Monoclonal antibody with high affinity to recognize natural human testis-expressed protein 101 and use thereof

Abstract

The application discloses a monoclonal antibody capable of high-affinity recognition of natural human testis-expressed protein 101 (TEX101). The monoclonal antibody can recognize natural human TEX101 with high affinity. The amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 5. The application establishes a human TEX101 protein double-antibody sandwich assay method by using the monoclonal antibody or an antigen-binding fragment thereof. The linear range of the method for human TEX101 protein determination is 0.1-500 ng / mL, and the minimum detection limit reaches 0.1 ng / mL. The method can be used for determination of the content of natural human TEX101 in semen samples and can assist accurate diagnosis of azoospermia patients.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay technology, specifically to a high-affinity monoclonal antibody that recognizes natural human testis-expressed protein (TEX101) and its application in immunoassay. Background Technology

[0002] Azoospermia is divided into two main categories: non-obstructive azoospermia (NOA) and obstructive azoospermia (OA). Currently, there are various methods for diagnosing and treating azoospermia, the most common being semen analysis, which includes parameters such as total sperm count, concentration, morphology, and motility. However, semen analysis can be inaccurate due to environmental factors and infections. Testicular biopsy is the gold standard for differentiating between OA and NOA, but it is an invasive procedure that may be accompanied by complications such as tissue damage, pain, bleeding, decreased testicular function, and even impaired fertility. Therefore, finding a more accurate and non-invasive method to differentiate azoospermia and predict the success rate of testicular microsurgical sperm retrieval in NOA is of significant clinical importance for assessing testicular spermatogenesis and developing subsequent treatment plans.

[0003] Seminal plasma is composed of secretions from the seminal vesicles, prostate, testes, epididymis, and urethra. The levels of sperm-specific proteins contained within it show a significant correlation with sperm count, making it an ideal biomarker for the auxiliary diagnosis of diseases such as azoospermia. Currently, clinically approved reagents for azoospermia testing all target sperm protein 10 (SP10). However, a small number of patients cannot be accurately diagnosed based on this single biomarker and require the use of other biomarkers for auxiliary diagnosis. Testis expressed protein 101 (TEX101) is a membrane glycoprotein that is mainly expressed on the surface of germ cells during spermatogenesis. It is shed into the seminal plasma during the later stages of sperm maturation in the testes. Therefore, its concentration level is significantly correlated with sperm count and can serve as an effective supplement to SP10 in the diagnosis of male infertility.

[0004] The human TEX101 gene is located on chromosome 19q13.31, encoding the TEX101 protein, which has a relative molecular weight of approximately 38 kDa. It includes one signal peptide sequence, one N-glycosylation site, four O-glycosylation sites, one short transmembrane domain, one GPI binding site, and one conserved urokinase-type plasminogen activator receptor domain. Therefore, the TEX101 protein is a conformationally complex, highly glycosylated protein, making it difficult to prepare high-affinity antibodies. Consequently, no TEX101 protein detection reagent has been approved for clinical use to date. In view of this, this invention will first prepare antibodies by immunizing animals with prokaryotically expressed TEX101 protein, and then use a double-antibody sandwich method to screen for high-affinity monoclonal antibodies that can recognize the natural TEX101 protein, providing key raw materials for the development of a TEX101 protein detection reagent. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a monoclonal antibody prepared using mouse hybridoma technology that can recognize natural human TEX101 protein with high affinity. The monoclonal antibody, as a detection antibody, can accurately determine the content of human TEX101 protein in semen samples through a double antibody sandwich method.

[0006] Therefore, a first aspect of the present invention relates to a high-affinity monoclonal antibody or antigen-binding fragment thereof for recognizing natural human TEX101 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein,

[0007] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2;

[0008] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3;

[0009] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4;

[0010] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6;

[0011] The amino acid sequence of the light chain CDR2 is LVS;

[0012] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7.

[0013] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0014] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody or a humanized antibody, which can recognize the natural human TEX101 protein with high affinity because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain.

[0015] A second aspect of the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described monoclonal antibody or an antigen-binding fragment thereof.

