Cortisol compound antibody and application
By developing cortisol complex antibodies, the problems of insufficient detection sensitivity and specificity in existing technologies have been solved, realizing high-sensitivity and low-cost cortisol sandwich method detection, which is suitable for clinical sample analysis.
Patent Information
- Application Number
- CN202511100796.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-07
- Publication Date
- 2025-11-18
AI Technical Summary
The lack of antibodies available for small molecule cortisol sandwich assays in current technologies leads to insufficient detection sensitivity and specificity, as well as high detection costs.
A cortisol complex antibody containing specific heavy and light chain CDR sequences was developed for the preparation of cortisol sandwich assay reagents. Highly specific and sensitive antibodies were obtained by preparing and screening hybridoma cell line 10D1.
It achieves high sensitivity, wide detection range and good specificity for cortisol detection, reduces detection costs, and is suitable for clinical sample analysis.
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Figure CN120965884A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a cortisol complex antibody and its uses, belonging to the field of antibody technology. Background Technology
[0002] Currently, cortisol (Cor) is an important adrenocortical hormone, mainly secreted by the zona fasciculata of the adrenal cortex, and plays a variety of physiological roles in the human body.
[0003] In peripheral blood, most cortisol exists bound to corticosteroid-binding globulin and albumin. The most important physiological functions of cortisol are raising blood glucose, anti-inflammation, and immunosuppression.
[0004] In terms of metabolism, cortisol raises blood sugar by promoting gluconeogenesis, lipolysis, and proteolysis, while inhibiting protein synthesis. In terms of anti-inflammation, cortisol can suppress inflammatory responses by inhibiting the chemotaxis and phagocytosis of inflammatory cells (such as leukocytes) and reducing the production of inflammatory factors. In terms of immunosuppression, cortisol can inhibit the proliferation and activity of lymphocytes and reduce antibody production.
[0005] In terms of secretory regulation, cortisol synthesis and secretion are regulated by the negative feedback mechanism of the hypothalamic-pituitary-adrenal cortex axis. When blood cortisol levels rise to a certain level, they exert a negative feedback inhibitory effect on the hypothalamus and pituitary gland, preventing excessive cortisol secretion. For example, when the adrenal cortex secretes too much cortisol, it inhibits the secretion of CRH from the hypothalamus and ACTH from the anterior pituitary gland, thereby reducing further cortisol secretion and maintaining a relatively stable cortisol level in the body.
[0006] Cortisol levels in the blood exhibit cyclical changes. They peak in the early morning, gradually decrease during the day, and reach their lowest point at night, approximately half the peak level. Therefore, the time of blood collection needs to be considered when analyzing test results.
[0007] Measuring blood cortisol levels can be used to diagnose disorders of the adrenal glands, pituitary gland, and hypothalamus. Excessive cortisol secretion can cause Cushing's syndrome, in which patients may experience significant weight gain in a short period, develop a round face, relatively thin limbs, and are prone to fractures. Insufficient cortisol secretion can cause Addison's disease, in which patients, due to a lack of sufficient cortisol to cope with stress such as infection, are prone to serious conditions such as shock.
[0008] Therefore, effective detection of cortisol plays a crucial role in diagnosing related diseases. Currently, cortisol assays mostly employ LCMS and immunoassay methods. While LCMS is accurate and sensitive, it is expensive and requires high-precision, costly equipment, making it difficult for most laboratories to implement. Immunoassays, on the other hand, are low-cost and convenient, making them suitable for widespread application. However, because cortisol is a small molecule compound with only one antibody binding site, most immunological detection methods on the market are competitive methods.
[0009] This invention provides raw materials and reagents for the sandwich method of cortisol detection. Compared with competing methods, the sandwich method has higher sensitivity, better specificity, and a wider detection range. There is a strong demand in the field for sandwich method detection of small molecule cortisol. However, there is a lack of antibodies in the prior art that can be used for sandwich method detection of small molecule cortisol. Summary of the Invention This invention provides a cortisol complex antibody and its uses, which can effectively solve the above-mentioned problems.
[0010] A cortisol complex antibody, the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1-3; the amino acid sequences of its light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4-6. In some embodiments, the cortisol complex antibody has the amino acid sequence of its heavy chain variable region as shown in SEQ ID NO:7 and the amino acid sequence of its light chain variable region as shown in SEQ ID NO:8.
