Antibody combination for detecting progesterone by sandwich method and application thereof

Abstract

The invention provides an antibody for detecting progesterone by a sandwich method. The antibody comprises a monoclonal antibody 4F5 and a monoclonal antibody 3D8, a heavy chain CDR1 sequence of the monoclonal antibody 4F5 is shown as SEQ ID NO: 1, a heavy chain CDR2 sequence of the monoclonal antibody 4F5 is shown as SEQ ID NO: 2, and a heavy chain CDR3 sequence of the monoclonal antibody 4F5 is shown as SEQ ID NO: 3; the sequence of the light chain CDR1 is as shown in SEQ ID NO: 4, the sequence of the light chain CDR2 is as shown in SEQ ID NO: 5, and the sequence of the light chain CDR3 is as shown in SEQ ID NO: 6; a heavy chain CDR1 sequence of the monoclonal antibody 3D8 is shown as SEQ ID NO: 11, a heavy chain CDR2 sequence of the monoclonal antibody 3D8 is shown as SEQ ID NO: 12, and a heavy chain CDR3 sequence of the monoclonal antibody 3D8 is shown as SEQ ID NO: 13; the sequence of the light chain CDR1 is as shown in SEQ ID NO: 14, the sequence of the light chain CDR2 is as shown in SEQ ID NO: 15, and the sequence of the light chain CDR3 is as shown in SEQ ID NO: 16. The antibody can solve the problems of insufficient sensitivity, narrow detection range and poor specificity of a small molecule compound progesterone competition method reagent, and provides a new method for rapid and accurate detection of progesterone.

Description

Technical Field

[0001] The present invention relates to an antibody combination for detecting progesterone by a sandwich method and application thereof, belonging to the technical field of antibodies. Background Art

[0002] Progesterone (PROG), also known as progesterone or luteinizing hormone, is the primary biologically active progestogen secreted by the ovaries. Its chemical formula is C21H30O2. Its primary physiological functions include: 1) promoting endometrial thickening, preparing for implantation of a fertilized egg and maintaining a stable pregnancy; 2) inhibiting uterine contractions, preventing premature birth; 3) promoting breast development, preparing for lactation; and 4) working together with estrogen to regulate the menstrual cycle. Progesterone testing is widely used clinically. Measuring progesterone levels during the luteal phase (the second half of the menstrual cycle) can assess normal luteal phase function. Luteal phase insufficiency can lead to infertility or early miscarriage. In early pregnancy, progesterone levels gradually increase, and testing can be used to assess pregnancy status. Low levels may indicate the risk of threatened miscarriage or ectopic pregnancy. In mid- to late pregnancy, progesterone is primarily secreted by the placenta, and its levels reflect placental function. In addition, testing progesterone levels can also assist in the diagnosis of endocrine diseases (such as polycystic ovary syndrome, adrenal diseases), the use of assisted reproductive technology, and drug monitoring to adjust drug dosages.

[0003] Currently, the main methods for detecting progesterone on the market include biochemical immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), immunochromatography, and chemiluminescence immunoassay. These methods all rely on competitive assays, where small molecules in the sample compete with labeled antigens for binding to a limited amount of antibodies. The concentration of the antigen in the sample is indirectly inferred by measuring the intensity of the labeled signal. Disadvantages of these methods include a generally narrow detection range; the signal is inversely proportional to concentration, which can lead to inaccurate detection of high-concentration samples; and weak anti-interference capabilities. Impurities in the sample may affect antigen-antibody binding, leading to an increase in false positive or false negative results. Sandwich assays can address the challenges of insufficient sensitivity, narrow detection range, and poor specificity of competitive assays for small molecule progesterone. However, antibodies for sandwich assays for progesterone are currently lacking in the existing technology. Summary of the Invention

[0004] The present invention provides an antibody combination for detecting progesterone by a sandwich method and application thereof, which can effectively solve the above problems.

