Monoclonal antibody for sandwich detection of testosterone as well as preparation method and application of monoclonal antibody

Abstract

The invention provides a monoclonal antibody for sandwich detection of testosterone as well as a preparation method and application thereof, and the preparation method comprises the following steps: step S1, preparing testosterone-3-(O-carboxymethyl) oxime, and activating the testosterone-3-(O-carboxymethyl) oxime; taking a testosterone antibody P2G1 after activation, and dropwise adding the activated testosterone-3-(O-carboxymethyl) oxime according to the proportion for reaction to prepare an immunogen; s2, immunizing an animal based on the prepared immunogen; s3, fusing splenocytes and myeloma cells of the immune animals into hybridoma cells, and selecting real hybridoma cells; and S4, separating and purifying the antibody from the screened hybridoma cells. The invention provides a preparation method of a raw material and a reagent for testosterone sandwich method detection, solves the problems of insufficient sensitivity, narrow detection range and poor specificity of a micromolecule compound testosterone competition method reagent, and provides a new thought and method for rapid and accurate testosterone detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody for sandwich detection of testosterone and a preparation method and application thereof. Background Art

[0002] Testosterone (Tes) is a steroid hormone containing 19 carbon atoms and a molecular weight of 288 Daltons, which is a small molecule. The male hormone in the male body is mainly testosterone, which is almost entirely synthesized by interstitial cells of the testicles. Its secretion is regulated by luteinizing hormone (LH) and negatively affects the regulation mechanism of the pituitary-hypothalamus. The adrenal glands of both men and women can synthesize some testosterone. In addition, the ovaries of women can also secrete a small amount of testosterone. The most important physiological function of testosterone is to promote the development and maturation of male sexual organs and maintain secondary sexual characteristics. In terms of metabolism, testosterone can promote muscle growth and protein synthesis, while inhibiting protein decomposition caused by glucocorticoids. Most of the testosterone in the blood circulation is bound to carrier proteins (such as sex hormone binding globulin (SHBG)). Women's ovaries can produce a small amount of testosterone, and physiological concentrations of androgens have no special effect on women. Increased androgen levels in women can lead to masculinization (depending on the degree of increase in androgen levels). Testing the testosterone content in women can help diagnose androgen syndrome (AGS), polycystic ovary (Stei n-Leventhal syndrome), and when ovarian tumors, adrenal tumors, adrenal hypoplasia or ovarian dysfunction are suspected. Testing the testosterone content in men can be used to diagnose diseases of insufficient testosterone production, such as gonadal hypoplasia, estrogen therapy, chromosomal abnormalities (such as Klinefelter syndrome) and cirrhosis. Therefore, accurately detecting its content in the body plays an important role in judging and evaluating the function of the gonads and the body.

[0003] At present, the main in vitro diagnostic test for hormones is the immunological test method, which is a method based on the specific reaction of antibodies and antigens. It can amplify and display the detected signal using isotopes, enzymes, chemiluminescent substances, etc. At present, the immunological test methods for testosterone include biochemical immunoturbidimetry, radioimmunoassay, fluorescent immunochromatography, chemiluminescence, etc. However, because testosterone is a small molecule compound with only one antibody binding site, the sandwich method cannot be used directly, and basically all of them are competitive methods. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a monoclonal antibody for sandwich detection of testosterone and a preparation method and application thereof.

[0005] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a method for preparing a monoclonal antibody for sandwich detection of testosterone.

[0006] Step S1, preparing testosterone-3-(O-carboxymethyl)oxime and activating it; after activation, taking testosterone antibody P2G1, adding the activated testosterone-3-(O-carboxymethyl)oxime dropwise according to the proportion to react, so as to prepare an immunogen;

[0007] Step S2, immunizing an animal based on the prepared immunogen;

[0008] Step S3, fusing the spleen cells of the immunized animal with myeloma cells to form hybridoma cells, and selecting true hybridoma cells;

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

[0010] Optionally, step S1 includes:

[0011] Weigh testosterone, dissolve it in methanol, and prepare a testosterone solution; weigh carboxymethoxyamine hemihydrochloride, dissolve it in pure water, and 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 collect the precipitate by centrifugation to obtain testosterone-3-(O-carboxymethyl)oxime;

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

[0013] Take the testosterone antibody P2G1, add activated testosterone-3-(O-carboxymethyl)oxime dropwise according to the proportion, react overnight, and after the reaction is completed, remove the unreacted free testosterone derivatives by ultrafiltration, and obtain the 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 them subcutaneously at multiple points. Emulsify the antigen with Freund's incomplete adjuvant and inject it subcutaneously at multiple points every 2 weeks. Strengthen the immunization twice and perform intraperitoneal injection for shock 3 days before fusion.

