Preparation of a monoclonal antibody of testosterone and its application

Abstract

The present application relates to the technical field of biology, and particularly to preparation and application of a testosterone monoclonal antibody, and provides a high-specificity and high-sensitivity testosterone monoclonal antibody, and a kit prepared by using the antibody can rapidly detect the content of testosterone in serum, and is suitable for immunological detection of testosterone hormone. The problems of low content of testosterone hormone in samples and high detection difficulty are solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the preparation and application of a testosterone monoclonal antibody. Background Technology

[0002] Testosterone is one of the main sex hormones in the human body. It is produced by the interstitial cells of the testes and transported to the periphery via capillaries or veins, or it infiltrates the seminiferous tubules, which produce sperm, through the myoid cells of the testes. In men, 95% of the testosterone in the bloodstream is secreted by the testes. In women, the ovaries and adrenal glands secrete small amounts of testosterone, but most of the testosterone in the bloodstream is produced by the metabolism of androstenedione. The determination of testosterone levels is mainly used as an auxiliary diagnostic tool for male diseases such as precocious puberty, delayed puberty, hypogonadism, azoospermia, or oligospermia. Testosterone generally cannot be stored in the body and is usually rapidly utilized or degraded, resulting in extremely low levels in the body, making it difficult to determine using conventional chemical methods. Currently, commonly used detection methods are liquid chromatography or ultraviolet spectrophotometry, but these two methods are cumbersome and time-consuming, limiting their widespread application in rapid clinical diagnosis. Using monoclonal antibodies is the best way to solve this problem.

[0003] Screening for small-molecule testosterone monoclonal antibodies from immune libraries is a widely used method. During antibody development, because small-molecule antigens lack immunogenicity, obtaining antibodies with specific antigenic determinants requires conjugation with a vector protein followed by animal immunization. Phage display technology integrates the complete set of antibody-encoding gene sequences from the immunized immune organs into a vector using molecular cloning techniques, forming an antibody library. This library is then displayed on the phage surface as a fusion protein, and specific antibodies can be obtained through multiple rounds of screening. Using phage display technology to screen for stable and highly specific testosterone monoclonal antibodies is of great significance for the clinical diagnosis of male diseases. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing a testosterone monoclonal antibody and its application. The monoclonal antibody provided by the present invention can be applied to serum sample detection, and can rapidly, specifically, and sensitively detect the concentration of testosterone in clinical serum, and can be used for clinical diagnosis.

[0005] The testosterone monoclonal antibody provided by this invention is obtained by conjugating a testosterone derivative (T-3-CMO) with a carrier protein, followed by animal immunization and phage display. Sequencing analysis revealed that the three CDR regions of its light chain have amino acid sequences as shown in SEQ ID NO:1-3, and the three CDR regions of its heavy chain have amino acid sequences as shown in SEQ ID NO:4-6.

[0006] Furthermore, the testosterone monoclonal antibody has a light chain variable region as shown in SEQ ID NO:7 and a heavy chain variable region as shown in SEQ ID NO:8;

[0007] The testosterone monoclonal antibody further includes a light chain constant region and a heavy chain constant region; the light chain constant region is of the κ type; and the heavy chain constant region is of the rabbit IgG type.

[0008] The testosterone monoclonal antibody provided by this invention is superior to other antibodies. Experimental results show that the testosterone monoclonal antibody described in this invention has better specificity. When this testosterone monoclonal antibody is applied to a kit, its accuracy, sensitivity, stability, precision, and recovery rate are found to be superior to kits made with other antibodies and commercially available kits.

[0009] This invention provides a nucleic acid encoding the aforementioned testosterone monoclonal antibody, specifically comprising:

[0010] The nucleic acid encoding the light chain variable region of the testosterone monoclonal antibody has the sequence shown in SEQ ID NO:9;

[0011] The nucleic acid encoding the heavy chain variable region of the testosterone monoclonal antibody has a sequence as shown in SEQ ID NO:10; or

[0012] Nucleotide sequences that encode the same protein as the nucleotide sequences shown in SEQ ID NO:9-10, but are different from the nucleotide sequences shown in SEQ ID NO:9-10 due to the degeneracy of the genetic code.

[0013] In this invention, the nucleic acid can be DNA, RNA, cDNA, or PNA. In this embodiment, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The RNA form is mRNA obtained by gene transcription, etc.

