Anti-human sperm protein 10 (SP10) monoclonal antibody pair combination and application thereof

Abstract

The application provides a pair of anti-human sperm SP10 protein monoclonal antibody combinations and application thereof, comprising anti-human sperm SP10 protein monoclonal antibodies Ab1 and Ab2; the amino acid sequences of the heavy chain CDR1 sequence, the CDR2 sequence and the CDR3 sequence of the monoclonal antibody Ab1 are shown in SEQ ID NO:2-4, the amino acid sequences of the light chain CDR1 sequence, the CDR2 sequence and the CDR3 sequence are shown in SEQ ID NO:6-8; the amino acid sequences of the heavy chain CDR1 sequence, the CDR2 sequence and the CDR3 sequence of the monoclonal antibody Ab2 are shown in SEQ ID NO:10-12, and the amino acid sequences of the light chain CDR1 sequence, the CDR2 sequence and the CDR3 sequence are shown in SEQ ID NO:14-16. The pair has the characteristics of high affinity and high specificity.

Description

TECHNICAL FIELD

[0001] The application relates to a pair of anti-human sperm SP10 protein monoclonal antibodies and application thereof, and belongs to the technical field of antibodies. BACKGROUND

[0002] The WHO Manual for the Examination and Processing of Human Semen (6th edition) points out that the main factors affecting male infertility include semen volume, total sperm count, sperm concentration, liquefaction time, semen pH value and sperm normal morphology rate, wherein the sperm concentration detection is an important index for diagnosing male infertility. The manual sets the reference lower limit of the sperm concentration to 15x10 6 When the sperm concentration is lower than 15x10 6 , it can be considered that there is difficulty in procreation.

[0003] At present, the semen detection methods mainly include a classic chromogenic micro counting method and a colorimetric method. The detection instruments using the micro counting method mainly include a blood cell counting pool, a Makler counting pool and a Cell-VU counting pool and the like, and these detection methods are highly dependent on professional semen analyzers, are complex in operation and are greatly affected by the semen samples themselves.

[0004] Human sperm acrosome protein SP-10 is a sperm-specific protein and plays an important role in the fertilization process. It is expressed by a human ACRV1 gene and exists in multiple subtypes. A typical SP-10 protein has a molecular weight of 28kD and is distributed in acrosomes, and is a highly conserved specific differentiation marker in the sperm development process. Numerous studies on the correlation between human sperm acrosome protein and semen routine parameters show that the semen concentration, sperm motility and sperm morphology are positively correlated with the acrosome protein SP-10. Therefore, the acrosome protein SP-10 can be used as a detection means for rapidly evaluating the semen quality, providing assistance for the diagnosis of infertility and providing pre-pregnancy guidance for couples of childbearing age.

[0005] The existing detection methods for the human sperm acrosome protein SP-10 mainly include a colloidal gold immunoreagent and an ELISA kit (human sperm SP10 protein ELISA detection kit). The colloidal gold method is a high-efficiency and rapid detection method, is simple in operation and is suitable for individual on-site rapid detection, but has low sensitivity, is sensitive to the environment and has a relatively high cost compared with the latex method. The ELISA method has high sensitivity, but has a long detection time, is complicated in operation and needs professional personnel and special equipment.

[0006] These detection methods all rely on monoclonal antibodies with high affinity and high specificity. Reported SP10 antibodies have several drawbacks: either insufficient specificity leading to cross-reactivity with reproductive tract pathogens (such as gonococci and chlamydia), or low affinity with a sensitivity of only 1 ng / ml. Therefore, developing high-affinity and high-specificity anti-human sperm SP10 protein monoclonal antibodies is fundamental to the preparation of high-performance immunoassay reagents. Summary of the Invention

[0007] This invention provides a pairing combination of monoclonal antibodies against human sperm SP10 protein and its application, which can effectively solve the above-mentioned problems.

[0008] A pair of monoclonal antibodies against human sperm SP10 protein, comprising monoclonal antibodies Ab1 and Ab2 against human sperm SP10 protein;

[0009] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences of the monoclonal antibody Ab1 are shown in SEQ ID NO:2-4, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:6-8.

[0010] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences of the monoclonal antibody Ab2 are shown in SEQ ID NO:10-12, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:14-16.