[0005] An antibody combination for sandwich detection of progesterone, comprising monoclonal antibody 4F5 and monoclonal antibody 3D8; The heavy chain CDR1 sequence of the monoclonal antibody 4F5 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; The heavy chain CDR1 sequence of the monoclonal antibody 3D8 is shown in SEQID NO:11, the heavy chain CDR2 sequence is shown in SEQID NO:12, and the heavy chain CDR3 sequence is shown in SEQID NO:13; the light chain CDR1 sequence is shown in SEQID NO:14, the light chain CDR2 sequence is shown in SEQID NO:15, and the light chain CDR3 sequence is shown in SEQID NO:16.

[0006] In some embodiments, the heavy chain variable region sequence of the monoclonal antibody 4F5 is shown in SEQ ID NO: 7, and the light chain variable region sequence is shown in SEQ ID NO: 8.

[0007] In some embodiments, the heavy chain sequence of the monoclonal antibody 4F5 is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:10.

[0008] In some embodiments, the heavy chain variable region sequence of the monoclonal antibody 3D8 is shown in SEQ ID NO: 17, and the light chain variable region sequence is shown in SEQ ID NO: 18.

[0009] In some embodiments, the heavy chain sequence of the monoclonal antibody 3D8 is shown in SEQ ID NO: 19, and the light chain sequence is shown in SEQ ID NO: 20.

[0010] A reagent for detecting progesterone by a sandwich method comprises the monoclonal antibody 4F5 and the monoclonal antibody 3D8.

[0011] A sandwich method test kit for detecting progesterone comprises the monoclonal antibody 4F5 and the monoclonal antibody 3D8.

[0012] A monoclonal antibody 4F5, whose 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.

[0013] In some embodiments, the monoclonal antibody 4F5 has a heavy chain variable region sequence as shown in SEQ ID NO: 7, and a light chain variable region sequence as shown in SEQ ID NO: 8.

[0014] A monoclonal antibody 3D8, whose heavy chain CDR1 sequence is shown in SEQID NO:11, the heavy chain CDR2 sequence is shown in SEQID NO:12, and the heavy chain CDR3 sequence is shown in SEQID NO:13; the light chain CDR1 sequence is shown in SEQID NO:14, the light chain CDR2 sequence is shown in SEQID NO:15, and the light chain CDR3 sequence is shown in SEQID NO:16.

[0015] A method for preparing an antibody combination for detecting progesterone by a sandwich method comprises activating progesterone and combining it with a progesterone antibody to form a complex as an immunogen, and then immunizing an animal to obtain the complex.

[0016] The beneficial effects of the present invention are: The present invention provides an antibody combination for the sandwich method detection of small molecule progesterone, which can solve the problems of insufficient sensitivity, narrow detection range and poor specificity of small molecule compound progesterone competition method reagents, and provide a new method for the rapid and accurate detection of progesterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the purification diagram of progesterone complex antibodies 4F5 and 3D8.

[0019] Figure 2 This is a diagram of ELISA detection of 4F5 antibody activity.

[0020] Figure 3 This is a diagram showing the activity of 3D8 antibody detected by ELISA.

[0021] Figure 4 This is the linear graph of the clinical detection of progesterone immunofluorescence sandwich assay reagent. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0023] Example 1: Preparation of Anti-Progesterone Complex Antibodies The reagents or kits involved in this application and their sources are as follows: Freund's Adjuvant, Complete (Cat. No. 77140, ThermoFisher); Freund's Adjuvant, Incomplete (Cat. No. 77145, ThermoFisher); HAT Media Supplement (50×) (Cat. No. 21060017, ThermoFisher); HT Media Supplement (50×) (Cat. No. H0111067030, ThermoFisher) ; PEG (Cat. No. P7181, Sigma); RPMI1640 (Cat. No. L210KJ, Shanghai Yuanpei Biotechnology); Fetal bovine serum FBS (C04001-500, Shanghai Xiaopeng Biotechnology); DMEM (Cat. No. L310KJ, Shanghai Yuanpei Biotechnology); Penicillin-Streptomycin (Cat. No. 15140122, Gibco); HRP-labeled goat anti-mouse antibody (Cat. No. D110087, Shanghai Sangon Biotechnology); ProteinAResin (Cat. No. SA023010, Changzhou Tiandirenhe Biotechnology Co., Ltd.