[0014] This invention provides an expression module comprising a promoter, a terminator, and the nucleic acid described in this invention.

[0015] Furthermore, the expression module includes the expression module composed of the nucleic acid described in this invention in a single or multiple tandem configuration with the promoter and terminator, and this invention does not limit this.

[0016] The present invention also provides a transcription unit containing the aforementioned nucleic acid or expression module, wherein the transcription unit refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include a promoter operatively linked to the nucleic acid sequence, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site, such as an enhancer of the promoter, a poly(A) signal, etc.

[0017] The present invention also provides a recombinant vector comprising:

[0018] The vector backbone and the nucleic acid described in this invention; or

[0019] The carrier skeleton and the expression module described in this invention.

[0020] The recombinant vector described in this invention refers to a nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in bacteria include promoters, ribosome binding sites, and possibly other sequences. Bacterial cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate itself into the genome. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0021] The present invention provides a host comprising at least one of the following: (I) to (III)

[0022] I) Secreting the testosterone monoclonal antibody described in this invention;

[0023] II) Genome integration of the nucleic acid or expression module described in this invention;

[0024] III) Transfection or transformation of the recombinant vector described in this invention.

[0025] In this invention, the recombinant vector is transfected or transformed into the host; the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0026] Furthermore, the host described in this invention includes bacteria, fungi, viruses, or animals. The bacteria include Gram-positive and Gram-negative bacteria; the Gram-positive bacteria include, but are not limited to, *Escherichia coli*. The fungi include molds, yeasts, and fungi; the yeasts include *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Candida*, etc. The viruses include, but are not limited to, adenoviruses, adeno-associated viruses, lentiviruses, and prions. The animals include humans, mice, rabbits, pigs, zebrafish, etc.

[0027] The present invention provides a method for preparing the aforementioned testosterone monoclonal antibody, comprising culturing the host described in the present invention to obtain the monoclonal antibody.

[0028] The present invention provides a conjugate formed by conjugating the testosterone monoclonal antibody with a mediator.

[0029] Specifically, the conjugate formed by coupling the testosterone monoclonal antibody of the present invention with a medium includes a radionuclide, a polypeptide toxin, a drug molecule, or a carrier. The polypeptide toxin includes plant toxins, bacterial toxins, and animal toxins. The drug molecule includes doxorubicin, daunorubicin, bleomycin, bleomycin, mitomycin, neomycin, methotrexate, etc. The carrier refers to any carrier capable of loading monoclonal antibodies, such as magnetic microparticles, test strips, polystyrene plates, or ELISA plates.

[0030] The present invention provides a complex formed by chemical or biological labeling of the testosterone monoclonal antibody.

[0031] Specifically, the testosterone monoclonal antibody complex of the present invention is formed by chemical labeling or biological labeling. The chemical labeling includes isotope labeling and fluorescein labeling; the biological labeling includes enzyme labeling and biotin labeling. Examples include fluorescein isothiocyanate, horseradish peroxidase, alkaline phosphatase, β-galactosidase, biotin, colloidal gold, etc.

[0032] The present invention provides the use of at least one of the following shown in a) to h) in the preparation of a testosterone detection kit.

[0033] a) The testosterone monoclonal antibody described in this invention;

[0034] b) The nucleic acid described in this invention;

[0035] c) The expression module described in this invention;

[0036] d) The recombinant vector described in this invention;

[0037] e) The host described in this invention;

[0038] f) The culture containing the testosterone monoclonal antibody prepared by the preparation method described in this invention;

[0039] g) The coupling compound described in this invention;

[0040] h), the complex described in this invention.

[0041] In some embodiments of the present invention, the testosterone detection kit includes the testosterone monoclonal antibody described in the present invention, and also includes coating buffer, washing solution, blocking solution and / or chromogenic solution.

[0042] This invention also provides a product for detecting testosterone, the raw materials of which include at least one of the following: a) to f)

[0043] a) The testosterone monoclonal antibody described in this invention;

[0044] b) The nucleic acid described in this invention;

[0045] c) The expression module described in this invention;

[0046] d) The recombinant vector described in this invention;

[0047] e) The host described in this invention;

[0048] f) The culture containing the testosterone monoclonal antibody prepared by the preparation method described in this invention.

[0049] g) The coupling compound described in this invention;

[0050] h), the complex described in this invention.