[0011] In some embodiments, the monoclonal antibodies against human sperm SP10 protein are paired together, wherein the heavy chain variable region sequence of monoclonal antibody Ab1 is as shown in SEQ ID NO:1, and the light chain variable region sequence is as shown in SEQ ID NO:5; and the heavy chain variable region sequence of monoclonal antibody Ab2 is as shown in SEQ ID NO:9, and the light chain variable region sequence is as shown in SEQ ID NO:13.

[0012] In some embodiments, the monoclonal antibodies against human sperm SP10 protein are paired together, wherein the heavy chain constant regions of the monoclonal antibodies Ab1 and Ab2 are the mouse IgG1 constant regions, and their amino acid sequences are SEQ ID NO:17.

[0013] In some embodiments, the monoclonal antibody pairing for anti-human sperm SP10 protein, wherein the light chain constant regions of the monoclonal antibodies Ab1 and Ab2 are mouse κ constant regions, and their amino acid sequences are SEQ ID NO:18. A reagent for detecting human sperm SP10 protein includes the aforementioned monoclonal antibody pairing for anti-human sperm SP10 protein.

[0014] In some embodiments, the detection reagent is a sandwich ELISA reagent, wherein: the monoclonal antibody Ab1 is a coating antibody, and the monoclonal antibody Ab2 is a detection antibody.

[0015] A test kit for detecting human sperm SP10 protein, comprising:

[0016] The conjugate pad contains a colored microsphere marker labeled with monoclonal antibody Ab2 and a mouse IgG marker;

[0017] A nitrocellulose membrane, wherein a detection line (T line) and a control line (C line) are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody Ab1, and the control line is coated with goat anti-mouse IgG polyclonal antibody.

[0018] In some embodiments, the concentration of monoclonal antibody Ab2 in the monoclonal antibody Ab2-labeled colored microsphere marker of the human sperm SP10 protein detection kit is 0.4-0.6 mg / mL; and the concentration of mouse IgG in the mouse IgG marker is 0.5-1.5 mg / mL.

[0019] A detection kit for human sperm SP10 protein, comprising the aforementioned detection kit card for human sperm SP10 protein.

[0020] The application of the monoclonal antibody pairing combination against human sperm SP10 protein in the preparation of reagents or kits for detecting human sperm SP10 protein.

[0021] The beneficial effects of this invention are:

[0022] This invention aims to provide a monoclonal antibody pairing scheme targeting the human sperm SP10 protein. This pairing scheme not only exhibits extremely high affinity but also excellent specificity, significantly surpassing the performance level of existing technologies. Through careful design and optimization, this monoclonal antibody pairing demonstrates outstanding performance in recognizing and binding to the human sperm SP10 protein, providing a more reliable and efficient tool for related research fields.

[0023] This invention also introduces a detection kit based on human sperm SP10 protein, using the latex method for detection. This kit cleverly combines the speed and simplicity of the colloidal gold method with the high sensitivity of the ELISA method, achieving a dual improvement in detection efficiency and accuracy. Specifically, compared to the traditional colloidal gold method, this kit significantly improves sensitivity, enabling more accurate capture of low concentrations of SP10 protein; and compared to the ELISA method, its operation is simpler, greatly shortening the detection time and reducing operational difficulty. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a flowchart of the detection operation of the detection card in Example 3.

[0026] Figure 2 This is a method for judging the detection results of the detection card in Example 3. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0028] The reagents or kits involved in the embodiments of this application and their sources are as follows:

[0029] SP10 recombinant protein (Jingjie Biotechnology); Freund's Adjuvant, Complete (ThermoFisher); Freund's Adjuvant, Incomplete (Thermo Fisher); HAT Media Supplement (50×) (Thermo Fisher); HT Media Supplement (50×) (ThermoFisher); PEG (sigma); RPMI 1640 (Shanghai Yuanpei Biotechnology); Fetal bovine serum FBS (Shanghai Xiaopeng Biotechnology); DMEM (Shanghai Yuanpei Biotechnology); Penicillin-Streptomycin (gibco); HRP-labeled goat anti-mouse antibody (Shanghai Sangon Biotech); Protein A Resin (Changzhou Tiandi Renhe Biotechnology Co., Ltd.).

