] A_head: Head area (pixels);

[0371] P_head: Head perimeter (pixels);

[0372] E_head: Head ellipticity.

[0373] (2) Sperm tail segmentation algorithm

[0374] Tail recognition formula:

[0375] L_tail ≥ L_min;

[0376] W_tail ≤ W_max;

[0377] S_tail = L_tail / W_tail ≥ S_min;

[0378] in:

[0379] L_tail: Tail length;

[0380] W_tail: Tail width;

[0381] S_tail: The aspect ratio of the tail.

[0382] (3) Green fluorescence expression analysis

[0383] Fluorescence intensity calculation formula:

[0384] FI_green = (1 / N)∑[I_green(x,y) - I_background];

[0385] FI_green: Average fluorescence intensity;

[0386] N: Number of pixels in the sperm head;

[0387] I_green(x,y): Pixel intensity of the green channel;

[0388] Determination of positive fluorescence:

[0389] FI_green ≥ T_positive = μ_negative + Kσ_negative;

[0390] in:

[0391] T_positive: Positive threshold;

[0392] μ_negative: Average fluorescence intensity of the negative control;

[0393] σ_negative: Standard deviation of fluorescence intensity in the negative control;

[0394] K: Confidence coefficient (usually taken as 2-3).

[0395] (4) Key statistical indicators

[0396] Total sperm count:

[0397] N_total = ∑[I_PI(i) ≥ T_PI];

[0398] Expressing sperm count:

[0399] N_expression = ∑[FI_green(i) ≥ T_positive];

[0400] Expression rate:

[0401] R_expression = (N_expression / N_total) × 100%;

[0402] Average fluorescence intensity:

[0403] MFI = (1 / N_expression)∑FI_green(i).

[0404] Example 5: Criteria for Interpreting Reagent Kit Detection Results

[0405] After testing sperm samples using this kit, key indicators need to be quantitatively analyzed to assess sperm function. The test results are primarily based on two core indicators: expression rate (localization rate) and average fluorescence intensity. By comparing with clinically validated reference ranges, the performance of sperm samples on each target protein can be categorized into three levels: excellent, good, and needing improvement.

[0406] The definitions of each level are as follows:

[0407] Excellent: The protein expression level is highly consistent with that of sperm with high fertilization potential, indicating that it has excellent self-motility and ability to combine with the egg.

[0408] Good: Protein expression levels are within an acceptable range, but there may be slight functional defects. It is recommended to combine this with other clinical indicators for comprehensive evaluation.

[0409] Needs improvement: The protein expression level is significantly lower than the reference range, suggesting that the protein may have a functional defect, which is a high-risk factor for decreased fertilization potential.

[0410] Table 3 below details the specific interpretation criteria for twelve key proteins.

[0411] Table 3: Interpretation Criteria for Fluorescence Detection Results of Key Sperm Proteins

[0412] ;

[0413] .

[0414] Comprehensive Interpretation: This table presents independent interpretation criteria for each protein. In actual diagnosis, a comprehensive assessment should be made by combining the expression profiles of multiple proteins. For example, even if a single protein is "good," if multiple key proteins simultaneously show "needs improvement," the overall risk to fertility potential is significantly increased.

[0415] Report generation: The accompanying analysis software will automatically calculate the various indicators for each sample and generate a comprehensive report including grade interpretation and clinical analysis based on this table.

[0416] Dynamic monitoring: This standard can also be used to monitor the efficacy of assisted reproductive treatment. After treatment, the indicator changes from "needs improvement" to "good / excellent", indicating that the treatment is effective.