[0011] In some embodiments, the heavy chain amino acid sequence of the cortisol complex antibody is shown in SEQ ID NO:9; and the light chain amino acid sequence is shown in SEQ ID NO:10.
[0012] A reagent for detecting cortisol, comprising the cortisol complex antibody.
[0013] A kit for detecting cortisol, comprising the cortisol complex antibody described above.
[0014] A non-disease diagnostic and therapeutic detection method for cortisol, comprising using the aforementioned cortisol complex antibody to perform sandwich immunoassay for cortisol.
[0015] The application of the cortisol complex antibody described above in the preparation of cortisol detection reagents.
[0016] A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the cortisol complex antibody described above.
[0017] An expression vector comprising the aforementioned nucleic acid molecule.
[0018] A host cell comprising the expression vector described above.
[0019] The beneficial effects of this invention are: The cortisol complex antibody of the present invention can be used to prepare a cortisol sandwich assay reagent for detecting clinical samples. The sensitivity, linear range, and specificity of the assay are all better than those of competing assay reagents. Attached Figure Description
[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.
[0021] Figure 1 Image showing the purification process of cortisol complex antibody 10D1.
[0022] Figure 2 The graph shows the activity of 10D1 antibody detected by ELISA.
[0023] Figure 3 Linearity graph for clinical detection of cortisol using the immunofluorescence sandwich assay. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.
[0025] Example 1: Preparation of anti-cortisol complex antibody The reagents or kits involved in this application and their sources are as follows: 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-Streptomycin (Catalog No. 15140122, Gibco); HRP-labeled Goat Anti-Mouse Antibody (Catalog No. D110087, Shanghai Sangon Biotech); Protein A Resin (Catalog No. SA023010, Changzhou Tiandi Renhe Biotechnology Co., Ltd.)
[0026] 1. Immunogen preparation Weigh 100 mg of cortisol and dissolve it in 1 mL of methanol. Weigh 100 mg of carboxymethoxyamine hemihydrochloride and dissolve it in 1 mL of pure water. Add the cortisol solution dropwise to the carboxymethoxyamine hemihydrochloride solution at room temperature while stirring vigorously. A white precipitate forms immediately. Add 5 mL of water, shake, and centrifuge to collect the precipitate. The precipitate is cortisol-3-(O-carboxymethyl)oxime.
[0027] Dissolve the above-mentioned cortisol-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 cortisol-3-(O-carboxymethyl)oxime.
[0028] Take cortisol antibody Ab1 (purchased from HaiTai Biotechnology, catalog number 2C2 / 2C2cc), and add activated cortisol-3-(O-carboxymethyl) oxime dropwise at a molar ratio of 1:24. React overnight at 4°C. After the reaction, remove unreacted free cortisol derivatives by ultrafiltration. This is the immunogen.
[0029] 2. Mouse immunization The above immunogen was dissolved and emulsified with an equal volume of Freund's Adjuvant, Complete (Thermo Fisher, catalog number: 77140). 6-8 week old SPF-grade Balb / c mice (Fuzhou Wu's Animal Experiment Center) were injected subcutaneously at multiple sites with 200 μg / mouse. Two weeks later, an equal volume of Freund's Adjuvant, Incomplete (Thermo Fisher, catalog number: 77145) was used to emulsify the antigen, and 100 μg / mouse was injected subcutaneously at multiple sites. Two booster immunizations were administered. Three days before fusion, 100 μg / mouse was injected intraperitoneally for a shock immunization.
[0030] 3. Preparation of feeder cells Balb / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and then, under aseptic conditions in a laminar flow hood, the abdominal skin was cut open to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium containing 1% penicillin-streptomycin was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, and the rinse fluid 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 (RPMI 1640 basal medium containing 10% fetal bovine serum and 1% penicillin-streptomycin). The cell concentration was adjusted to 1×10⁶ cells / mL. 5 Add 150 μL / well to a 96-well plate and incubate overnight at 37°C with 5% CO2.
[0031] Penicillin-Streptomycin: Gibco, Product No.: 15140122.