[0016] A third aspect of the present invention relates to an expression vector comprising the above-described nucleic acid molecules, said expression vector being capable of expressing the above-described monoclonal antibody or its antigen-binding fragment.

[0017] The fourth aspect of the present invention relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned monoclonal antibody or its antigen-binding fragment, and further, it can be a mammalian cell recombinant, an insect cell recombinant, a yeast recombinant or a bacterial recombinant.

[0018] The fifth aspect of this invention relates to a monoclonal antibody mouse hybridoma cell line that secretes the aforementioned monoclonal antibody capable of recognizing the natural human TEX101 protein with high affinity. Further, the monoclonal antibody mouse hybridoma cell line is mouse hybridoma cell line 1B62 with accession number CGMCC No. 46850.

[0019] The sixth aspect of this invention relates to the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for detecting human TEX101.

[0020] A seventh aspect of the present invention relates to a method for detecting human TEX101, the method comprising the step of using the aforementioned monoclonal antibody or its antigen-binding fragment as the detection antibody to achieve accurate determination of human TEX101 content. Furthermore, the method for detecting human TEX101 is a double-antibody sandwich detection method, wherein the monoclonal antibody or its antigen-binding fragment is used as the detection antibody.

[0021] The eighth aspect of this invention relates to a kit for detecting human TEX101, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment. Furthermore, the kit is a double-antibody sandwich type kit, wherein the monoclonal antibody or its antigen-binding fragment is used as the detection antibody.

[0022] Instructions for the Preservation of Biological Materials

[0023] The mouse hybridoma cell line 1B62 of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with registration number CGMCC No. 46850, deposit date May 22, 2026, and classified as: Mouse Hybridoma Cell Line. The address of the China General Microbiological Culture Collection Center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0024] Figure 1 This is an SDS-PAGE electrophoresis image of the mature peptide of human TEX101 protein expressed in prokaryotes, where M is the molecular weight standard, 1 is pG-TEX101 protein, and 2 is pE-TEX101 protein.

[0025] Figure 2 This is the result of the titer assay for rabbit anti-human TEX101 protein polyclonal antibody.

[0026] Figure 3 This is the result of TEX101 protein assay in human semen samples.

[0027] Figure 4 This is a diagram showing the identification results of the anti-human TEX101 monoclonal antibody 1B62 subtype. Detailed Implementation

[0028] The purpose of this invention is to provide a monoclonal antibody prepared using mouse hybridoma technology that can recognize natural human TEX101 protein with high affinity. The monoclonal antibody is used as a detection antibody to accurately determine the content of human TEX101 protein through a double antibody sandwich method.

[0029] The specific preparation process is as follows: First, the mature peptide (26-222aa) of isoform 2 of human TEX101 protein is expressed in prokaryotes to obtain pG-TEX101 and pE-TEX101 proteins. Rabbits were immunized with pE-TEX101 protein as an immunogen to prepare rabbit anti-human TEX101 polyclonal antibodies. Simultaneously, mice were immunized with pE-TEX101 protein as an immunogen to prepare monoclonal antibodies. Using pG-TEX101 protein as a coating antigen, monoclonal antibodies with high affinity for TEX101 protein were screened using an indirect enzyme-linked immunosorbent assay (ELISA). Since natural human TEX101 protein is a conformationally complex, highly glycosylated protein, it is necessary to screen for monoclonal antibodies that can recognize natural human TEX101 protein to establish an accurate detection method. Therefore, this embodiment uses normal human semen samples containing a high concentration of natural TEX101 protein and employs a double-antibody sandwich method combined with an indirect method to screen for high-affinity monoclonal antibodies that specifically recognize natural human TEX101 protein. The mouse hybridoma cell line that secretes this monoclonal antibody is named mouse hybridoma cell line 1B62 or 1B62 in this patent application. The inventors deposited this mouse hybridoma cell line at the China General Microbiological Culture Collection Center (CGMCC) on May 22, 2026, with accession number CGMCC No. 46850, and classified it as mouse hybridoma cell line.

[0030] Next, the inventors used the prepared high-affinity monoclonal antibody 1B62, which recognizes natural human TEX101 protein, as the detection antibody, and established a method for determining the content of human TEX101 protein based on the double antibody sandwich principle.