Claims

1. A fluorescently labeled kit for assessing sperm function and fertilizing potential, characterized in that, Kit A and Kit B, Kit A is the following components: Antibody components: including specific antibodies of rabbit anti-human DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, MIF; Labeling system: fluorescently labeled secondary antibody, nuclear fluorescent dye; Solid support: sperm protein slide, the sperm protein slide is a multi-well glass slide, and the well is pre-coated with positive and negative control human sperm samples; Kit B is the following components: Concentrated washing solution, mounting medium, fixative; The specific sequences of the antibodies of rabbit anti-human DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF are as follows: (a) DEFB126 antibody The sequence of the light chain of the DEFB126 antibody is shown in SEQ ID NO. 1, the variable region VL of the light chain of the DEFB126 antibody is shown in SEQ ID NO. 2, the complementarity determining region CDR-L1 of the light chain of the DEFB126 antibody is shown in SEQ ID NO. 3, the complementarity determining region CDR-L2 of the light chain of the DEFB126 antibody is shown in SEQ ID NO. 4, the complementarity determining region CDR-L3 of the light chain of the DEFB126 antibody is shown in SEQ ID NO. 5; the sequence of the heavy chain of the DEFB126 antibody is shown in SEQ ID NO. 6, the variable region VH of the heavy chain of the DEFB126 antibody is shown in SEQ ID NO. 7, the complementarity determining region CDR-H1 of the heavy chain of the DEFB126 antibody is shown in SEQ ID NO. 8, the complementarity determining region CDR-H2 of the heavy chain of the DEFB126 antibody is shown in SEQ ID NO. 9, and the complementarity determining region CDR-H3 of the heavy chain of the DEFB126 antibody is shown in SEQ ID NO. 10; (b) CATSPER2 antibody The CATSPER2 antibody light chain sequence is shown as SEQ ID NO. 11, the CATSPER2 antibody light chain variable region VL is shown as SEQ ID NO. 12, the CATSPER2 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 13, the CATSPER2 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 14, the CATSPER2 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 15; the CATSPER2 antibody heavy chain sequence is shown as SEQ ID NO. 16, the CATSPER2 antibody heavy chain variable region VH is shown as SEQ ID NO. 17, the CATSPER2 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 18, the CATSPER2 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 19, the CATSPER2 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 20; (c) ACROSIN antibody The ACROSIN antibody light chain sequence is shown as SEQ ID NO. 21, the ACROSIN antibody light chain variable region VL is shown as SEQ ID NO. 22, the ACROSIN antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 23, the ACROSIN antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 24, the ACROSIN antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 25; the ACROSIN antibody heavy chain sequence is shown as SEQ ID NO. 26, the ACROSIN antibody heavy chain variable region VH is shown as SEQ ID NO. 27, the ACROSIN antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 28, the ACROSIN antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 29, the ACROSIN antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 30; (d) IZUMO1 antibody The IZUMOl antibody light chain sequence is shown as SEQ ID NO. 31, the IZUMOl antibody light chain variable region VL is shown as SEQ ID NO. 32, the IZUMOl antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 33, the IZUMOl antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 34, the IZUMOl antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 35; the IZUMOl antibody heavy chain sequence is shown as SEQ ID NO. 36, the IZUMOl antibody heavy chain variable region VH is shown as SEQ ID NO. 37, the IZUMOl antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 38, the IZUMOl antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 39, the IZUMOl antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 40; (e) SPACA6 antibody The SPACA6 antibody light chain sequence is shown as SEQ ID NO. 41, the SPACA6 antibody light chain variable region VL is shown as SEQ ID NO. 42, the SPACA6 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 43, the SPACA6 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 44, the SPACA6 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 45; the SPACA6 antibody heavy chain sequence is shown as SEQ ID NO. 46, the SPACA6 antibody heavy chain variable region VH is shown as SEQ ID NO. 47, the SPACA6 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 48, the SPACA6 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 49, the SPACA6 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 50; (f) SPACA6 antibody The SPACA6 antibody light chain sequence is shown as SEQ ID NO. 51, the SPACA6 antibody light chain variable region VL is shown as SEQ ID NO. 52, the SPACA6 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 53, the SPACA6 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 54, the SPACA6 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 55; the SPACA6 antibody heavy chain sequence is shown as SEQ ID NO. 56, the SPACA6 antibody heavy chain variable region VH is shown as SEQ ID NO. 57, the SPACA6 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 58, the SPACA6 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 59, the SPACA6 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 60; (g) GRIM-19 antibody The GRIM-19 antibody light chain sequence is shown as SEQ ID NO. 61, the GRIM-19 antibody light chain variable region VL is shown as SEQ ID NO. 62, the GRIM-19 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 63, the GRIM-19 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 64, the GRIM-19 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 65; the GRIM-19 antibody heavy chain sequence is shown as SEQ ID NO. 66, the GRIM-19 antibody heavy chain variable region VH is shown as SEQ ID NO. 67, the GRIM-19 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 68, the GRIM-19 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 69, the GRIM-19 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 70; (h) ODF2 antibody The ODF2 antibody light chain sequence is shown as SEQ ID NO. 71, the ODF2 antibody light chain variable region VL is shown as SEQ ID NO. 72, the ODF2 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 73, the ODF2 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 74, the ODF2 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 75; the ODF2 heavy chain sequence is shown as SEQ ID NO. 76, the ODF2 antibody heavy chain variable region VH is shown as SEQ ID NO. 77, the ODF2 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 78, the ODF2 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 79, the ODF2 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 80; (i) PMCA4 antibody The PMCA4 antibody light chain sequence is shown as SEQ ID NO. 81, the PMCA4 antibody light chain variable region VL is shown as SEQ ID NO. 82, the PMCA4 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 83, the PMCA4 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 84, the PMCA4 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 85; the PMCA4 heavy chain sequence is shown as SEQ ID NO. 86, the PMCA4 antibody heavy chain variable region VH is shown as SEQ ID NO. 87, the PMCA4 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 88, the PMCA4 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 89, the PMCA4 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 90; (j) GPX5 antibody The GPX5 antibody light chain sequence is shown as SEQ ID NO. 91, the GPX5 antibody light chain variable region VL is shown as SEQ ID NO. 92, the GPX5 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 93, the GPX5 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 94, the GPX5 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 95; the GPX5 heavy chain sequence is shown as SEQ ID NO. 96, the GPX5 antibody heavy chain variable region VH is shown as SEQ ID NO. 97, the GPX5 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 98, the GPX5 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 99, the GPX5 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 100; (k) HPBP1 antibody The sequence of the light chain of the HPBP1 antibody is shown as SEQ ID NO. 101, the variable region VL of the light chain of the HPBP1 antibody is shown as SEQ ID NO. 102, the CDR-L1 of the light chain of the HPBP1 antibody is shown as SEQ ID NO. 103, the CDR-L2 of the light chain of the HPBP1 antibody is shown as SEQ ID NO. 104, and the CDR-L3 of the light chain of the HPBP1 antibody is shown as SEQ ID NO. 105; the sequence of the heavy chain of the HPBP1 antibody is shown as SEQ ID NO. 106, the variable region VH of the heavy chain of the HPBP1 antibody is shown as SEQ ID NO. 107, the CDR-H1 of the heavy chain of the HPBP1 antibody is shown as SEQ ID NO. 108, the CDR-H2 of the heavy chain of the HPBP1 antibody is shown as SEQ ID NO. 109, and the CDR-H3 of the heavy chain of the HPBP1 antibody is shown as SEQ ID NO. 110; (l) MIF antibody sequence The sequence of the light chain of the MIF antibody is shown as SEQ ID NO. 111, the variable region VL of the light chain of the MIF antibody is shown as SEQ ID NO. 112, the CDR-L1 of the light chain of the MIF antibody is shown as SEQ ID NO. 113, the CDR-L2 of the light chain of the MIF antibody is shown as SEQ ID NO. 114, and the CDR-L3 of the light chain of the MIF antibody is shown as SEQ ID NO. 115; the sequence of the heavy chain of the MIF antibody is shown as SEQ ID NO. 116, the variable region VH of the heavy chain of the MIF antibody is shown as SEQ ID NO. 117, the CDR-H1 of the heavy chain of the MIF antibody is shown as SEQ ID NO. 118, the CDR-H2 of the heavy chain of the MIF antibody is shown as SEQ ID NO. 119, and the CDR-H3 of the heavy chain of the MIF antibody is shown as SEQ ID NO.