[0024] 1. Immunogen Preparation Weigh 100 mg of progesterone (purchased from Aladdin, product number P276608) and dissolve it in 1 mL of methanol. Weigh 100 mg of carboxymethyloxyamine hemihydrochloride and dissolve it in 1 mL of pure water. Add the progesterone solution dropwise to the carboxymethyloxyamine hemihydrochloride solution at room temperature and stir vigorously. A white precipitate will immediately form. Add 5 mL of water, shake, and collect the precipitate by centrifugation. The precipitate is progesterone-3-(O-carboxymethyl)oxime.

[0025] Dissolve the above-mentioned progesterone-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 it under reduced pressure. The dried powder is dissolved in 10 mL of DMSO. This is the activated progesterone-3-(O-carboxymethyl)oxime.

[0026] Take progesterone antibody Ab1 (purchased from Shanghai Modis, catalog number HM1222) and add activated progesterone-3-(O-carboxymethyl)oxime dropwise at a molar ratio of 1:24. Incubate at 4°C overnight. After the reaction, remove unreacted free progesterone derivatives by ultrafiltration. This is the immunogen.

[0027] 2. Mouse immunization After dissolving the above immunogen, the mixture was emulsified evenly with an equal volume of Freund's Adjuvant, Complete (ThermoFisher, Catalog No. 77140). 6-8 week old SPF Balb / c mice (Fuzhou Wu's Animal Experimental Center) were selected and injected subcutaneously at multiple points with 200 μg / mouse. Two weeks later, the antigen was emulsified with an equal volume of Freund's Adjuvant, Incomplete (ThermoFisher, Catalog No. 77145) and injected subcutaneously at multiple points with 100 μg / mouse. Boost immunization was performed twice, and 100 μg / mouse was injected intraperitoneally for shock 3 days before fusion.

[0028] 3. Preparation of feeder cells Balb / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, mice were sacrificed by cervical distension and the whole body was disinfected by immersion in 75% alcohol for 5 minutes. Aseptically, the abdominal skin was cut open with scissors to expose the peritoneum in a clean bench. 5 mL of RPMI1640 basal culture medium containing 1% penicillin-streptomycin was injected into the peritoneal cavity with a syringe. The cells were rinsed repeatedly, and the rinsing fluid was recovered and centrifuged at 1000 rpm for 5 minutes. The pellet was then resuspended in RPMI1640 complete culture medium (RPMI1640 basal culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) containing 1% HAT to adjust the cell concentration to 1×10 5 Add 150 μL / well of a 96-well plate and culture overnight at 37°C in 5% CO2.

[0029] Penicillin-Streptomycin: Gibco, Cat. No. 15140122.

[0030] RPMI1640 basal culture medium: Shanghai Yuanpei Biological, product number: L210KJ.

[0031] HAT culture medium: HAT Media Supplement (50×), ThermoFisher, product number: 21060017.

[0032] 4. Preparation of immune spleen cells Three days after the final immunization of these mice, the spleens were removed under sterile conditions, placed on a plate, rinsed once with RPMI 1640 basal medium, and ground and filtered on a nylon mesh placed in a small beaker to prepare a cell suspension. The cells were centrifuged, the supernatant discarded, and the suspension resuspended in RPMI 1640 basal medium. This process was repeated three times and the cells were counted.

[0033] 5. Cell fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium (Shanghai Yuanpei Biological, catalog number: L310KJ) and 1 mL of 50% PEG (M12000, Sigma, catalog number: P7181) and preheat them in a 37°C water bath; (2) Mouse myeloma cells Sp2 / 0 (2×10 7 Add the above immune spleen cell suspension (1 × 108 cells) to a 50 mL centrifuge tube, mix thoroughly, and add serum-free DMEM to 40 mL. Centrifuge for 10 minutes, pour off the supernatant, and mix thoroughly. (3) Place the centrifuge tube in 37°C pre-warmed water, take 0.7 mL of 37°C pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of 37°C pre-warmed serum-free culture medium; (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and pour off the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum (FBS). Mix thoroughly with a pipette and add 2 drops per well to the wells of the 96-well cell culture plate containing the feeder cells. Incubate in a 37°C, 7% CO2 incubator.

[0034] Fetal bovine serum: Shanghai Xiaopeng Biological, catalog number: C04001-500.

