A monoclonal antibody for detecting testosterone in a sandwich and a preparation method and application thereof

Abstract

The application provides a kind of sandwich detection testosterone monoclonal antibody and its preparation method and application, comprising: step S1, preparation testosterone-3-(O-carboxymethyl) oxime, and it is activated;After activation, take testosterone antibody P2G1, according to proportion, drop the activated testosterone-3-(O-carboxymethyl) oxime is reacted to prepare immunogen;Step S2, based on the prepared immunogen, immunization animal;Step S3, the spleen cell of immunization animal and myeloma cell are fused into hybridoma cell, and the real hybridoma cell is selected;Step S4, antibody is separated and purified from the screened hybridoma cell.The application provides a kind of preparation method of raw material and reagent for testosterone sandwich method detection, solves the problem that the sensitivity of small molecule compound testosterone competition method reagent is insufficient, detection range is narrow, specificity is poor, provides a new thought and method for the rapid, accurate detection of testosterone.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a sandwich-type monoclonal antibody for detecting testosterone, its preparation method, and its application. Background Technology

[0002] Testosterone (Tes) is a 19-carbon steroid hormone with a molecular weight of 288 Daltons, making it a small molecule. In men, testosterone is the primary male hormone, almost entirely synthesized by the interstitial cells of the testes. Its secretion is regulated by luteinizing hormone (LH) and negatively influences the pituitary-hypothalamic regulatory mechanism. Both male and female adrenal glands synthesize some testosterone, and the female ovaries also secrete small amounts. The most important physiological function of testosterone is to promote the development and maturation of male sex organs and maintain secondary sexual characteristics. Metabolically, testosterone promotes muscle growth and protein synthesis while inhibiting protein breakdown induced by glucocorticoids. The vast majority of testosterone in the bloodstream is bound to carrier proteins (such as sex hormone-binding globulin (SHBG)). The female ovaries produce small amounts of testosterone; physiological levels of androgens have no specific effect on women. Elevated androgen levels in women can lead to masculinization (depending on the degree of increase in androgen levels). Measuring testosterone levels in women is helpful in diagnosing androgen syndrome (AGS), polycystic ovary syndrome (Stene-Leventhal syndrome), and in cases of suspected ovarian tumors, adrenal tumors, adrenal hypoplasia, or ovarian dysfunction. Measuring testosterone levels in men can be used to diagnose conditions of insufficient testosterone production, such as gonadal hypoplasia, estrogen therapy, chromosomal abnormalities (e.g., Klineffelter syndrome), and cirrhosis. Therefore, accurate measurement of testosterone levels in the body plays a crucial role in assessing gonadal and bodily functions.

[0003] Currently, the main in vitro diagnostic methods for hormones are immunological detection methods. These methods are based on the specific reaction between antibodies and antigens, and can amplify and display the detected signal using isotopes, enzymes, chemiluminescent substances, etc. Current immunological detection methods for testosterone include biochemical immunoturbidimetry, radioimmunoassay, fluorescence immunochromatography, and chemiluminescence. However, because testosterone is a small molecule compound with only one antibody binding site, it cannot be directly detected using sandwich methods; therefore, most methods are competitive assays. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a sandwich-type monoclonal antibody for detecting testosterone, its preparation method, and its application.

[0005] To achieve the above-mentioned objectives, the present invention provides a method for preparing a sandwich-type monoclonal antibody for detecting testosterone.

[0006] Step S1: Prepare testosterone-3-(O-carboxymethyl) oxime and activate it; after activation, take testosterone antibody P2G1, add activated testosterone-3-(O-carboxymethyl) oxime dropwise according to the ratio to prepare immunogen;

[0007] Step S2: Immunize animals based on the prepared immunogen;

[0008] Step S3: Fuse spleen cells and myeloma cells of immunized animals into hybridoma cells, and select the true hybridoma cells;

[0009] Step S4: Isolate and purify antibodies from the selected hybridoma cells.