[0031] The double-antibody sandwich assay method for human TEX101 protein established in this invention has a linear detection range of 0.1-500 ng / mL and a limit of detection of 0.1 ng / mL, achieving accurate quantitative determination of TEX101 protein.

[0032] The inventors amplified and sequenced the gene sequence of the monoclonal antibody 1B62 secreted by the mouse hybridoma cell line CGMCC No. 46850. Then, using public software from the National Center for Biotechnology Information (NCBI) website, they analyzed the immunoglobulin domain sequence of this monoclonal antibody and found that its heavy chain variable region has 115 amino acids, with the following sequence: VQLQQSGAELVKPGASVKLSCKAS GYTFTSYY VYWVKQRPGQGLEWIGE IDPRNGVSNFNEKFKTKATLTVDKSSNTAYMQLSSLTSEDSAVYYC AGRFDAMEY WGQGTTVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 25-32 amino acids with the amino acid sequence GYTFTSYY (SEQ ID NO.2); CDR2 is located at 50-57 amino acids with the amino acid sequence IDPRNGVS (SEQ ID NO.3); and CDR3 is located at 96-104 amino acids with the amino acid sequence AGRFDAMEY (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSH MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEEAPSWKS (SEQ ID NO. 5) has three CDR regions indicated by underscores. CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSH (SEQ ID NO. 6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO. 7).

[0033] As is well known in the art, although the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens, conserved amino acid substitution is a biotechnological means in protein engineering to maintain the functional properties of proteins by replacing amino acid residues of the same family with similar physicochemical properties. This method mainly involves the directional substitution between amino acids of the same family, thereby ensuring that the binding affinity and specificity of the protein do not change significantly after substitution. In this patent application, the conserved amino acid substitution includes the substitution between aromatic amino acids Phe, Trp, and Tyr; the substitution between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; the substitution between polar amino acids Gln and Asn; the substitution between basic amino acids Lys, Arg, and His; the substitution between acidic amino acids Asp and Glu; and the substitution between hydroxyl amino acids Ser and Thr. The conserved substitution of a single amino acid in the amino acid sequences of the heavy chain CDR region and the light chain CDR region should not change the structure of the protein. Therefore, the conserved substitution of a single amino acid in the above-mentioned regions may still have the property of binding to the corresponding antigen. Therefore, monoclonal antibodies or their antigen-binding fragments obtained by making a conserved substitution of one amino acid in heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize natural human TEX101 protein with high affinity.

[0034] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing natural human TEX101 protein with high affinity from the monoclonal antibodies of the present invention, such as, but not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the region in the antibody structure that can bind to the antigen, consisting of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragments can be prepared as follows: for example, after enzymatic digestion with papain, antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind the corresponding antigen, they can be used to prepare kits for detecting human TEX101.

[0035] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. The single-chain antibody can be expressed, for example, in *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of high affinity recognition of natural human TEX101 protein, and therefore can be used to prepare kits for detecting human TEX101.

[0036] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of the monoclonal antibody that recognizes the natural human TEX101 protein with high affinity, based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector capable of expressing a monoclonal antibody or its antigen-binding fragment that recognizes the natural human TEX101 protein with high affinity. Those skilled in the art can also introduce the synthesized nucleic acid molecules or constructed expression vectors into host cells such as mammalian cells, insect cells, yeast cells, or bacterial cells to obtain mammalian cell recombinants, insect cell recombinants, yeast recombinants, or bacterial recombinants, and then express these recombinants to produce the antibody or its antigen-binding fragment of the present invention. The antibody or its antigen-binding fragment expressed in this way can recognize the natural human TEX101 protein with high affinity; therefore, the aforementioned nucleic acid molecules, expression vectors, and mammalian cell recombinants, yeast recombinants, or bacterial recombinants are within the scope of protection of the claims of this invention. Furthermore, the above-described techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0037] As described above, the antibody or its antigen-binding fragment of the present invention can recognize natural human TEX101 protein with high affinity, and therefore can be used to prepare a kit for detecting human TEX101. The kit can be any kit that utilizes the antibody or its antigen-binding fragment of the present invention to react with natural human TEX101 protein, such as, but not limited to, double antibody sandwich kits. Specific kits include, but are not limited to, kits using enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence, Western blotting, and immunohistochemistry.