[0051] Furthermore, the product of the present invention also includes solvents and / or excipients, which are applied in the product to maintain the stability of the raw materials or to help the raw materials perform their function.

[0052] Furthermore, the solvents include, but are not limited to, TB buffer, PBS buffer, Tris buffer, KCl buffer, or NaCl buffer. The excipients include, but are not limited to, proteins, carriers, antioxidants, surfactants, and / or protease inhibitors; the proteins include, but are not limited to, BSA; the carriers include, but are not limited to, auxiliary carriers for viral packaging, auxiliary carriers for phage packaging, and / or auxiliary carriers for eukaryotic or prokaryotic host cell genome integration, which are not limited in this invention; the antioxidants include, but are not limited to, DTT or β-mercaptoethanol. The surfactants include, but are not limited to, Triton-X-100. The protease inhibitors include, but are not limited to, PMSF.

[0053] The present invention also provides a method for detecting testosterone, including testing a sample using the product described in the present invention.

[0054] This invention utilizes phage display technology to screen and obtain a monoclonal antibody against testosterone. This monoclonal antibody exhibits high specificity and detection sensitivity, overcoming the challenges of low testosterone levels and difficult detection in samples. Repeated detection using this monoclonal antibody demonstrates good stability and repeatability, as well as high precision and accuracy, making it suitable for widespread clinical diagnosis of male diseases. Attached Figure Description

[0055] Figure 1 Image of RNA extracted from spleen via agarose gel electrophoresis;

[0056] Figure 2 Agarose gel electrophoresis image of VL gene PCR products;

[0057] Figure 3 Agarose gel electrophoresis image of VH gene PCR products;

[0058] Figure 4 Agarose gel electrophoresis image of PCR products of the scFv gene;

[0059] Figure 5 Agarose gel electrophoresis image showing antibody library recombination rate and bacterial culture PCR identification;

[0060] Figure 6 The images show SDS-PAGE electrophoresis results of the purified recombinant antibody after reduction and without reduction.

[0061] Figure 7 This paper presents a correlation analysis between the antibody (clone 2) of this invention and the antibody from a reference manufacturer;

[0062] Figure 8 Correlation analysis of candidate antibody (clone 3) with reference antibody from the manufacturer is shown.

[0063] Figure 9 The high-performance liquid chromatography (HPLC) chromatogram of the antibody of this invention is shown. Detailed Implementation

[0064] This invention provides a method for preparing a testosterone monoclonal antibody and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0065] The amino acid sequences of the CDR region of the testosterone monoclonal antibody described in this invention are as follows:

[0066] Light chain CDR1: ESVYNNNA (as shown in SEQ ID NO:1);

[0067] Light chain CDR 2: GAS (as shown in SEQ ID NO:2);

[0068] Light chain CDR 3: AGVFIGSSDAYG (as shown in SEQ ID NO:3);

[0069] Heavy chain CDR1: GFAFSSYY (as shown in SEQ ID NO:4);

[0070] Heavy chain CDR2: IYPSKGRT (as shown in SEQ ID NO:5);

[0071] Heavy chain CDR3: ARTYVGYGSATIDYGMDL (as shown in SEQ ID NO:6).

[0072] The amino acid sequence of the variable region of the light chain of the testosterone monoclonal antibody is: DPVLTQTPSPVSAGVGGTVSVSCQSSESVYNNNALSWYQQKPGQPPKLLIY GASTLASGVPSRFKGNGSGTRFTLTINDVQCDDAATYYCAGVFIGSSDAYGFGGGTKVEIK (as shown in SEQ ID NO:7).

[0073] The amino acid sequence of the variable region of the heavy chain of the testosterone monoclonal antibody is: QEQLKESGGGLVQPGGSLTLSCKASGFAFSSYYITWVRQAPGKGLEWIGTI YPSKGRTYYATWVNGRFTMSSDNAQKTVDLRMNSLTAADRATYFCARTYVGYGSATIDYGMDLWGPGTLVTVSS (as shown in SEQ ID NO:8).

[0074] The nucleic acid sequence encoding the variable region of the light chain of the testosterone monoclonal antibody is: GACCCTGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGGTGTGGGGAGGCACAGTCAGCGTCAGTTGCCAGTCCAGTGAGAGTGTTTATAATAACAATGCCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTAATCTATGGTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAATGGATCTGGGACTCGGTTCACTCTCACCATCAACGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGTTTTTATTGGTAGTAGTGATGCGTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (as shown in SEQ ID NO: 9).