[0010] Optionally, step S1 includes:

[0011] Weigh out testosterone and dissolve it in methanol to prepare a testosterone solution; weigh out carboxymethoxyamine hemihydrochloride and dissolve it in pure water to prepare a carboxymethoxyamine hemihydrochloride solution; add the testosterone solution dropwise to the carboxymethoxyamine hemihydrochloride solution at room temperature and stir vigorously to form a white precipitate; add water, shake, and centrifuge to collect the precipitate, which is testosterone-3-(O-carboxymethyl)oxime;

[0012] Testosterone-3-(O-carboxymethyl) oxime was dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The mixture was stirred at room temperature, and the reaction product was washed with water and dried under reduced pressure. The dried powder was dissolved in DMSO to prepare activated testosterone-3-(O-carboxymethyl) oxime.

[0013] Take testosterone antibody P2G1, add activated testosterone-3-(O-carboxymethyl) oxime according to the ratio, react overnight, and after the reaction is completed, remove unreacted free testosterone derivatives by ultrafiltration, and obtain immunogen after ultrafiltration.

[0014] Optionally, step S2 includes:

[0015] After dissolving the immunogen, emulsify it evenly with an equal volume of Freund's complete adjuvant. Take mice and inject it subcutaneously at multiple points. Two weeks later, emulsify the antigen with Freund's incomplete adjuvant and inject it subcutaneously at multiple points. Administer two booster immunizations. Three days before fusion, administer a shock immunization via intraperitoneal injection.

[0016] Optionally, step S3 includes:

[0017] Preparation of feeder cells: 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. RPMI 1640 basal culture medium containing penicillin-streptomycin was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, the rinsing solution was collected, centrifuged, and the pellet was resuspended in RPMI 1640 complete culture medium containing HAT. After adjusting the cell concentration, the pellet was added to 96-well plates and cultured.

[0018] Preparation of immune spleen cells: Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, ground and filtered on a nylon mesh in a small beaker to prepare a cell suspension; 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.

[0019] Cell fusion: (1) Take HAT culture medium, DMEM serum-free culture medium and 50% PEG solution respectively for pre-warming; (2) Take mouse myeloma cells and the above immune spleen cell suspension respectively into centrifuge tubes and mix well, and add pre-warmed DMEM serum-free culture medium to the target volume; after centrifugation, discard the supernatant and mix well; (3) After mixing, place the centrifuge tube in pre-warmed water, take pre-warmed 50% PEG solution into the centrifuge tube and let it stand for 90 seconds; after standing, add pre-warmed DMEM serum-free culture medium dropwise; (4) Add pre-warmed DMEM serum-free culture medium to the target volume, centrifuge, discard the supernatant; after discarding the supernatant, add HAT culture medium containing fetal bovine serum, mix well, and dropwise into the wells of 4 96-well cell culture plates containing feeder cells for culture;

[0020] Hybridoma cell selection and culture: The cells were cultured in HAT medium on days 1, 3, 5 and 7 after fusion. After the culture was completed, the live hybridoma cells were selected to obtain true hybridoma cells.

[0021] Optionally, step S4 includes:

[0022] Supernatant was collected from each culture well, and the OD490 values ​​of the supernatant and the complex containing testosterone small molecules and testosterone antibodies, and the OD490 values ​​of the supernatant and testosterone antibodies were detected by indirect ELISA. Cell lines with an OD490 difference greater than 1.5 were considered as having good reactivity. Feeder cells were prepared and plated the day before cloning. The cell lines with good reactivity were mixed by pipetting, and the cells in each well were diluted to one cell per well using HT medium. The cells were cultured at 37°C and 5% CO2 for 7-10 days. Antibody detection was performed when visible clones appeared. Wells with only a single clone were marked under an inverted microscope, and the monoclonal antibody hybridoma cell line 15G5 was obtained through preliminary screening. The obtained monoclonal antibody hybridoma cell line 15G5 was then used for antibody expression and purification to obtain the monoclonal antibody.

[0023] On the other hand, the present invention provides a sandwich-type monoclonal antibody for detecting testosterone, wherein the sandwich-type monoclonal antibody for detecting testosterone is prepared according to the above method.