[0038] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0039] Example 1: Prokaryotic expression of mature human TEX101 protein peptide

[0040] The amino acid sequence of human TEX101 protein was searched in the GenBank database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ). Two isoforms of human TEX101 protein were found: isoform 1 and isoform 2. The NCBI reference sequence for isoform 1 is NP_113639, with a total length of 267 amino acids; the NCBI reference sequence for isoform 2 is NP_001123483, with a total length of 249 amino acids. The amino acid sequences of the two isoforms were compared using the BLAST (protein-protein BLAST) function on the NCBI website. The results showed that isoform 1 has 18 more amino acids at the N-terminus compared to isoform 2, while the remaining 249 amino acids are identical to isoform 2. After the signal peptide and C-terminal amino acids of isoform 2 are removed, it is transformed into a mature peptide of approximately 21 kD, namely 26-222 amino acids, with the amino acid sequence LYCQKGLSMTVEADPANMFNWTTEEVETCDKGALCQETILIIKAGTETAILATKGCIPEGEEAITIVQHSSPPGLIVTSYSNYCEDSFCNDKDSLSQFWEFSETTASTVSTTLHCPTCVALGTCFSAPSLPCPNGTTRCYQGKLEITGGGIESSVEVKGCTAMIGCRLMSGILAVGPMFVREACPHQLLTQPRKTEN (SEQ ID NO. 8). The mature peptide of TEX101 isoform 2 is the TEX101 protein that is shed into the seminal plasma during the later stages of sperm maturation, making it an ideal biomarker detectable in seminal plasma. Therefore, this invention expresses the mature peptide of TEX101 protein.

[0041] First, based on the genetic code preferences of *E. coli*, the optimized nucleotide sequence of the mature peptide (26-222aa) of TEX101 protein suitable for expression in the *E. coli* expression system was deduced, as shown in SEQ ID NO. 9. Then, Beijing Qingke Biotechnology Co., Ltd. synthesized the optimized nucleotide sequence of the mature human TEX101 protein. Using conventional biological techniques, this sequence was ligated into the pGEX-4T-2 and pET-28a vectors, respectively, to construct the recombinant expression vectors pGEX-TEX and pET-TEX. Both vectors had BamHI as the upstream restriction enzyme site and EcoRI as the downstream restriction enzyme site. The pGEX-TEX recombinant expression vector was transformed into *E. coli* HB101, and the pET-TEX recombinant expression vector was transformed into *E. coli* BL21(DE3). Expression was induced by IPTG at 37℃. The pG-TEX101 and pE-TEX101 proteins were then purified by GST or Ni column, respectively. The purified proteins were identified by SDS-PAGE gel electrophoresis. The results are as follows: Figure 1As shown, 1 is the pG-TEX101 protein, with a molecular weight of approximately 48 kDa; 2 is the pE-TEX101 protein, with a molecular weight of approximately 26 kDa.

[0042] Example 2: Preparation of rabbit anti-human TEX101 protein polyclonal antibody