[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 killed by pulling their necks, and the whole body was immersed in 75% alcohol. In a clean bench, the abdominal skin was cut open with scissors under sterile operation to expose the peritoneum. RPMI 1640 basal culture medium containing penicillin-streptomycin was injected into the peritoneal cavity with a syringe, and the cells were repeatedly rinsed, the rinse solution was recovered, centrifuged, and the precipitate was retained and resuspended in RPMI 1640 complete culture medium containing HAT. After adjusting the cell concentration, the cells were added to a 96-well plate 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 plate, rinsed once with RPMI 1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension; centrifuged, discarded the supernatant, resuspended in RPMI 1640 basal culture medium, repeated three times, and counted;

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

[0020] Selection and culture of hybridoma cells: On the 1st, 3rd, 5th and 7th days after cell fusion, the culture medium is replaced with HAT culture medium. After the culture is completed, the living hybridoma cells are selected to obtain true hybridoma cells.

[0021] Optionally, step S4 includes:

[0022] The supernatant of each culture well is aspirated, and the OD490 values ​​of the supernatant and the complex containing the testosterone small molecule and the testosterone antibody, and the OD490 values ​​of the supernatant and the testosterone antibody are detected by the indirect ELISA method, and the antibody cell strain in the culture wells with the difference of the two OD490 values ​​greater than 1.5 is used as the antibody cell strain with better reactivity; the feeder cells are prepared and plated one day before cloning; the antibody cell strain with better reactivity is blown and mixed by a pipette, and the cells in the well are diluted to 1 cell in each well with HT culture medium; the cells are cultured at 37° C. and 5% CO2 for 7-10 days in a humidified environment, and the antibodies can be detected when clones visible to the naked eye appear; the cells are observed under an inverted microscope, and the wells with only a single clone growing are marked, and finally the monoclonal antibody hybridoma cell strain 15G5 is obtained by preliminary screening; the obtained monoclonal antibody hybridoma cell strain 15G5 is subjected to antibody expression and purification to obtain the monoclonal antibody.

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

[0024] Optionally, the heavy chain CDR1 sequence of the monoclonal antibody for sandwich detection of 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, and 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 a use of the above-mentioned monoclonal antibody for sandwich detection of testosterone in the testosterone sandwich method detection.

[0026] The advantages and beneficial effects of the present invention over the prior art are as follows:

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

[0028] 2. The present 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 by the present invention is obtained by injecting hybridoma cells into the peritoneal cavity of mice, taking ascites, and obtaining purified testosterone 15G5 antibody by A column purification and ultrafiltration concentration, and performing ELISA activity determination. The present invention uses the testosterone complex antibody 15G5 as a raw material to prepare a progesterone sandwich method detection reagent, detects clinical samples, and the sensitivity and detection linear range of the detection samples are better than the competitive method, and tests the temperature verification of the reagent and the 37-degree thermal acceleration stability verification. The deviations of the test results at different temperatures are small, and the stability of the reagent at 37°C is good. The preparation method of a testosterone complex antibody 15G5 provided by the present invention can be used as a raw material for testosterone sandwich immunofluorescence detection, and a testosterone sandwich immunofluorescence reagent is prepared using the complex antibody as a raw material. The problem of low sensitivity, poor detection linearity, and narrow detection range of the small molecule testosterone competition method is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

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

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

[0032] Figure 3 It is a line graph of clinical detection of testosterone immunofluorescence competitive reagent when the goat anti-mouse secondary antibody is used in the immunofluorescence reagent in the embodiment of the present invention;

[0033] Figure 4 This is a line graph of clinical detection of testosterone immunofluorescence competitive reagent when the antibody 15G5 in the embodiment of the present invention is used as an immunofluorescence reagent. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1

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

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

[0038] Freund's complete adjuvant, Complete (Cat. No. 77140, Thermo Fisher); Freund's incomplete adjuvant, Incomplete (Cat. No. 77145, Thermo Fisher); HAT Media Supplement (50×) (Cat. No. 21060017, Thermo Fisher); HT Media Supplement (50×) (Cat. No. H0111067030, Thermo Fisher); PEG (Cat. No. P7181, Sigma); RPMI 1640 (Cat. No. L210KJ, Shanghai Yuanpei Biological); Fetal bovine serum FBS (C04001-500, Shanghai Xiaopeng Biological); DMEM (Cat. No. L310KJ, Shanghai Yuanpei Biological); Penicillin-St reptomycin (Cat. No. 15140122, Gibco); HRP-labeled goat anti-mouse antibody (Cat. No. D110087, Shanghai Shenggong Biotechnology); Protein AResin (Cat. No. SA023010, Changzhou Tiandirenhe Biotechnology Co., Ltd.).