[0024] Optionally, the heavy chain CDR1 sequence of the sandwich monoclonal antibody for detecting testosterone is shown in SEQ ID NO:1, the heavy chain CDR2 sequence is shown in SEQ ID NO:2, the heavy chain CDR3 sequence is shown in SEQ ID NO:3, the light chain CDR1 sequence is shown in SEQ ID NO:4, the light chain CDR2 sequence is shown in SEQ ID NO:5; the light chain CDR3 sequence is shown in SEQ ID NO:6, the heavy chain variable region sequence is shown in SEQ ID NO:7, the light chain variable region sequence is shown in SEQ ID NO:8, the antibody heavy chain sequence is shown in SEQ ID NO:9, and the antibody light chain sequence is shown in SEQ ID NO:10.

[0025] On the other hand, the present invention provides an application of the above-mentioned monoclonal antibody for sandwich detection of testosterone in the sandwich method for testosterone detection.

[0026] The advantages and beneficial effects of this invention compared to the prior art are as follows:

[0027] 1. This invention provides a method for preparing raw materials and reagents for the sandwich method of testosterone detection, which solves the problems of insufficient sensitivity, narrow detection range and poor specificity of small molecule compound testosterone competitive reagents, and provides a new idea and method for rapid and accurate detection of testosterone.

[0028] 2. This invention provides an amino acid sequence of a testosterone complex antibody 15G5, wherein the heavy chain amino acid sequence of the testosterone antibody is SEQ ID NO:9 and the light chain amino acid sequence is SEQ ID NO:10. The testosterone complex antibody designed in this invention is obtained by injecting mouse peritoneal hybridoma cells and collecting ascites fluid. The purified testosterone 15G5 antibody is obtained through A-column purification and ultrafiltration concentration for ELISA activity assay. This invention uses testosterone complex antibody 15G5 as a raw material to prepare a progesterone sandwich assay reagent. The sensitivity and linearity of the reagent in clinical samples are better than those of the competitive assay. Temperature verification and thermally accelerated stability verification at 37°C were also tested. The deviation of the test results at different temperatures was small, and the reagent showed good stability at 37°C. This invention provides a method for preparing testosterone complex antibody 15G5, which can be used as a raw material for testosterone sandwich immunofluorescence detection. A testosterone sandwich immunofluorescence reagent was prepared using this complex antibody as a raw material. This solves the problems of low sensitivity, poor linearity, and narrow detection range of the small molecule compound testosterone competitive assay. Attached Figure Description

[0029] 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 on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is an electrophoresis image of the purified protein from the 15G5 antibody in an embodiment of the present invention;

[0031] Figure 2 This is a graph showing the results of ELISA detection of 15G5 antibody activity in an embodiment of the present invention;

[0032] Figure 3 This is a linear graph of clinical detection of testosterone using the immunofluorescence competitive assay when goat anti-mouse secondary antibody is applied to immunofluorescence reagent in an embodiment of the present invention.

[0033] Figure 4 This is a linear graph of the clinical detection of testosterone using the immunofluorescence competitive assay when antibody 15G5 is applied to the immunofluorescence reagent in an embodiment of the present invention. Detailed Implementation

[0034] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0035] Example 1

[0036] A method for preparing a sandwich monoclonal antibody for detecting testosterone:

[0037] The reagents or kits involved in this embodiment and their sources are as follows:

[0038] Freund's Adjuvant Complete (Catalog No. 77140, Thermo Fisher); Freund's Incomplete Adjuvant (Catalog No. 77145, Thermo Fisher); HAT Media Supplement (50×) (Catalog No. 21060017, Thermo Fisher); HT Media Supplement (50×) (Catalog No. H0111067030, Thermo Fisher); PEG (Catalog No. P7181, Sigma); RPMI 1640 (Catalog No. L210KJ, Shanghai Yuanpei Biotechnology); Fetal Bovine Serum (FBS) (C04001-500, Shanghai Xiaopeng Biotechnology); DMEM (Catalog No. L310KJ, Shanghai Yuanpei Biotechnology); Penicillin-St reptomycin (catalog number 15140122, Gibco); HRP-labeled goat anti-mouse antibody (catalog number D110087, Shanghai Sangon Biotech); Protein AResin (catalog number SA023010, Changzhou Tiandi Renhe Biotechnology Co., Ltd.).