[0030] Example 1: Preparation of monoclonal antibody against human sperm SP10 protein

[0031] 1. Mouse immunization

[0032] SP10 recombinant protein was used as the antigen. After dissolution, it was emulsified with an equal volume of Freund's complete adjuvant. 6-8 week old SPF-grade Balb / c mice (Fuzhou Wu's Animal Experiment Center) were injected subcutaneously at multiple sites with 200 μg / mouse. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites with 100 μg / mouse. Two booster immunizations were given. Three days before fusion, intraperitoneal injection was given as a shock.

[0033] 2. Preparation of feeder cells

[0034] BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mice were euthanized by cervical retraction, immersed in 75% alcohol, and under aseptic conditions in a laminar flow hood, the abdominal skin was cut open with scissors to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium containing 1% penicillin-streptomycin was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, and the rinsing solution was collected. The cells were centrifuged at 1000 rpm for 5 minutes, and the pellet was collected and resuspended in RPMI 1640 complete culture medium containing 1% HAT. The cell concentration was adjusted to 1×10⁵ cells / mL, and 150 μL / well was added to 96-well plates. The cells were incubated overnight at 37°C with 5% CO₂.

[0035] 3. Preparation of immune spleen cells

[0036] Three days after the last immunization of mice, the spleen was removed under aseptic conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, and then ground and filtered on a nylon mesh in a small beaker to prepare a cell suspension. The suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and the cells were counted.

[0037] 4. Cell fusion

[0038] (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12000) and place them in a 37°C water bath for preheating;

[0039] (2) Mouse myeloma cells Sp2 / 0 (2-5×10⁻⁵) were collected respectively. 7 (10) of the above-mentioned immune spleen cells 8 Add the suspension to a 50mL centrifuge tube, mix well, and add DMEM serum-free culture medium to a final volume of 40mL. Centrifuge for 10 minutes, discard the supernatant, and mix well.

[0040] (3) Place the centrifuge tube in pre-warmed water at 37°C, take 0.7 mL of pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of pre-warmed serum-free culture medium at 37°C;

[0041] (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%–20% fetal bovine serum. Mix well with a pipette and add 2 drops to each of the four wells of a 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.

[0042] 5. Selective culture of hybridoma cells

[0043] The cells were cultured in the aforementioned HAT medium on days 1, 3, 5, and 7 after fusion. The surviving cells were hybridoma cells, while the non-hybridoma cells died, thus selecting the true hybrid cells.

[0044] 6. Detection of specific antibodies and cloning of hybridoma cells

[0045] Collect the supernatant from each culture well and use indirect ELISA to detect culture wells containing the specific recognition protein SP10. Wells with an OD490 value greater than 2 in the supernatant ELISA are considered positive hybridoma cell lines. The day before cloning, prepare feeder cells according to step 3 and plate them. Use a pipette to mix the selected positive hybridoma cells to be cloned, and dilute the cells in each well to 1 cell per well using HT medium. Incubate at 37°C and 5% CO2 for 7-10 days. Antibody detection is indicated when visible clones appear. Observe under an inverted microscope and mark the wells with only a single clone growing. Finally, preliminary screening yields monoclonal antibody hybridoma cell lines 25E11 and 35G5.

[0046] 7. Antibody sequencing

[0047] Cloned 25E11 cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 25E11, identified as Ab1, is as follows:

[0048] Ab1

[0049] The heavy chain variable region sequence is: QVKLQESGPALVKPSQTLSLTCTISPWSASQKALVWTRQAGQEPVAWLGDLSTQSMTFYKPAIRSRLSINKRGSKNQVSLSLLNVTPEDAAVYYCTREILSMQSHNYGWDFWGRGLLVTVSS (SEQ ID NO:1)

[0050] CDR1:TISPWSASQKALV (SEQ ID NO:2);

[0051] CDR2:DLSTQSMTF (SEQ ID NO:3);

[0052] CDR3:TREILSMQSHNYGWDF (SEQ ID NO:4)

[0053] The light chain variable region sequence is: QTVLTQPPQVSPAIGQHVTISCSGRSGNIGDMDVPWYKDHPGSAPKTLIYQSHIRKPGVQDRPSGTKSGATGSITLTAIQSEDEANWYCSTIDHNRLTVIFGSSTRITVL (SEQ ID NO:5)