[0039] 1. Preparation of immunogen

[0040] Weigh 100mg testosterone, dissolve in 1mL methanol, make testosterone solution. Weigh 100mg carboxymethyloxyamine hemihydrochloride, dissolve in 1mL pure water, make carboxymethyloxyamine hemihydrochloride solution. Testosterone solution is added dropwise to carboxymethyloxyamine hemihydrochloride solution at room temperature, and stirred vigorously. White precipitate is formed immediately. Add 5mL water, shake, and collect precipitate by centrifugation. The precipitate is testosterone-3-(O-carboxymethyl) oxime.

[0041] Take the above testosterone-3-(O-carboxymethyl) oxime and dissolve it in 10mL of dichloromethane, add 100mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 50mg of N-hydroxysuccinimide, and stir at room temperature for 4 hours. The reaction product is washed with water 3 times and dried under reduced pressure, and the dried powder is dissolved in 10mL of DMSO. That is the activated testosterone-3-(O-carboxymethyl) oxime.

[0042] Take the testosterone antibody P2G1 (purchased from ThermoFisher, catalog number MIT0102, mouse antibody), P2G1: activated testosterone-3-(O-carboxymethyl) oxime at a mass ratio of 1:24, add activated testosterone-3-(O-carboxymethyl) oxime dropwise. React at 4°C overnight. After the reaction is completed, ultrafiltration is performed to remove the unreacted free testosterone derivatives, and the ultrafiltration is the immunogen.

[0043] 2. Mouse immunization

[0044] Dissolve the above immunogens and emulsify them 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 points. Emulsify the antigen with Freund's incomplete adjuvant at intervals of 2 weeks and inject 100 μg / mouse subcutaneously at multiple points. Strengthen the immunization twice and perform shock by intraperitoneal injection 3 days before fusion.

[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 killed by pulling the neck, and the whole body was immersed in 75% alcohol. Under sterile operation in a clean bench, the abdominal skin was cut open with scissors to expose the peritoneum. 5 mL of RPMI1640 basal culture medium containing 1% penicillin-streptomycin was injected into the peritoneal cavity with a syringe, and the cells were repeatedly rinsed. The rinsing fluid was recovered and centrifuged at 1000 rpm for 5 minutes. The precipitate was retained and resuspended in RPMI1640 complete culture medium containing 1% HAT to adjust the cell concentration to 1×10 5 / mL, added to 96-well plate, 150 μL / well, 37°C, 5% CO2 overnight.

[0047] 4. Preparation of immune spleen cells

[0048] Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a plate, rinsed once with RPMI1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension. Centrifuged, the supernatant was discarded, and the cell suspension was resuspended in RPMI1640 basal culture medium. This process was repeated three times and 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°C water bath for preheating;

[0051] (2) Mouse myeloma cells Sp2 / 0 (2-5×10 7 ), the above immune spleen cells (10 8Add the suspension to a 50 mL centrifuge tube and mix well, then add DMEM serum-free culture medium to 40 mL. Centrifuge for 10 minutes, pour off the supernatant, and mix well;

[0052] (3) After mixing, place the centrifuge tube in 37°C pre-warmed water, take 0.7 mL of pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately after the standing period, add 15 mL of 37°C pre-warmed DMEM serum-free culture medium;

[0053] (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and pour off the supernatant. After pouring off the supernatant, add 40 mL of HAT culture medium containing 15% to 20% fetal bovine serum. Mix well with a pipette, and drop 2 drops into the small wells of 4 96-well cell culture plates containing feeder cells, and culture in an incubator at 37°C and 7% CO2.

[0054] 6. Selection and culture of hybridoma cells

[0055] On the 1st, 3rd, 5th and 7th days after cell fusion, the culture medium is replaced with the aforementioned HAT culture medium. After the culture is completed, the living hybridoma cells are selected to obtain true hybridoma cells.