[0039] 1. Immunogen preparation

[0040] Weigh 100 mg of testosterone and dissolve it in 1 mL of methanol to prepare a testosterone solution. Weigh 100 mg of carboxymethoxyamine hemihydrochloride and dissolve it in 1 mL of pure water to prepare a carboxymethoxyamine hemihydrochloride solution. Add the testosterone solution dropwise to the carboxymethoxyamine hemihydrochloride solution at room temperature while stirring vigorously. A white precipitate immediately forms. Add 5 mL of water, shake, and centrifuge to collect the precipitate. The precipitate is testosterone-3-(O-carboxymethyl)oxime.

[0041] Dissolve the above-mentioned testosterone-3-(O-carboxymethyl)oxime in 10 mL of dichloromethane, add 100 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 50 mg of N-hydroxysuccinimide, and stir at room temperature for 4 hours. Wash the reaction product three times with water and dry under reduced pressure. Dissolve the dried powder in 10 mL of DMSO. This is the activated testosterone-3-(O-carboxymethyl)oxime.

[0042] Testosterone antibody P2G1 (purchased from Thermo Fisher, catalog number MIT0102, mouse antibody) was added dropwise with activated testosterone-3-(O-carboxymethyl) oxime at a molar ratio of 1:24. The reaction was carried out overnight at 4°C. After the reaction, unreacted free testosterone derivatives were removed by ultrafiltration, and the ultrafiltration product was the immunogen.

[0043] 2. Mouse immunization

[0044] After dissolving the above immunogen, emulsify it evenly with an equal volume of Freund's complete adjuvant. Take 6-8 week old SPF-grade Balb / c mice (Fuzhou Wu's Animal Experiment Center) and inject 200 μg / mouse subcutaneously at multiple sites. Two weeks later, emulsify the antigen with Freund's incomplete adjuvant and inject 100 μg / mouse subcutaneously at multiple sites. Administer two booster immunizations. Three days before fusion, administer a shock immunization via intraperitoneal injection.

[0045] 3. Preparation of feeder cells

[0046] 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 intraperitoneally 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 resuspended in RPMI 1640 complete culture medium containing 1% HAT. The cell concentration was adjusted to 1×10⁻⁶ cells / mL. 5 Add 150 μL / well to a 96-well plate and incubate overnight at 37°C with 5% CO2.

[0047] 4. Preparation of immune spleen cells

[0048] 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.

[0049] 5. Cell fusion

[0050] (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℃ water bath for preheating;

[0051] (2) Mouse myeloma cells Sp2 / 0 (2-5×10⁻⁵) were collected respectively. 7 (10) of the above-mentioned immune spleen cells 8Add 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.

[0052] (3) After mixing, 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 after standing, add 15 mL of pre-warmed DMEM serum-free culture medium at 37°C;

[0053] (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. After discarding 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 that already contains feeder cells. Incubate at 37°C and 7% CO2.

[0054] 6. Selective culture of hybridoma cells

[0055] The cells were cultured in the aforementioned HAT medium on days 1, 3, 5, and 7 after fusion. After the culture was completed, the live hybridoma cells were selected to obtain true hybridoma cells.

[0056] 7. Detection of specific antibodies and cloning of hybridoma cells

[0057] Supernatant was collected from each culture well, and the OD490 values ​​of the supernatant and the complex containing testosterone small molecules and testosterone antibodies, and the OD490 values ​​of the supernatant and testosterone antibodies were detected by indirect ELISA. The antibody cell lines in the culture wells with a difference of OD490 greater than 1.5 were considered as antibody cell lines with better reactivity. The day before cloning, feeder cells were prepared and plated according to step 3. The antibody cell lines with better reactivity were mixed by pipetting, and the cells in the wells were diluted with HT medium to 1 cell per well. The cells were cultured at 37°C and 5% CO2 for 7-10 days. The antibody was detected when the clones were visible to the naked eye. The wells with only a single clone were marked under an inverted microscope. Finally, the monoclonal antibody hybridoma cell line 15G5 was obtained through preliminary screening.