[0054] CDR1:SGRSGNIGDMDVP (SEQ ID NO:6);

[0055] CDR2:YQSHIRKP (SEQ ID NO:7);

[0056] CDR3:STIDHNRLTVI (SEQ ID NO:8)

[0057] Cloned 35G5 cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 35G5, as shown below, is Ab2:

[0058] Ab2

[0059] The heavy chain variable region sequence is as follows:

[0060] QVQLKTSGPGLVTPSQSISLTCHVSGSFITSWDVHWVRQPNGRGLVWLGVIWTYGSHNYNSAIMSRISLSKDNSKNQVFLRMNSIQTDDTAMYYCWHAQYKYEDVPYWGQGTLVTVSA (SEQ ID NO:9)

[0061] CDR1:HVSGSFITSWDVH (SEQ ID NO:10);

[0062] CDR2:VIWTYGSHN (SEQ ID NO:11);

[0063] CDR3:WHAQYKYEDVPY(SEQ ID NO:12)

[0064] The light chain variable region sequence is as follows:

[0065] DIVMTQSHKFMGTSVGNRVSLTCKSTQDVGTSVHWYQQKPGQSPKILLYWQTSKHTGVPDRFTSSGSGTDFTITLSNVQSEDLADYFCQHYSNWPYTFGGGTKLEIKRA (SEQ ID NO: 13)

[0066] CDR1:KSTQDVGTSVH (SEQ ID NO:14);

[0067] CDR2:YWQTSKHT (SEQ ID NO:15);

[0068] CDR3:QHYSNWPYT (SEQ ID NO:16).

[0069] The heavy and light chains of Ab1 and Ab2 can be fused with the constant region sequence of any antibody to form a complete antibody sequence, preferably the mouse light chain κ constant region and the mouse IgG1 constant region sequence.

[0070] The mouse IgG1 constant region sequence is as follows:

[0071] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:17)

[0072] The mouse light chain κ constant region sequence is as follows:

[0073] DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 18)

[0074] 8. Antibody expression and purification

[0075] The monoclonal antibodies Ab1 and Ab2 were synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and inserted into the PTT5 vector. Following the Thermo Fisher FreeStyle™ 293 Expression System User Manual, the vector containing the recombinant protein sequence was transfected into HEK293-F cells, cultured, and the cell culture supernatant was collected.

[0076] The above operations are well known to those skilled in the art, and other plasmid construction, cell transfection, and culture methods in the public domain can also be used to obtain the above single-chain antibodies.

[0077] Separation and purification

[0078] Cell supernatant from HEK293-F cells expressing protein A was fed into a protein A affinity chromatography column equilibrated with 20 mM phosphate buffer (pH 7.4) at a linear flow rate of 200–400 cm⁻¹ / h for enrichment of the target protein. After loading, elution was performed with 20 mM acetate buffer (pH 3.5), and the eluent was collected.

[0079] The affinity chromatography elution fractions were collected, and the recombinant protein was concentrated by ultrafiltration and buffer replacement using a 30kD ultrafiltration tube. The recombinant protein was then stored in 20mM phosphate buffer containing 100mM sodium chloride at pH 7.4.

[0080] The above operations are well known to those skilled in the art, and other purification and separation methods in the public domain can also be used to obtain the above recombinant protein.

[0081] Example 2: Determination of antibody-paired detection limit

[0082] In this experiment, a sandwich ELISA detection technique was employed. Specifically, Ab1 was first used as the coating antibody, which specifically binds to the target protein SP10. Then, HRP-labeled Ab2 was used as the primary antibody to further form a sandwich structure with the SP10 protein, thereby achieving the detection objective.

[0083] During the experiment, SP10 protein was serially diluted 5-fold using PBS buffer, starting at a concentration of 1 μg / ml and then decreasing sequentially to determine the optimal detection range. The diluted SP10 protein was added to pre-coated Ab1-coated detection wells and incubated at a suitable temperature for 1 hour to ensure sufficient antibody-antigen binding.

[0084] After incubation, HRP-labeled Ab2 is reacted with the SP10 protein bound to the wells. This step is to generate a colorimetric reaction through the enzymatic activity of HRP, thus facilitating value reading. After HRP-labeled Ab2 binds to the SP10 protein, it produces a color change under the catalysis of the substrate. The intensity of the color is directly proportional to the concentration of SP10 protein; therefore, the content of SP10 protein can be quantified by reading the absorbance (OD value).