[0075] The nucleic acid sequence encoding the variable region of the heavy chain of the testosterone monoclonal antibody is: CAGGAGCAGCTGAAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGGGGGATCCCTGACACTCTCCTGCAAAGCCTCTGGATTCGCCTTCAGTAGCTACTACATAACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGCACCATTTATCCTAGTAAAGGTCGAACATACTACGCGACCTGGGTGAATGGCCGATTCACCATGTCCAGCGACAACGCCCAGAAGACTGTGGATCTTCGAATGAACAGTCTGACAGCCGCGGACAGGGCCACCTATTTCTGTGCAAGAACCTATGTTGGTTATGGTTCAGCTACGATTGACTACGGCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCA (as shown in SEQ ID NO:10).

[0076] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0077] The present invention will be further illustrated below with reference to the embodiments:

[0078] Example 1: Preparation for Phage Platform Screening

[0079] 1. Immunizing animals with recombinant immunogen

[0080] New Zealand white rabbits were immunized three times with the prepared antigen T-3-CMO-KLH. The immunization cycle was 30 days, with multiple subcutaneous injections on the back. For the first immunization, 1 mg of the immunogen was emulsified with an equal volume of Freund's complete adjuvant and then immunized. For the subsequent three immunizations, 0.5 mg of the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant and then immunized. On day 10 after the third immunization, blood was collected from the marginal ear vein, incubated at 37°C for 1 hour, and then centrifuged at 6000 rpm for 10 minutes. The supernatant (antiserum) was collected for immunization efficacy testing, and dominant immunized animals were selected for monoclonal antibody preparation.

[0081] 2. Antiserum titer detection

[0082] Prepare the detection plates. Add anti-rabbit secondary antibody (RaIgG-Fc) to a final concentration of 4 μg / ml in 0.05 mol / L Cb (pH 9.6) coating buffer, mix well, and add 100 μL / well to the luminescent plate. Incubate overnight at 2-8℃. After coating, block the detection plate with irrelevant proteins. Serial dilutions of the test serum were started at 1:200, 50 μL / well. Serum titers were detected using a reverse indirect method (rabbits #1, #2, and #3). The results are shown in Table 1. The results show that the antiserum titer was high. Based on these results, animal #1 was given a booster immunization. Three days later, the animal was sacrificed, and spleen cells were extracted. Total RNA was extracted from the spleen tissue using the Trizol method (e.g., ...). Figure 1 (As shown), cDNA is synthesized by reverse transcription.

[0083] Table 1

[0084]

[0085] Example 2: scFv gene splicing and phage screening construction

[0086] 1. scFv gene splicing

[0087] PCR was used to amplify the variable regions of the light and heavy chains of the antibody, respectively. The PCR reaction procedure is shown in Table 2.

[0088] Table 2

[0089]

[0090] PCR products were recovered via 1% agarose gel electrophoresis, such as... Figure 2 and Figure 3 As shown. The PCR-amplified light chain variable region and heavy chain variable region were spliced ​​together using overlap-PCR to form scFv. The product was recovered by 1% agarose gel electrophoresis and stored at -20℃. Figure 4 As shown.

[0091] 2. Construction and screening of phage single-chain antibody libraries:

[0092] The phage vector pcomb3XSS and the purified ScFv fragment were digested with SfiI to construct a recombinant plasmid. The recombinant plasmid was electroporated into TG1 competent cells to construct a rabbit-derived immune single-chain antibody library (antibody library recombination rate was identified by bacterial PCR as follows). Figure 5 The primary phage single-chain antibody library was prepared; the primary phage single-chain antibody library was enriched and screened for 3 rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; single clones with good gradient (e.g., clone 2) were selected to prepare single-clone phage supernatant, and positive clones were identified by Phage-ELISA to obtain positive sequences.

[0093] After library construction and screening, several variable region sequences were identified. A competitive assay was used to verify antibody performance, and gradient calibrators and clinical testing were conducted to determine antibody specificity. The results of eight clones are shown in Table 3.

[0094] Table 3

[0095]

[0096] The results show that clone 2 is superior to other antibodies and has a better clinical gradient than the control. The clinical gradient of clone 3 is comparable to the control. Clone 2 will be selected as the preferred clone and clone 3 as the candidate clone for further validation.