[0058] 8. Antibody sequencing

[0059] Cloned 15G5 antibody cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 15G5 was as follows:

[0060] Heavy chain CDR1 sequence: IATDS (SEQ ID NO:1)

[0061] Heavy chain CDR2 sequence: VDACTSLWHMQESC (SEQ ID NO:2)

[0062] Heavy chain CDR3 sequence: KGFYDCTTS (SEQ ID NO:3)

[0063] Light chain CDR1 sequence: PEYSGDQH (SEQ ID NO:4)

[0064] Light chain CDR2 sequence: ATSYM (SEQ ID NO:5)

[0065] Light chain CDR3 sequence: CKGGDTE (SEQ ID NO:6)

[0066] The heavy chain variable region sequence is as follows:

[0067] QVQLQQSGAELVKPGTSVKLSCKASGYNFIIATDSWVKQRPGQGLEWI

[0068] GVDACTSLWHMQESCKATLTVDKSSSTAYMQLSGLASADSAVYYCTRKGFYDCTTSWGQGTTLTVSS(SEQ ID NO:7)

[0069] The light chain variable region sequence is as follows:

[0070] DVVMTQSTPSLSVSLGDRVTISCPEYSGDQHWYQQKPDGTVKLLIYATS

[0071] YMRVPSRFSASGSGTDFSLTISNLEQEDFATYFCCKGGDTEFGGGTKLEIK(SEQ ID NO:8)

[0072] The antibody heavy chain sequence is as follows:

[0073] QVQLQQSGAELVKPGTSVKLSCKASGYNFIIATDSWVKQRPGQGLEWI

[0074] GVDACTSLWHMQESCKATLTVDKSSSTAYMQLSGLASADSAVYYCTRK

[0075] GFYDCTTSWGQGTTLTVSSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGY

[0076] FPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN

[0077] VAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVL

[0078] MISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTL

[0079] RVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVY

[0080] VLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPV

[0081] LDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:9)

[0082] The antibody light chain sequence is as follows:

[0083] DVVMTQSTPSLSVSLGDRVTISCPEYSGDQHWYQQKPDGTVKLLIYATS

[0084] YMRVPSRFSASGSGTDFSLTISNLEQEDFATYFCCKGGDTEFGGGTKLEI

[0085] KRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ

[0086] NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)

[0087] 9. Antibody expression and purification

[0088] Balb / c mice were injected intraperitoneally with 0.5 ml of liquid paraffin. Ten days later, the selected monoclonal antibody hybridoma cell line 15G5 was inoculated into 1×10⁻⁶ cells. 6 The fluid was injected into the peritoneal cavity of Balb / c mice. After about 10 days, the mice's abdomens began to swell. The mice were then euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and the ascites fluid was extracted in a single procedure.

[0089] Ascites supernatant was purified by protein A affinity chromatography, following the Cytiva handbook: Affinity Chromatography, Vol. 1: Antibodies. Anti-testosterone complex monoclonal antibody 15G5 was prepared from the purified sample. The purified SDS-PAGE image is shown below. Figure 1 The purified antibody has clear and single heavy and light chain bands, indicating a high degree of purification.

[0090] The above operations are known to those skilled in the art, and other plasmid construction, cell transfection, culture methods, separation and purification techniques in the known field can also be used to obtain the above monoclonal antibodies.

[0091] Example 2

[0092] ELISA detection of antibody 15G5 activity:

[0093] 1. Coat each well with 100 ng of testosterone P2G1 antibody and 10 ng of testosterone small molecule complex in coating solution (10 mM phosphate buffer, pH 7.4, pH 9.4), cover the wells, and incubate overnight at 2-8°C.

[0094] 2. Aspirate the liquid from each well and wash each well once with 200 μL of washing buffer (containing 0.05% Tween 20 mM phosphate-buffered saline). After washing, invert the plate and gently tap it on absorbent paper to remove any remaining liquid.

[0095] 3. At room temperature, add 200 μL of blocking buffer (10% skim milk) to each well and block for 1 hour.

[0096] 4. Suction, invert the perforated plate and gently pat it on absorbent paper to remove residual liquid.