[0043] Rabbit anti-human TEX101 polyclonal antibodies were prepared using the pE-TEX101 protein prepared in Example 1 as an immunogen. The specific preparation process is as follows: One healthy New Zealand white rabbit was selected, and 1.0 mg of pE-TEX101 protein prepared in Example 1 was mixed with 1.0 mL of Freund's complete adjuvant. After thorough emulsification with a stirrer, 0.2 mL of the mixture was injected subcutaneously at both sides of the rabbit's spine. Four weeks later, 1.0 mg of pE-TEX101 protein was mixed with 1.0 mL of Freund's incomplete adjuvant. After thorough emulsification with a stirrer, a second immunization was performed at different points on the same site. A third booster immunization was performed four weeks later. One week later, blood was collected from the heart, centrifuged at 5000 rpm for 15 minutes, and the serum was aliquoted and stored at -20°C for later use. The titer of polyclonal antibodies was determined using an indirect enzyme-linked immunosorbent assay (ELISA). The specific procedure was as follows: ELISA plates were coated with pG-TEX101 protein prepared in Example 1 at a concentration of 2.0 μg / mL, 150 μL per well, and incubated overnight at 4°C. The plates were washed twice with washing buffer. 200 μL / well blocking buffer was added, and the plates were blocked at room temperature for 6 hours. The plates were then washed five times with washing buffer. Rabbit serum was serially diluted with PBS to a dilution factor of 0.2 × 10⁻⁶. 4 0.4×10 4 0.8×10 4 1.6×10 4 3.2×10 4 6.4×10 4 12.8×10 4 25.6×10 4 and 51.2×10 4 Add 100 μL of sample to each well; incubate at 37°C for 45 min; wash the plate 5 times with washing buffer, 200 μL per well; incubate with HRP-labeled goat anti-rabbit secondary antibody at 37°C for 45 min; wash the plate 5 times with washing buffer, 200 μL per well; add 100 μL of freshly prepared substrate solution to each well, and incubate at 37°C for 10 min; stop the reaction by adding 50 μL of 2 M H2SO4 to each well. Measure the absorbance of each well (A450nm) using a microplate reader at a wavelength of 450 nm, and read the values ​​within 10 minutes after termination. Results are as follows. Figure 2 As shown, using pre-immunization rabbit serum as a negative control, the prepared rabbit anti-human TEX101 protein polyclonal antibody achieved a titer of 1:256000. The prepared polyclonal antibody exhibits high affinity for the TEX101 protein.

[0044] Example 3: Preparation of high-affinity monoclonal antibody against human TEX101

[0045] Using the pE-TEX101 protein prepared in Example 1 as the immunogen, 6-8 week old female BALB / c mice were immunized with 100 µg of antigen per mouse plus an equal volume of Freund's complete adjuvant, thoroughly emulsified, and injected subcutaneously and intraperitoneally into the back. A second immunization was performed 4 weeks later, with 50 µg of antigen per mouse plus incomplete Freund's adjuvant, thoroughly emulsified, and injected subcutaneously and intraperitoneally into the back. A third immunization was performed 8 weeks later. One week after the third immunization, blood was collected from the tail vein of the mice to detect the titer of the immune serum. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection. Three days later, spleen cells were harvested for fusion. Spleen cells were prepared from the spleens of the immunized mice to form a spleen cell suspension. Spleen cells and SP20 myeloma cells were fused at a ratio of 9:1 using standard procedures. When the fused cells covered approximately 60% of the bottom of the well, the cell culture supernatant was collected. High-affinity positive clones were screened using the enzyme-linked immunosorbent assay (ELISA) as described in Example 2. Clones with an A450nm value greater than 3.0 were defined as high-affinity positive clones. Five high-affinity positive clones were screened: 1B62, 1E113, 2F912, 3A46, and 4H101. The mouse hybridoma cell lines from these five high-affinity positive clones were further expanded, and 1×10⁻⁶ cells were harvested from each clone. 6 One cell was injected into the peritoneal cavity of mice, and ascites fluid was collected 2 weeks later. The antibody was purified by conventional methods using a protein G column. The purified antibody was aliquoted into 1 mg vials and stored at -20°C.

[0046] Example 4: Screening of high-affinity monoclonal antibodies for recognizing natural human TEX101 protein

[0047] Because the natural human TEX101 protein is a highly glycosylated protein with a complex conformation, it is necessary to screen for monoclonal antibodies that can recognize the natural human TEX101 protein in order to establish an accurate detection method. This embodiment uses normal human semen samples and employs a double-antibody sandwich method combined with an indirect method to screen for high-affinity monoclonal antibodies that can specifically recognize the natural human TEX101 protein.