[0097] Example 3: Expression and purification of recombinant monoclonal antibodies

[0098] 1. Construction of stable cell lines

[0099] Recombinant antibody genes were obtained by ligating the constant and variable regions of the heavy chain and the light chain separately using overlap-PCR. The heavy chain antibody gene and the light chain antibody gene were then digested with HindIII / XbaI and HindIII / XbaI, respectively, and ligated into the expression vector PCMV3. After transfection into competent DH5α cells, the cells were activated by shaking, and positive clones were selected for sequencing to extract the plasmid. Once the sequencing was successful, the cells were transfected into CHO-S cells.

[0100] 2. Antibody SPA purification and SDS-PAGE column equilibration

[0101] Purification of monoclonal antibodies: Protein A affinity packing material was purchased from Stofavente. 10 mL of the above-preserved cell supernatant was purified with 2 mL of packing material. The eluted antibodies were desalted with Sephadex G-25 and the purity of the monoclonal antibodies was determined by SDS-PAGE. The purity of the target antibodies was all above 90%.

[0102] Collected proteins were pooled and analyzed by SDS-PAGE, such as... Figure 6As shown. This indicates that the purified antibody has high purity and high concentration. The physicochemical properties of the antibody (including antibody size, presence of antibody aggregates, and size of the aggregates, etc.) are as follows: Figure 9 As shown.

[0103] Example 4: Application of the Recombinant Rabbit Monoclonal Antibody Kit

[0104] The recombinant rabbit monoclonal antibody T prepared in this invention is used in a kit, along with other components, to detect antigens in samples. Specific performance evaluation is as follows:

[0105] 1. Accuracy Measurement

[0106] Eighty-seven clinical samples were analyzed using a commercially available reagent kit. The T concentration in the samples was calculated using the system, and correlation analysis was performed with a reference manufacturer. The results are shown below. Figure 7 , Figure 8 .

[0107] According to the test results, the linear equation obtained by clone 2 is: y = 1.0425x - 0.0244, and the correlation coefficient R is... 2 =0.9936; the linear equation obtained from clone 3 is: y = 1.0244x + 0.5542, with a correlation coefficient R. 2 =0.9879. The results indicate that the T recombinant rabbit monoclonal antibody provided by the dominant antibody (clone 2) of this invention has high accuracy in detecting testosterone in clinical samples, while the clone 3 antibody is slightly inferior to the primary antibody.

[0108] 2. Sensitivity Performance Evaluation

[0109] Five clinical samples with values ​​close to 0 were selected, and each sample was repeated three times over a total of four days, resulting in 60 data points. Some of the data are shown in Table 4. The antibody detection values ​​of both strains were lower than the blank line (0.1 ng / mL), indicating that the antibodies had high sensitivity and met the requirements for in vitro diagnostics.

[0110] Table 4

[0111]

[0112] 3. Stability Analysis

[0113] Based on the above specificity and sensitivity analysis, the antibody can be used in in vitro diagnostic kits. To verify antibody stability, the working solution of the antibody component was stored at 37℃ and 2-8℃ for 8 days, respectively. After being removed and stored overnight at 2-8℃, it was then subjected to magnetic particle platform detection to monitor the signal value reduction of clinical, quality control, and calibrator samples. The results showed that clone 2 antibody had the best stability, followed by clone 3. The stability of both antibodies was better than that of the positive control. The reduction data are shown in Table 5.

[0114] Table 5

[0115]

[0116] The results above show that the recombinant monoclonal antibody prepared by this invention has reached a level that is highly consistent with the detection results of mainstream manufacturers in the market, and exhibits advantages such as high sensitivity and excellent stability. When used as a detection antibody in a kit, its detection performance meets the requirements of the kit according to the evaluation, which is of great significance for subsequent applications and clinical diagnosis.

[0117] 4. Precision assessment

[0118] The precision evaluation conducted in this study was intra-assay precision. Samples at high, medium, and low levels within the detection range were analyzed using the same batch of reagents. Each sample in each batch was measured 20 times, and the mean, SD, and coefficient of variation (CV) were calculated. The intra-assay CV for all three levels was less than 5%, as shown in Table 6, indicating good reproducibility of the kit. In a cross-sectional comparison of the three antibodies, clone 3 antibody showed the worst variation in the low-value region, lower than the control.