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

[0057] The supernatant of each culture well was aspirated, and the OD490 values ​​of the supernatant and the complex containing the testosterone small molecule and the testosterone antibody, and the OD490 values ​​of the supernatant and the testosterone antibody were detected by indirect ELISA method, and the antibody cell strain in the culture wells with the difference of two OD490 values ​​greater than 1.5 was used as the antibody cell strain with better reactivity; the feeder layer cells were prepared and plated according to step 3 one day before cloning; the antibody cell strain with better reactivity was mixed by pipetting, and the cells in the wells were diluted to 1 cell in each well with HT culture medium; the cells were cultured at 37° C. and 5% CO2 in a humidified environment for 7-10 days, and the antibodies could be detected when clones visible to the naked eye appeared; the cells were observed under an inverted microscope, and the wells with only single clones growing were marked, and finally the monoclonal antibody hybridoma cell line 15G5 was obtained by preliminary screening.

[0058] 8. Antibody Sequencing

[0059] The cloned 15G5 antibody cells were sent to Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the sequenced monoclonal antibody 15G5 is 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:

[0067] QVQLQQSGAELVKPGTSVKLSCKASGYNFIIATDSWVKQRPGQGLEWI

[0068] GVDACTSLWHMQESCKATLTVDKSSSTAYMQLSGLASADSAVYYCTRKGFYDCTTSWGQGTTLTVSS(SEQ ID NO:7)

[0069] The light chain variable region sequence is:

[0070] DVVMTQSTPSLSVSLGDRVTISCPEYSGDQHWYQQKPDGTVKLLIYATS

[0071] YMRVPSRFSASGSGTDFSLTISNLEQEDFATYFCCKGGDTEFGGGTKLEIK(SEQ ID NO:8)

[0072] The antibody heavy chain sequence is:

[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:

[0083] DVVMTQSTPSLSVSLGDRVTISCPEYSGDQHWYQQKPDGTVKLLIYATS

[0084] YMRVPSRFSASGSGTDFSLTISNLEQEDFATYFCCKGGDTEFGGGTKLEI

[0085] KRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ

[0086] NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)

[0087] 9. Antibody expression and purification

[0088] Balb / c mice were intraperitoneally injected with 0.5 ml of liquid paraffin. Ten days later, the selected monoclonal antibody hybridoma cell line 15G5 was inoculated with 1×10 6 The mouse abdomen will begin to swell after about 10 days, and the mouse will be killed by dislocating the cervical vertebrae, and then immersed in 75% alcohol for 5 minutes for disinfection, and the ascites will be extracted once.

[0089] The ascites supernatant was separated and purified by protein A affinity chromatography according to the Cytiva handbook: Affinity Chromatography, Vol 1: Antibodies. Anti-testosterone complex monoclonal antibody 15G5 was prepared by purification. The purified SDS-PAGE image is shown in Figure 1 The heavy and light chain bands of the purified antibodies are clear and single, and the purification is relatively high.

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

[0091] Example 2

[0092] ELISA to detect the activity of antibody 15G5:

[0093] 1. Each well was coated with 100 ng of testosterone P2G1 antibody, 100 ng of testosterone P2G1 antibody + 10 ng of testosterone small molecule complex, and the well plate was coated with coating solution (10 mM phosphate buffer, pH 7.4, pH 9.4), and the well plate was covered and incubated at 2-8°C overnight.

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

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

[0096] 4. Aspirate, invert the plate and tap gently on absorbent paper to remove any residual liquid.

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

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

[0099] 7. Add 10,000-fold diluted HRP-labeled goat anti-mouse antibody (Cat. No. D110087, Shanghai Sangon Biotechnology), 50-100ul per well, incubate at 37°C for 30min, and wash 6 times.

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

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

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

[0103]

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

[0105] Example 3

[0106] 1. Testosterone competition reagents are used in immunofluorescence reagents

[0107] The testosterone hapten Testo-3-BSA (purchased from Shanghai Modis, item number LA814) and the goat anti-mouse secondary antibody (purchased from Sangon Biotechnology, item number D111024) were streaked on the nitrocellulose membrane, with the dosage of 0.066 μg / cm2 and 0.1 μg / cm2, respectively, and dried at 55°C for 3 days. At the same time, the testosterone P2G1 antibody was coupled and labeled with fluorescent microspheres, sprayed on the sample pad, with the dosage of 0.026 μg testosterone P2G1 / cm2 binding pad, and vacuum-dried for 3 hours to assemble into a reagent strip. Subsequently, different concentrations of clinical samples were added in an environment of normal temperature and humidity (25°C, humidity 50%-60%), and the fluorescence values ​​were read and detected. The results are shown in Table 2 and Figure 3 .