[0085] The final detection results are shown in Table 1. It can be seen that as the concentration of SP10 protein decreases, the absorbance also gradually decreases, indicating that the ELISA detection system has a good linear response range. This method can detect not only high concentrations of SP10 protein but also sensitively detect low concentrations, thus providing reliable experimental data for the quantitative analysis of SP10 protein.

[0086] Table 1

[0087]

[0088] The detailed experimental data presented in Table 1 clearly show that the test wells without SP10 showed negative reactions, indicating that there is no non-specific binding between Ab1 and Ab2. This important finding provides a solid theoretical and practical basis for the subsequent development of immunoassay reagents based on the sandwich assay principle using this combination antibody.

[0089] As the amount of SP10 protein added gradually decreased, the OD450 reading obtained by ELISA also continued to decrease. This trend fully demonstrates that Ab2 has the ability to specifically recognize the complex formed by the binding of SP10 and Ab1.

[0090] Furthermore, the test results showed that the OD450 readings and the concentration of SP10 exhibited a good correlation, which means that the antibody combination can accurately and sensitively reflect the actual concentration of SP10 in the test sample, with extremely high sensitivity and a detection limit of 0.00256 ng / ml.

[0091] Based on these significant experimental results, it can be determined that this antibody combination has broad application prospects and high practical value in the development of immunoassay reagents based on the sandwich method principle.

[0092] Example 3: Preparation of Human Sperm SP10 Protein Detection Kit (Latex Method)

[0093] The kit consists of an SP10 protein detection card, a sample processing tube (containing sample diluent), a semen collection cup, and a semen collection device.

[0094] The SP10 protein detection card consists of: a binding pad (containing Ab2-labeled colored microsphere markers and mouse IgG markers); a test line: Ab1; and a control line: goat anti-mouse IgG polyclonal antibody.

[0095] Sample processing tubes: Tris Buffer, 1% Tween 80, 200mM sodium chloride, 0.05% preservative.

[0096] The preparation method of the test card is as follows:

[0097] Goat anti-mouse IgG polyclonal antibody was diluted to 1.2 mg / mL using coating buffer, and the first mouse anti-human SP10 monoclonal antibody (Ab1) was diluted to 0.8 mg / mL, serving as the C-line coating solution and T-line coating solution, respectively. Using a continuous stripping apparatus, each coating solution was evenly spread onto a nitrocellulose membrane at a spray rate of 1 μL / cm to form the control line (C-line) and the detection line (T-line), and dried overnight at 55°C. The second mouse anti-human SP10 monoclonal antibody (Ab2) was labeled at a concentration of 0.5 mg / mL, and the mouse IgG was labeled at a concentration of 1.0 mg / mL, preparing mouse anti-human SP10 monoclonal antibody-colored microsphere markers and mouse IgG markers, respectively. Spraying solutions were prepared and applied to the conjugate pad treatment pad at a spray rate of 2.0 μL / cm, and dried overnight at 37°C. The prepared process samples were then assembled into a kit.

[0098] The detection procedure for this kit is as follows: Figure 1 As shown, the result judgment method is as follows: Figure 2 As shown.

[0099] Example 4: Human Sperm SP10 Protein Detection Kit (Latex Method) for Clinical Sample Detection

[0100] The kit prepared in Example 3 was used to test 295 semen samples, of which 77 were negative (98% concordance rate) and 218 were positive (99% concordance rate).

[0101] Example 5: Detection Limit of Human Sperm SP10 Protein Detection Kit (Latex Method)

[0102] Semen samples were taken from healthy individuals and the concentration was adjusted to 10 × 10 using microscopic calculations. 6 pcs / ml, 15×10 6 cells / ml, 200×10 6 316×10⁻⁶ cells / ml 6 The sample size was measured at 100 cells / ml using the kit prepared in Example 3. The results were negative, positive, positive, and positive, respectively. This indicates that the detection limit of this kit meets the WHO Manual of Laboratory Tests for Human Semen Analysis (6th Edition) requirement of 15 × 10⁻⁶ cells / ml. 6 The lower reference limit for sperm concentration detection is specified as sperm count / ml.