[0035] 6. Selection and culture of hybridoma cells On the 1st, 3rd, 5th and 7th day after cell fusion, the culture medium is replaced with HAT culture medium containing 15%-20% fetal bovine serum. The cells that survive are hybridoma cells, while non-hybridoma cells die, thus selecting true hybridoma cells.

[0036] 7. Detection of specific antibodies and hybridoma cell cloning The supernatant from each culture well was aspirated and detected using an indirect ELISA assay to identify wells containing a complex of progesterone-specific antibodies and small-molecule progesterone, but not progesterone-specific antibodies. This screened for antibody cell lines whose OD490 difference between the ELISA for recognizing the complex and the antibody alone was greater than 1.5. The day before cloning, feeder cells were prepared and plated according to step 3. The wells were mixed using a pipette and the cells in the wells were diluted to a single cell per well using HT culture medium. The cells were cultured at 37°C in a humidified atmosphere with 5% CO2 for 7-10 days. Antibodies were detected when visible colonies appeared. Under an inverted microscope, wells containing only single clones were marked. This preliminary screening yielded the monoclonal antibody hybridoma cell lines 4F5 and 3D8.

[0037] 8. Antibody Sequencing The cloned 4F5 and 3D8 antibody cells were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 4F5 was as follows: Heavy chain CDR1 sequence: TEMSVR (SEQ ID NO: 1) Heavy chain CDR2 sequence: IFSVTDQLKP (SEQ ID NO: 2) Heavy chain CDR3 sequence: YDGVYCNEP (SEQ ID NO: 3) Light chain CDR1 sequence: LYIQSRNAS (SEQ ID NO: 4) Light chain CDR2 sequence: GHSLRA (SEQ ID NO: 5) Light chain CDR3 sequence: TWPTNLA (SEQ ID NO: 6) The heavy chain variable region sequence is: QVQLQQPGAELVKPGASVKMSCKASGYNFTEMSVRWVKQRPGRGLEWIGIFSVTDQLKPKATLTVDKSSSTAYIQLSGLASEDSAVYYCTRYDGVYCNEPWGQGTTLTVSS (SEQ ID NO: 7) The light chain variable region sequence is: DIVMTQSTSSLSVSLGDRVTITCLYIQSRNASWYQQKPDGTVKLLIYGHSLRAVPSRFSASGSGTDYSLTISNLEQEDFATYFCTWPTNLAFGGGTKLEIK (SEQ ID NO: 8) The antibody heavy chain sequence is: QVQLQQPGAELVKPGASVKMSCKASGYNFTEMSVRWVKQRPGRGLEWIGIFSVTDQLKPKATLTVDKSSSTAYIQLSGLASEDSAVYYCTRYDGVYCNEPWGQGTTLTVSS STPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKG SVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQID NO:9) The antibody light chain sequence is: DIVMTQSTSSLSVSLGDRVTITCLYIQSRNASWYQQKPDGTVKLLIYGHSLRAVPSRFSASGSGTDYSLTISNLEQEDFATYFCTWPTNLAFGGGTKLEIKRT DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQID NO:10) The amino acid sequence of monoclonal antibody 3D8 is as follows: Heavy chain CDR1 sequence: VHLEHT (SEQ ID NO: 11) Heavy chain CDR2 sequence: CEQTAVPE (SEQ ID NO: 12) Heavy chain CDR3 sequence: YMDTLGACANE (SEQ ID NO: 13) Light chain CDR1 sequence: LNQISDNE (SEQ ID NO: 14) Light chain CDR2 sequence: YSGHL (SEQ ID NO: 15) Light chain CDR3 sequence: TPLNFQG (SEQ ID NO: 16) The heavy chain variable region sequence is: QIQLQQSGPELVKPGASVKLSCKASGYTFVHLEHTWVKQKPGQGLEWIGCEQTAVPEKATLTVDKSSSTAYMQLSGLASADSAVYFCTRYMDTLGACANEWGQGTTLTVSS(SEQ ID NO:17) The light chain variable region sequence is: DIQMTQTTPSLSVSLGDRVTISCLNQISDNEWYQQKPDGTVKLLIYYSGHLRVPSRFSGSGTGTDFSLTISNLEQEDFATYFCTPLNFQGFGGGTKLEIK(SEQ ID NO:18) The antibody heavy chain sequence is: QIQLQQSGPELVKPGASVKLSCKASGYTFVHLEHTWVKQKPGQGLEWIGCEQTAVPEKATLTVDKSSSTAYMQLSGLASADSAVYFCTRYMDTLGACANEWGQGTTLTVSSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:19) The antibody light chain sequence is: DIQMTQTTPSLSVSLGDRVTISCLNQISDNEWYQQKPDGTVKLLIYYSGHLRVPSRFSGSGTGTDFSLTISNLEQEDFATYFCTPLNFQGFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:20) 9. Antibody expression and purification Balb / c mice were intraperitoneally injected with 0.5 ml of liquid paraffin. Ten days later, the selected hybridoma cell lines were inoculated with 1×10 6 Place the solution into the abdominal cavity of a Balb / c mouse. After about 10 days, the mouse's abdomen will begin to swell. The mouse will be killed by dislocating the cervical vertebrae and disinfected by soaking in 75% alcohol for 5 minutes. Ascites will then be extracted once.