[0108] Table 2 Testosterone immunofluorescence competitive method clinical test results

[0109] Sample No. 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 as immunofluorescence reagent

[0111] The antibody 15G5 and the testosterone hapten Testo-3-BSA were streaked on the nitrocellulose membrane, with the dosage of 0.033 μg / cm2 and 0.066 μg / cm2, respectively, and dried at 55°C for 3 days. At the same time, the testosterone P2G1 antibody was coupled and labeled with fluorescent microspheres, sprayed on the sample pad, with the dosage of 0.026 μg testosterone P2G1 antibody / cm2 binding pad, and vacuum-dried for 3 hours to assemble into a testosterone sandwich immunofluorescence reagent strip. Subsequently, different concentrations of clinical samples (serum containing testosterone) were added in an environment of normal temperature and humidity (25°C, humidity 50%-60%), and the fluorescence values ​​were read and detected. The results are shown in Table 3 and Figure 4 .

[0112] Table 3 Testosterone immunofluorescence sandwich method clinical test results

[0113]

[0114]

[0115] Compare Table 2 with Table 3 and Figure 3 and Figure 4 For the same labeled antibody, the testosterone competition method has a good linear gradient in the testosterone sample concentration of 0.44-6.56ng / L, but there is basically no gradient in the samples below 0.44ng / L and above 6.56ng / L; while the testosterone sandwich method has a good linear gradient in the range of 0.08-9.37ng / L, and the linear R2 value is higher than that of the competition method. The testosterone sandwich method has great advantages over the competition method in sensitivity and detection linear range.

[0116] Example 4

[0117] Testosterone sandwich immunofluorescence reagent strip temperature verification:

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

[0119] Table 4 Testosterone sandwich immunofluorescence reagent strip temperature verification

[0120]

[0121] From Table 4, it can be seen that the deviation of the low, medium and high samples tested by the testosterone sandwich immunofluorescence reagent strip at low temperature and high temperature 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] Testosterone sandwich immunofluorescence reagent strip heating stability verification:

[0124] The testosterone sandwich immunofluorescence reagent strips were wrapped and sealed with aluminum foil. The reagents were taken out at 0, 1, 2, and 3 weeks respectively and placed in a 37°C incubator. All were taken out at 4 weeks. Clinical samples of low, medium, and high concentrations were tested at room temperature and humidity. Each concentration sample was repeated three times, the fluorescence value was read, and the average of the three times was taken. The results are shown in Table 3.

[0125] It can be seen from Table 5 that after being placed at 37°C for 4 weeks, the T / C value data of the testosterone sandwich immunofluorescence reagent strip test is stable, with a deviation within 15%, indicating that the testosterone sandwich immunofluorescence reagent strip is relatively stable and is stable when placed at 37°C for 4 weeks.

[0126] Table 5 Testosterone sandwich immunofluorescence reagent strip 37℃ heating stability verification

[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 in the protection scope of the present invention.

Claims

1. A method for preparing a monoclonal antibody for sandwich detection of testosterone, characterized in that: include: Step S1, preparing testosterone-3-(O-carboxymethyl)oxime and activating it; After activation, testosterone antibody P2G1 is taken, and activated testosterone-3-(O-carboxymethyl)oxime is added dropwise according to the proportion to react to prepare an immunogen; Step S2, immunizing an animal based on the prepared immunogen; Step S3, fusing the spleen cells of the immunized animal with myeloma cells to form hybridoma cells, and selecting true hybridoma cells; Step S4: Isolate and purify antibodies from the screened hybridoma cells.

2. The method for preparing a monoclonal antibody for sandwich detection of testosterone according to claim 1, characterized in that: Step S1 includes: Weigh testosterone, dissolve it in methanol, and prepare a testosterone solution; weigh carboxymethoxyamine hemihydrochloride, dissolve it in pure water, and 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 collect the precipitate by centrifugation to obtain testosterone-3-(O-carboxymethyl)oxime; Testosterone-3-(O-carboxymethyl)oxime was dissolved in dichloromethane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature. The reaction product was washed with water and then dried under reduced pressure. The dried powder was dissolved in DMSO to prepare activated testosterone-3-(O-carboxymethyl)oxime. Take the testosterone antibody P2G1, add activated testosterone-3-(O-carboxymethyl)oxime dropwise according to the proportion, react overnight, and after the reaction is completed, remove the unreacted free testosterone derivatives by ultrafiltration, and obtain the immunogen after ultrafiltration.