[0103] Example 6: Cross-reactivity of Human Sperm SP10 Protein Detection Kit (Latex Method)

[0104] The test microorganisms were diluted with a negative matrix and detected using the detection kit prepared in Example 3. The results are shown in Table 2 below:

[0105] Table 2

[0106]

[0107] All the above-mentioned microbial standard strains were purchased from Beina Biotechnology. The test results indicate that the kit provided in this embodiment of the invention has strong resistance to cross-interference, and microorganisms that may appear in semen will not affect the test results.

[0108] Example 7: Interference Resistance of Human Sperm SP10 Protein Detection Kit (Latex Method)

[0109] The interfering analytes were diluted with a negative matrix and detected using a detection kit. The results are shown in Table 3 below:

[0110] Table 3

[0111]

[0112] Table 3 shows that these interfering substances are components of semen or other foreign interfering or contaminating substances that may appear in the sample. The test results demonstrate that the kit provided by this invention has strong anti-interference capabilities; any interfering substances or contaminants that may be present in the semen sample will not affect the test results.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A pair of monoclonal antibodies against human sperm SP10 protein, characterized in that, The monoclonal antibodies Ab1 and Ab2 against human sperm SP10 protein; The amino acid sequences of the heavy chain CDR1 sequence, CDR2 sequence and CDR3 sequence of the monoclonal antibody Ab1 are shown as SEQ ID NO: 2-4, and the amino acid sequences of the light chain CDR1 sequence, CDR2 sequence and CDR3 sequence are shown as SEQ ID NO: 6-8; The amino acid sequences of the heavy chain CDR1 sequence, CDR2 sequence and CDR3 sequence of the monoclonal antibody Ab2 are shown as SEQ ID NO: 10-12, and the amino acid sequences of the light chain CDR1 sequence, CDR2 sequence and CDR3 sequence are shown as SEQ ID NO: 14-16.

2. The pair of monoclonal antibodies against human sperm SP10 protein according to claim 1, characterized in that, The amino acid sequences of the heavy chain CDR1 sequence, CDR2 sequence and CDR3 sequence of the monoclonal antibody Ab1 are shown as SEQ ID NO: 2-4, and the amino acid sequences of the light chain CDR1 sequence, CDR2 sequence and CDR3 sequence are shown as SEQ ID NO: 6-8; 3. The paired set of monoclonal antibodies against human sperm SP10 protein according to claim 1, characterized in that, The heavy chain constant region of the monoclonal antibodies Ab1 and Ab2 is a mouse IgG1 constant region, and the amino acid sequence is SEQ ID NO:

17.

4. The paired set of monoclonal antibodies against human sperm SP10 protein according to claim 1, characterized in that, The light chain constant region of the monoclonal antibodies Ab1 and Ab2 is a mouse κ constant region, and the amino acid sequence is SEQ ID NO:

18.

5. A test reagent for human sperm SP10 protein, characterized by, The monoclonal antibody pair of any one of claims 1-4 against human sperm SP10 protein.

6. The detection reagent according to claim 5, characterized in that, The detection reagent is a sandwich ELISA reagent, wherein the monoclonal antibody Ab1 is a coating antibody, and the monoclonal antibody Ab2 is a detection antibody.

7. A test kit for human sperm SP10 protein, characterized by comprising a monoclonal antibody against human sperm SP10 protein and a monoclonal antibody against human sperm SP10 protein. The kit comprises: The binding pad comprises a monoclonal antibody Ab2 labeled color microsphere marker and a mouse IgG marker; The nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line); the detection line is coated with the monoclonal antibody Ab1, and the quality control line is coated with a goat anti-mouse IgG polyclonal antibody.

8. The test kit for human sperm SP10 protein according to claim 7, characterized by, The labeling concentration of the monoclonal antibody Ab2 in the monoclonal antibody Ab2 labeled color microsphere marker is 0.4-0.6 mg / mL, and the labeling concentration of the mouse IgG in the mouse IgG marker is 0.5-1.5 mg / mL.

9. A test kit for human sperm SP10 protein, characterized by, The kit comprises:

10. Use of the monoclonal antibody pair of any one of claims 1-4 against human sperm SP10 protein in the preparation of a reagent or kit for detecting human sperm SP10 protein.

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