[0097] 5. Add 100 μL of diluted antibody 15G5 solution to the blocking buffer. Incubate at room temperature for 1 hour.

[0098] 6. Aspirate the liquid from each well and wash each well 6 times with 200 μL of washing buffer.

[0099] 7. Add 10,000 times diluted HRP-labeled goat anti-mouse antibody (catalog number D110087, Shanghai Sangon Biotech), 50-100 μL per well, incubate at 37°C for 30 min, and wash 6 times.

[0100] 8. Add 100 μL of freshly prepared substrate development solution (purchased from Sangon Biotech, catalog number D110098) to each well, incubate at 37°C for 15 min.

[0101] 9. The reaction was terminated with 2 mol / L H2SO4, and the OD450 value was read on an ELISA reader. The results are shown in Table 1 below. Figure 2 .

[0102] Table 1. ELISA of antibody 15G5 with testosterone P2G1 antibody and testosterone P2G1 + testosterone small molecule complex

[0103]

[0104] From Table 1 and Figure 2 From the top view: there is basically no binding between the testosterone P2G1 antibody and the 15G5 antibody. As the concentration of the complex increases, the OD450 increases only slightly. However, the binding of the testosterone P2G1 antibody + testosterone small molecule complex to the 15G5 antibody shows a good linear correlation, indicating that the 15G5 antibody specifically binds to the testosterone P2G1 antibody + testosterone small molecule complex and has good activity, which can be used for the development of testosterone reagent sandwich method.

[0105] Example 3

[0106] 1. Testosterone competitive assay reagents are used in immunofluorescence reagents.

[0107] Testosterone hapten Testo-3-BSA (purchased from Shanghai Modis, catalog number LA814) and goat anti-mouse secondary antibody (purchased from Sangon Biotech, catalog number D111024) were streaked onto nitrocellulose membranes at concentrations of 0.066 μg / cm² and 0.1 μg / cm², respectively. The membranes were dried at 55°C for 3 days. Simultaneously, testosterone P2G1 antibody was conjugated to fluorescent microspheres and sprayed onto sample pads at a concentration of 0.026 μg / cm² of binding pad. The membranes were then vacuum-dried for 3 hours to assemble the test strips. Subsequently, different concentrations of clinical samples were added to the strips at room temperature and humidity (25°C, 50%-60% humidity), and the fluorescence values ​​were read. The results are shown in Table 2 below. Figure 3 .

[0108] Table 2. Clinical Detection Results of Testosterone Immunofluorescence Competitive Assay

[0109] Sample number Concentration value (ng / L) T-line value C-line value T / T+C 1 0.08 62463 13376 0.824 2 0.15 62346 14124 0.815 3 0.23 63356 14576 0.813 4 0.44 50654 22318 0.694 5 0.53 48769 23613 0.674 6 0.72 45361 25698 0.638 7 0.89 41097 25893 0.613 8 1.03 39468 28904 0.577 9 2.32 30164 34724 0.465 10 3.12 25634 35568 0.419 11 4.56 20087 35573 0.361 12 5.19 16925 40789 0.293 13 6.56 15793 43265 0.267 14 8.28 16537 42756 0.279 15 9.37 15897 45237 0.260

[0110] 2. Antibody 15G5 is used in immunofluorescence reagents.

[0111] Antibody 15G5 and testosterone hapten Testo-3-BSA were streaked onto nitrocellulose membranes at concentrations of 0.033 μg / cm and 0.066 μg / cm, respectively. The membranes were dried at 55°C for 3 days. Simultaneously, testosterone P2G1 antibody was conjugated to fluorescent microspheres and sprayed onto a sample pad at a concentration of 0.026 μg testosterone P2G1 antibody / cm binding pad. The microspheres were then vacuum-dried for 3 hours to assemble a testosterone sandwich immunofluorescence strip. Subsequently, different concentrations of clinical samples (testosterone-containing serum) were added to the strips at room temperature and humidity (25°C, 50%-60% humidity), and the fluorescence values ​​were recorded. The results are shown in Table 3 below. Figure 4 .