[0048] First, semen samples were collected from men of normal fertility. After liquefaction at room temperature for 1 hour, the samples were centrifuged at 13000 g for 15 minutes, and the supernatant was collected. The supernatant was diluted 1:100 with physiological saline containing 2.0 mg / mL dithiothreitol and 0.5 mg / mL Trixton X-100 to prepare the sample to be tested. The specific operation of the double antibody sandwich method combined with the indirect method was as follows: the rabbit anti-human TEX101 protein polyclonal antibody prepared in Example 2 was used to coat the ELISA plate at a concentration of 2.5 μg / mL, with 150 μL coated per well, and incubated overnight at 4°C; the plate was washed twice with washing buffer; 200 μL / well blocking buffer was added and the plate was blocked at room temperature for 6 hours; the plate was washed 5 times with washing buffer. The first step involved adding 100 μL of male semen sample to each well, incubating at 37°C for 120 min, and washing the plate 5 times with washing buffer, with 200 μL added to each well. The second step involved adding 100 μL of each of the five high-affinity monoclonal antibodies against human TEX101 prepared in Example 3, diluted 1:1000, incubating at 37°C for 60 min, and washing the plate 5 times with washing buffer, with 200 μL added to each well. The third step involved adding HRP-labeled goat anti-mouse secondary antibody and incubating at 37°C for 30 min; washing the plate 5 times with washing buffer, with 200 μL added to each well. Finally, 50 μL each of chromogenic solutions A and B were added, and after incubation at 37°C for 10 minutes, 50 μL of 2 M H2SO4 was added to each well to terminate the reaction. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm (A450nm), and the readings were taken within 10 minutes after termination. The results are shown in Table 1. Monoclonal antibodies 1B62, 1E113 and 3A46 can recognize natural human TEX101 protein, with 1B62 and 3A46 showing high detection values; monoclonal antibodies 2F912 and 4H101 can hardly recognize natural human TEX101 protein.

[0049] Table 1. Detection performance of high-affinity monoclonal antibodies against human TEX101 against natural proteins.

[0050]

[0051] Next, the semen samples from normal individuals were serially diluted with physiological saline at ratios of 1:100, 1:1000, 1:5000, and 1:10000. High-affinity monoclonal antibodies 1B62 and 3A46 against human TEX101 were used as detection antibodies. The samples were tested using a combination of the aforementioned double-antibody sandwich method and an indirect method. The results are shown in Table 2. Monoclonal antibody 1B62 showed higher affinity for natural TEX101 protein, remaining detectable even at a 5000-fold dilution, and the detected value was significantly higher than that of monoclonal antibody 3A46.

[0052] Table 2. Detection performance of monoclonal antibodies 1B62 and 3A46 against native TEX101 protein (A450nm)

[0053]

[0054] Example 5: Establishment of a double-antibody sandwich assay method for human TEX101 protein

[0055] The purpose of this embodiment is to establish a double-antibody sandwich method for the specific detection of human TEX101 protein by using the rabbit anti-TEX101 polyclonal antibody prepared in this invention as the capture antibody and the high-affinity monoclonal antibody 1B62 recognizing natural TEX101 protein as the detection antibody. The specific method is as follows: An ELISA plate was coated with rabbit anti-TEX101 polyclonal antibody at a concentration of 2.5 μg / mL, with 120 μL added to each well. The plate was incubated overnight at 4°C, and washed twice with washing buffer. 150 μL / well of blocking buffer was added, and the plate was incubated overnight at 4°C. The buffer was then discarded and the plate was air-dried. The pG-TEX101 protein prepared in Example 1 was serially diluted with physiological saline to concentrations of 1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1, and 0.05 ng / mL. Add 100 μL of pG-TEX101 protein sample at different dilutions to the test wells and incubate at 37℃ for 120 min. Wash the plate 5 times with washing buffer, 300 μL per well. Then add HRP-labeled monoclonal antibody 1B62 and incubate at 37℃ for 60 min. Wash the plate 5 times with washing buffer, 300 μL per well. Finally, add 50 μL each of chromogenic solutions A and B, incubate at 37℃ for 10 min, and then add 50 μL of 2 M H2SO4 to each well to stop the reaction. Measure the absorbance of each well at 450 nm using a microplate reader (A450 nm). Read the values ​​within 10 minutes after termination. Each concentration of sample was tested three times, and the average value was calculated. The results are shown in Table 3. Plot the logarithm of protein concentration on the ordinate and the measured A450 nm value on the abscissa. The detectable linear range is 0.1-500 ng / mL, and the limit of detection is 0.1 ng / mL.