3. The method for preparing a monoclonal antibody for sandwich detection of testosterone according to claim 1, characterized in that: Step S2 comprises: After dissolving the immunogen, emulsify it evenly with an equal volume of Freund's complete adjuvant. Take mice and inject them subcutaneously at multiple points. Emulsify the antigen with Freund's incomplete adjuvant and inject it subcutaneously at multiple points every 2 weeks. Strengthen the immunization twice and perform intraperitoneal injection for shock 3 days before fusion.

4. The method for preparing a monoclonal antibody for sandwich detection of testosterone according to claim 1, characterized in that: Step S3 includes: Preparation of feeder cells: BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mice were killed by pulling their necks, and the whole body was immersed in 75% alcohol. In a clean bench, the abdominal skin was cut open with scissors under sterile operation to expose the peritoneum. RPMI1640 basic culture medium containing penicillin-streptomycin was injected into the peritoneal cavity with a syringe, and the cells were repeatedly rinsed, the rinse solution was recovered, centrifuged, and the precipitate was retained and resuspended in RPMI1640 complete culture medium containing HAT. After adjusting the cell concentration, the cells were added to a 96-well plate and cultured. Preparation of immune spleen cells: Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a plate, rinsed once with RPMI1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension; centrifuged, discarded the supernatant, resuspended in RPMI1640 basal culture medium, repeated three times, and counted; Cell fusion: (1) Pre-warming HAT culture medium, DMEM serum-free culture medium and 50% PEG solution respectively; (2) Pre-warming mouse myeloma cells and the above immune spleen cell suspension respectively, adding them to a centrifuge tube, mixing evenly, and adding pre-warmed DMEM serum-free culture medium to the target volume; after centrifugation, pour out the supernatant and mix evenly; (3) After mixing evenly, place the centrifuge tube in pre-warmed water, take the pre-warmed 50% PEG solution into the centrifuge tube, and let it stand for 90 seconds; after the standing period, add pre-warmed DMEM serum-free culture medium dropwise; (4) Add pre-warmed DMEM serum-free culture medium to the target volume, centrifuge, and pour out the supernatant; after pouring out the supernatant, add HAT culture medium containing fetal bovine serum, mix evenly, and dropwise add to the small holes of 4 96-well cell culture plates containing feeder cells for culture; Selection and culture of hybridoma cells: On the 1st, 3rd, 5th and 7th days after cell fusion, the culture medium is replaced with HAT culture medium. After the culture is completed, the living hybridoma cells are selected to obtain true hybridoma cells.

5. The method for preparing a monoclonal antibody for sandwich detection of testosterone according to claim 1, characterized in that: Step S4 comprises: The supernatant of each culture well is aspirated, and the OD490 values ​​of the supernatant and the complex containing the testosterone small molecule and the testosterone antibody, and the OD490 values ​​of the supernatant and the testosterone antibody are detected by indirect ELISA method, and the antibody cell strain in the culture wells with the difference of two OD490 values ​​greater than 1.5 is used as the antibody cell strain with better reactivity; feeder cells are prepared and plated one day before cloning; the antibody cell strain with better reactivity is blown and mixed by a pipette, and the cells in the well are diluted to 1 cell in each well with HT culture medium; 37° C., 5% CO2 humidified culture is carried out for 7-10 days, and the antibody can be detected when the clones visible to the naked eye appear; observation is made under an inverted microscope, and the wells with only single clone growth are marked, and finally the monoclonal antibody hybridoma cell strain 15G5 is obtained by preliminary screening; the obtained monoclonal antibody hybridoma cell strain 15G5 is subjected to antibody expression and purification to obtain the monoclonal antibody.

6. A monoclonal antibody for sandwich detection of testosterone, characterized in that: The monoclonal antibody for sandwich detection of testosterone is prepared according to the method according to any one of claims 1-5.

7. The monoclonal antibody for sandwich detection of testosterone according to claim 6, characterized in that: The heavy chain CDR1 sequence of the monoclonal antibody for sandwich detection of 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, and 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.

8. Use of the monoclonal antibody for sandwich detection of testosterone as claimed in claim 6 in the detection of testosterone by sandwich method.

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