[0112] Table 3. Clinical detection results of testosterone using the immunofluorescence sandwich method.

[0113]

[0114]

[0115] Comparison Table 2 and Table 3 and Figure 3 and Figure 4 With the same labeled antibody, the testosterone competitive assay showed a good linear gradient in testosterone sample concentrations of 0.44-6.56 ng / L, but almost no gradient in samples below 0.44 ng / L and above 6.56 ng / L. In contrast, the testosterone sandwich assay showed a good linear gradient in the range of 0.08-9.37 ng / L, and the R² value of the linearity was higher than that of the competitive assay. The testosterone sandwich assay has a significant advantage over the competitive assay in terms of sensitivity and detection linearity range.

[0116] Example 4

[0117] Temperature verification of testosterone sandwich immunofluorescence reagent strips:

[0118] The testosterone sandwich immunofluorescence reagent strips were placed in low-temperature and normal-humidity (20℃, 50%-60% humidity) and high-temperature and normal-humidity (30℃, 50%-60% humidity) environments to detect clinical samples of low, medium and high concentrations. Each concentration sample was tested three times, and the fluorescence values ​​were read. The average value of the three tests was taken, and the deviation values ​​between normal temperature and low temperature and between normal temperature and high temperature were calculated. The results are shown in Table 4 below.

[0119] Table 4 Temperature Verification of Testosterone Sandwich Immunofluorescence Reagent Strips

[0120]

[0121] As shown in Table 4, the deviation of the testosterone sandwich immunofluorescence reagent strip in low, medium and high temperature tests is within 5%, which meets the requirement of deviation within 15%, and its high and low temperature performance is very good.

[0122] Example 5

[0123] Validation of the heating stability of testosterone sandwich immunofluorescence reagent strips:

[0124] Testosterone sandwich immunofluorescence reagent strips were packaged in aluminum foil and sealed. Reagents were taken out at weeks 0, 1, 2 and 3 and placed in a 37℃ incubator. All strips were taken out at week 4 and tested at room temperature and humidity for low, medium and high concentrations of clinical samples. Each concentration sample was tested three times, and the fluorescence values ​​were read. The average value of the three tests was taken. The results are shown in Table 3 below.

[0125] As shown in Table 5, after being placed at 37℃ for 4 weeks, the T / C values ​​of the testosterone sandwich immunofluorescence reagent strips were stable with a deviation of less than 15%, indicating that the testosterone sandwich immunofluorescence reagent strips are relatively stable and remain stable after being placed at 37℃ for 4 weeks.

[0126] Table 5. Stability verification of testosterone sandwich immunofluorescence reagent strips at 37℃.

[0127]

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against testosterone, characterized in that, The heavy chain CDR1 sequence is shown in SEQ ID NO:1, the heavy chain CDR2 sequence is shown in SEQ ID NO:2, and the heavy chain CDR3 sequence is shown in SEQ ID NO:3; the light chain CDR1 sequence is shown in SEQ ID NO:4, the light chain CDR2 sequence is shown in SEQ ID NO:5, and the light chain CDR3 sequence is shown in SEQ ID NO:

6.

2. The monoclonal antibody against testosterone according to claim 1, characterized in that, Its heavy chain variable region sequence is shown in SEQ ID NO:7; the light chain variable region sequence is shown in SEQ ID NO:

8.

3. The monoclonal antibody against testosterone according to claim 1, characterized in that, Its heavy chain sequence is shown in SEQ ID NO:9; its light chain sequence is shown in SEQ ID NO:

10.

4. The monoclonal antibody against testosterone according to claim 1, characterized in that, It was prepared by immunizing animals with a testosterone-testosterone antibody-P2G1 complex as an immunogen.

5. A sandwich reagent for detecting testosterone, characterized in that, Monoclonal antibodies against testosterone as described in any one of claims 1 to 4.

6. A sandwich test kit for detecting testosterone, characterized in that, Monoclonal antibodies against testosterone as described in any one of claims 1 to 4.

7. The use of a monoclonal antibody of testosterone as described in any one of claims 1 to 4 in the non-disease diagnosis of testosterone sandwich assay.

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