[0056] Table 3. Limit of Detection for Double Antibody Sandwich Detection Method of Human TEX101 Protein

[0057]

[0058] Example 6: Determination of human TEX101 protein content in semen samples

[0059] The human TEX101 protein double-antibody sandwich assay method established above was used to simultaneously test semen samples from 10 patients with azoospermia and 10 normal healthy men. The specific testing procedures were the same as described above. Results are as follows... Figure 3As shown, the average human TEX101 protein level in 10 semen samples from patients with azoospermia was 109.65 ± 13.72 ng / mL, while the average human TEX101 protein level in 10 semen samples from normal healthy men was 3247.30 ± 386.29 ng / mL. The difference between the two groups was significant (p < 0.0001), indicating that the method of the present invention can be used for the specific detection of human TEX101 protein and the accurate diagnosis of azoospermia.

[0060] Example 7: Identification of the 1B62 subtype of the anti-human TEX101 monoclonal antibody

[0061] The heavy and light chain isotypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a, batch number 052725) from Antaiji (Beijing) Biotechnology Co., Ltd. 100 μL of supernatant from mouse hybridoma cell line 1B62 was added to the sample wells of the rapid mouse antibody subtype detection card, and the results were observed and recorded after standing for 5-10 min. The results are as follows: Figure 4 As shown, the anti-human TEX101 monoclonal antibody 1B62 is mouse IgG2b subtype, and the antibody light chain is Igκ subtype.

[0062] Example 8: Determination of the amino acid sequence of the variable region of anti-human TEX101 monoclonal antibody 1B62

[0063] Mouse hybridoma cell line 1B62, secreting anti-human TEX101 monoclonal antibody, was cultured. Total RNA was extracted from the hybridoma cells using the Trizol method, and cDNA was reverse transcribed. PCR amplification was then performed using primers for the Fab fragment of the mouse monoclonal antibody synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are available in *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen. Amplification conditions were as follows: preheating at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The PCR product was ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data was compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website.

[0064] Sequence analysis revealed that the heavy chain variable region contains 115 amino acids, with the following sequence: VQLQQSGAELVKPGASVKLSCKAS GYTFTSYY VYWVKQRPGQGLEWIGE IDPRNGVS NFNEKFKTKATLTVDKSSNTAYMQLSSLTSEDSAVYYC AGRFDAMEYWGQGTTVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 25-32 amino acids with the amino acid sequence GYTFTSYY (SEQ ID NO.2); CDR2 is located at 50-57 amino acids with the amino acid sequence IDPRNGVS (SEQ ID NO.3); and CDR3 is located at 96-104 amino acids with the amino acid sequence AGRFDAMEY (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVST SGYSH MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEEAPSWKS (SEQ ID NO.5) has three CDR regions indicated by underscores: CDR1 is located at 27-36 aa with the amino acid sequence KSVSTSGYSH (SEQ ID NO.6); CDR2 is located at 54-56 aa with the amino acid sequence LVS; and CDR3 is located at 93-100 aa with the amino acid sequence QHIRELTR (SEQ ID NO.7).

Claims

1. A high-affinity monoclonal antibody or antigen-binding fragment thereof recognizing natural human TEX101, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by mouse hybridoma cell line 1B62 with accession number CGMCC No.46850.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

5. A nucleic acid molecule, characterized in that, It comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A recombinant, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The recombinant according to claim 7, characterized in that, It can be a mammalian cell recombinant, an insect cell recombinant, a yeast recombinant, or a bacterial recombinant.

9. A mouse hybridoma cell line that secretes a high-affinity monoclonal antibody recognizing natural human TEX101, characterized in that, It is the mouse hybridoma cell line 1B62 with accession number CGMCC No.46850.

10. The use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a kit for detecting human TEX101.

11. A method for detecting human TEX101, characterized in that, The step includes using the monoclonal antibody or its antigen-binding fragment as a detection antibody according to any one of claims 1 to 4.

12. A reagent kit for detecting human TEX101, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

13. The kit of claim 12, characterized in that, It is a double-antibody sandwich type kit, in which the monoclonal antibody or its antigen-binding fragment serves as the detection antibody.

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