[0119] Table 6

[0120]

[0121] 5. Recovery rate performance evaluation

[0122] Three high-value samples were selected and diluted using a low-value / matrix sample at a ratio of 1:9. The volume of the high-value sample should not exceed 10% of the total volume. Recovery rates were evaluated using controls, clone 2, and clone 3. The recovery rates of the three positive control samples were 97.33%, 96.98%, and 96.95%, respectively; the recovery rates of the three clone 2 antibody samples were 97.09%, 97.32%, and 96.66%, respectively; and the recovery rates of the three clone 3 antibody samples were 94.09%, 95.32%, and 94.66%, respectively. In summary, the recovery deviations of the control and clone 2 antibody samples were both less than 5%, and the recovery deviation of the clone 3 antibody sample was less than 10%. The accuracy of the recovery of all three antibodies met the requirements.

[0123] In summary, the clone 2 antibody outperformed the candidate clone 3 antibody in all assessment levels, and the clone 2 antibody also showed superior stability compared to the control. These results demonstrate that the recombinant monoclonal antibody prepared in this invention has achieved a level of high consistency with the detection results of mainstream manufacturers in the market, exhibiting advantages such as high sensitivity and excellent stability. Its application as a detection antibody in clinical diagnostic kits has met all kit requirements based on evaluations, playing a crucial role in future applications and clinical diagnosis.

[0124] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A testosterone monoclonal antibody, characterized in that, The amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NO:1~3, and the amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NO:4~6.

2. The testosterone monoclonal antibody according to claim 1, characterized in that, It has a light chain variable region as shown in SEQ ID NO:7 and a heavy chain variable region as shown in SEQ ID NO:

8.

3. The testosterone monoclonal antibody according to claim 1 or 2, characterized in that, Its light chain constant region is κ type; its heavy chain constant region is rabbit IgG type.

4. The nucleic acid encoding the testosterone monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that, The nucleic acid encoding the light chain variable region of the testosterone monoclonal antibody has the sequence shown in SEQ ID NO:9; The nucleic acid encoding the heavy chain variable region of the testosterone monoclonal antibody has the sequence shown in SEQ ID NO:

10.

6. An expression module, characterized in that, It includes a promoter, a terminator, and the nucleic acid as described in claim 4 or 5.

7. A recombinant vector, characterized in that, include: The vector backbone and the nucleic acid as described in claim 4 or 5; or The carrier skeleton and the expression module as described in claim 6.

8. The host, characterized in that, It includes at least one of the following: (I) to (III) I) Secreting the testosterone monoclonal antibody as described in any one of claims 1 to 3; II) Genome integration of the nucleic acid as described in claim 4 or 5 or the expression module as described in claim 6; III) Transfection or transformation of the recombinant vector as described in claim 7.

9. The method for preparing the testosterone monoclonal antibody according to any one of claims 1 to 3, characterized in that, This includes culturing the host as described in claim 8 to obtain the testosterone monoclonal antibody.

10. The conjugate formed by conjugating the testosterone monoclonal antibody according to any one of claims 1 to 3 with a medium; The medium is magnetic microparticles, test strips, polystyrene plates, or enzyme-linked immunosorbent assay (ELISA) plates.

11. The complex formed by chemical or biological labeling of the testosterone monoclonal antibody according to any one of claims 1 to 3.

12. The use of at least one of the following shown in a) to h) in the preparation of a testosterone detection kit: a) The testosterone monoclonal antibody according to any one of claims 1 to 3; b) The nucleic acid as described in claim 4 or 5; c) The expression module as described in claim 6; d) The recombinant vector as described in claim 7; e) The host as described in claim 8; f) A culture containing the testosterone monoclonal antibody prepared by the preparation method according to claim 9; g) The coupling as described in claim 10; h) The complex according to claim 11.

13. A product for detecting testosterone, characterized in that, Its raw materials include at least one of the following: a) to h): a) The testosterone monoclonal antibody according to any one of claims 1 to 3; b) The nucleic acid as described in claim 4 or 5; c) The expression module as described in claim 6; d) The recombinant vector as described in claim 7; e) The host as described in claim 8; f) A culture containing the testosterone monoclonal antibody prepared by the preparation method according to claim 9; g) The coupling as described in claim 10; h) The complex according to claim 11.

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