[0038] Ascites supernatant was purified by protein A affinity chromatography according to the Cytiva handbook: Affinity Chromatography, Vol 1: Antibodies. Anti-progesterone complex monoclonal antibodies 4F5 and 3D8 were purified. The purified SDS-PAGE images are shown in Figure 2. Figure 1 The purified antibody heavy and light chain bands are clear and single, with high purity.

[0039] The above operations are well known to those skilled in the art. Other plasmid construction, cell transfection, culture methods, separation and purification techniques known in the art may also be used to obtain the above monoclonal antibodies.

[0040] Example 2: ELISA detection of progesterone complex antibody activity 1. Coat the plate with 100 ng of progesterone Ab1 antibody, 100 ng of progesterone Ab1 + 10 ng of progesterone small molecule complex, and coating solution (10 mM phosphate buffer, pH 7.4, pH 9.4) per well. Cover the plate and incubate overnight at 2–8°C.

[0041] 2. Aspirate the liquid from each well and wash once with 200µL of wash buffer (20mM phosphate-buffered saline containing 0.05% Tween) per well. After washing, invert the plate and tap gently on absorbent paper to remove any remaining liquid.

[0042] 3. Add 200 µL of blocking buffer (10% skim milk) to each well and block for 1 hour at room temperature.

[0043] 4. Aspirate, invert the plate and tap gently on absorbent paper to remove any remaining liquid.

[0044] 5. Add 100 μL of 4F5 and 3D8 solutions diluted at different concentrations to the blocking buffer and incubate at room temperature for 1 hour.

[0045] 6. Aspirate the liquid from each well and add 200µL of wash buffer to each well and wash six times.

[0046] 7. Add 10,000-fold diluted HRP-labeled goat anti-mouse antibody (Cat. No. D110087, Shanghai Sangon Biotechnology), 50-100 μL per well, incubate at 37°C for 30 min, and wash 6 times.

[0047] 8. Add 100 μL of freshly prepared substrate color development solution (purchased from Sangon Biotechnology, product number D110098) to each well and incubate at 37°C for 15 minutes.

[0048] 9. Terminate the reaction with 2 mol / L H2SO4 and read the OD on an enzyme-linked immunosorbent assay reader. 450 The results are shown in Table 1 and Figure 2 .

[0049] Table 1 ELISA of 4F5 and 3D8 with progesterone antibody Ab1 and progesterone antibody Ab1 + progesterone complex

[0050] From Table 1 and Figure 2 Above: Progesterone 4F5 and 3D8 antibodies have almost no binding with the complex antibody. As the concentration of the complex increases, OD 450 There is only a small increase, while the complex of progesterone Ab1 antibody + progesterone binds to the complex antibody and has a good linear correlation, indicating that this complex antibody specifically binds to the complex of progesterone Ab1 antibody + progesterone and has good activity, and can be used for the development of progesterone reagent sandwich method.