120.

2. The fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, characterized in that, The antibody diluent is phosphate buffer, bovine serum albumin, and sodium azide.

3. The fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, wherein, The second fluorescently labeled antibody is a fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG antibody.

4. The fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, wherein, The concentrated washing solution is 0.3M phosphate buffer at pH 7.

4.

5. The fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, wherein, The mounting agent is mounting glycerol.

6. The fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, wherein, The nuclear dye is propidium iodide (PI) solution.

7. A method of using the fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 1, characterized by, The method comprises the following steps: (1) Sample pretreatment and smear preparation The treated semen sample is applied to a multi-well glass slide, and morphological preliminary detection is performed using an optical microscope; (2) Incubation of the first antibody After the smears obtained in step (1) are fixed, specific first antibodies against DEFB126, CATSPER2, ACROSIN, IZUMO1, SPACA6, GRIM-19, ODF2, PMCA4, GPX5, HPBP1, and MIF are incubated; (3) Washing: the smears obtained after the incubation of the first antibody in step (2) are removed, washed once with 1x washing solution, then sequentially immersed in 3 portions of 1x washing solution, each for 5 minutes, finally immersed in deionized water for 30 seconds, and dried. (4) Incubate the smear after washing and drying in step (3) with the second antibody labeled with FITC; (5) Re-washing: use the steps in step (3) to re-wash and dry the smear obtained after incubating the second antibody in step (4); (6) Use propidium iodide (PI) solution to re-stain the nucleus of the smear after washing and drying in step (5), and incubate in the dark at room temperature; (7) Final washing: use the steps in step (3) to wash and dry the smear after incubating the re-staining of the nucleus in step (6); (8) Mount the smear obtained in step (7) and take images by confocal microscope after mounting; (9) Determine the qualitative expression of each target protein on sperm by software.

8. The method of using the fluorescently labeled kit for assessing sperm function and fertilizing potential according to claim 7, wherein, 1× washing solution is 0.3M phosphate buffer solution with pH 7.4.

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