[0051] Example 3: 1. Application of progesterone complex antibodies 4F5 and 3D8 in immunofluorescence reagents The progesterone complex antibodies 4F5 and 3D8 were mixed and streaked on the nitrocellulose membrane at a dosage of 0.035μg / cm2 and 0.028μg / cm2, respectively, as the T line. The progesterone hapten PROG-BSA was streaked on the nitrocellulose membrane at a dosage of 0.3μg / cm2 as the C line. The membrane was dried at 55℃ for 4 days. At the same time, the progesterone Ab1 antibody was conjugated and labeled with fluorescent microspheres and sprayed on the sample pad at a dosage of 0.016μg progesterone Ab1 antibody / cm2 conjugation pad. The membrane was vacuum-dried for 3 hours and assembled into a reagent strip. Subsequently, different concentrations of clinical samples were added in an environment of normal temperature and humidity (25℃, humidity 50%-60%), and the fluorescence values were read. The results are shown in Table 2 and Table 3. Figure 4 .

[0052] Table 2 Clinical test results of progesterone immunofluorescence sandwich method

[0053] From Table 2 and Figure 4It can be seen that when the sandwich immunoassay reagent is composed of two progesterone complex antibodies and progesterone Ab1, the fluorescence value increases with the increase of sample concentration, the detection sensitivity is high, and the overall linearity is good in the clinical sample range of 0.06-47.19 ng / mL.

[0054] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An antibody combination for sandwich detection of progesterone, characterized in that: Including monoclonal antibody 4F5 and monoclonal antibody 3D8; The heavy chain CDR1 sequence of the monoclonal antibody 4F5 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; The heavy chain CDR1 sequence of the monoclonal antibody 3D8 is shown in SEQID NO:11, the heavy chain CDR2 sequence is shown in SEQID NO:12, and the heavy chain CDR3 sequence is shown in SEQID NO:13; the light chain CDR1 sequence is shown in SEQID NO:14, the light chain CDR2 sequence is shown in SEQID NO:15, and the light chain CDR3 sequence is shown in SEQID NO:

16.

2. The antibody combination for detecting progesterone by sandwich method according to claim 1, characterized in that: The heavy chain variable region sequence of the monoclonal antibody 4F5 is shown in SEQ ID NO: 7, and the light chain variable region sequence is shown in SEQ ID NO:

8.

3. The antibody combination for detecting progesterone by sandwich method according to claim 1, characterized in that: The heavy chain sequence of the monoclonal antibody 4F5 is shown in SEQ ID NO: 9, and the light chain sequence is shown in SEQ ID NO:

10.

4. The antibody combination for detecting progesterone by sandwich method according to claim 1, characterized in that: The heavy chain variable region sequence of the monoclonal antibody 3D8 is shown in SEQ ID NO: 17, and the light chain variable region sequence is shown in SEQ ID NO:

18.

5. The antibody combination for detecting progesterone by sandwich method according to claim 1, characterized in that: The heavy chain sequence of the monoclonal antibody 3D8 is shown in SEQ ID NO: 19, and the light chain sequence is shown in SEQ ID NO:

20.

6. A reagent for detecting progesterone by sandwich method, characterized in that: The antibody combination according to any one of claims 1 to 5.

7. A sandwich method for detecting progesterone kit, characterized in that: The antibody combination according to any one of claims 1 to 5.

8. A monoclonal antibody 4F5, characterized in that Its heavy chain CDR1 sequence is shown in SEQID NO:1, the heavy chain CDR2 sequence is shown in SEQID NO:2, and the heavy chain CDR3 sequence is shown in SEQID NO:3; the light chain CDR1 sequence is shown in SEQID NO:4, the light chain CDR2 sequence is shown in SEQID NO:5, and the light chain CDR3 sequence is shown in SEQID NO:

6.

9. A monoclonal antibody 3D8, characterized in that Its heavy chain CDR1 sequence is shown in SEQID NO:11, its heavy chain CDR2 sequence is shown in SEQID NO:12, and its heavy chain CDR3 sequence is shown in SEQID NO:13; its light chain CDR1 sequence is shown in SEQID NO:14, its light chain CDR2 sequence is shown in SEQID NO:15, and its light chain CDR3 sequence is shown in SEQID NO:

16.

10. A method for preparing the antibody combination for sandwich detection of progesterone according to claims 1 to 5, characterized in that: The activated progesterone is combined with the progesterone antibody to form a complex which is used as the immunogen and then immunized to animals to obtain the immunogen.

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