[0032] RPMI1640 basic culture medium: Shanghai Yuanpei Biotechnology, catalog number: L210KJ.
[0033] HAT culture medium: HAT Media Supplement (50×), Thermo Fisher, catalog number: 21060017.
[0034] 4. Preparation of immune spleen cells Three days after the last immunization of the mice, the spleens were removed under sterile conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, and then ground and filtered on a nylon mesh (the nylon mesh was placed in a small beaker) to prepare a cell suspension. The cells were centrifuged, the supernatant was discarded, and the suspension was resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and the cells were counted.
[0035] 5. Cell fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium (Shanghai Yuanpei Biotechnology, catalog number: L310KJ) and 1 mL of 50% PEG (M12000, Sigma, catalog number: P7181) and place them in a 37°C water bath for preheating; (2) Take mouse myeloma cells Sp2 / 0 (2×10⁻⁶) respectively 7 Add the above-mentioned immune spleen cell suspension (1×10⁸ cells) 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. (3) Place the centrifuge tube in water preheated to 37°C, take 0.7 mL of 50% PEG solution preheated to 37°C, and let it stand for 90 seconds. Immediately add 15 mL of serum-free culture medium preheated to 37°C; (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum (FBS). Mix well with a pipette and add 2 drops to each well of the 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.
[0036] Fetal bovine serum: Shanghai Xiaopeng Biotechnology, product number: C04001-500.
[0037] 6. Selective culture of hybridoma cells On days 1, 3, 5, and 7 after cell fusion, the culture medium was changed with HAT medium containing 15%-20% fetal bovine serum. The cells that survived were hybridoma cells, while the non-hybridoma cells died, and the true hybridoma cells were selected.
[0038] 7. Detection of specific antibodies and cloning of hybridoma cells Supernatant from each culture well was aspirated, and indirect ELISA was used to detect wells containing a complex that specifically recognizes cortisol antibodies and small molecule cortisol, while not recognizing cortisol antibodies. Cell lines with an OD490 difference greater than 1.5 between the ELISA results recognizing the complex and those recognizing the individual antibody were screened. The day before cloning, feeder cells were prepared and plated according to step 3. The reactive antibody cell lines were mixed using a pipette, and the cells in each well were diluted to one cell per well using HT medium. The cells were incubated at 37°C and 5% CO2 for 7-10 days. Antibody detection was initiated when visible clones appeared. Wells with only a single clone were marked under an inverted microscope. Finally, the monoclonal antibody hybridoma cell line 10D1 was preliminarily obtained through screening.
[0039] 8. Antibody sequencing Cloned 10D1 antibody cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 10D1 was obtained as follows: Heavy chain CDR1 sequence: PSTQNL (SEQ ID NO:1) Heavy chain CDR2 sequence: GLETPQWCAH (SEQ ID NO:2) Heavy chain CDR3 sequence: MSFEPCLAN (SEQ ID NO:3) Light chain CDR1 sequence: QSALIPDF (SEQ ID NO:4) Light chain CDR2 sequence: DYVSRK (SEQ ID NO:5) Light chain CDR3 sequence: WPLQEIR (SEQ ID NO:6) The heavy chain variable region sequence is: QVQLQQPGAELVKPGASVKMSCKASGYNFPSTQNLWVKQRPGRGLEWIGGLETPQWCAHKATLTVDKSSSTAYIQLSGLASEDSAVYYCTRMSFEPCLANWGQGTTLTVSS (SEQ ID NO:7) The light chain variable region sequence is: DIVMTQSTSSLSVSLGDRVTITCQSALIPDFWYQQKPDGTVKLLIYDYVSRKVPSRFSASGSGTDYSLTISNLEQEDFATYFCWPLQEIRFGGGTKLEIK (SEQ ID NO:8) The antibody heavy chain sequence is as follows: QVQLQQPGAELVKPGASVKMSCKASGYNFPSTQNLWVKQRPGRGLEWIGGLETPQWCAHKATLTVDKSSSTAYIQLSGLASEDSAVYYCTRMSFEPCLANWGQGTTLTV SSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ IDNO:9) The antibody light chain sequence is: DIVMTQSTSSLSVSLGDRVTITCQSALIPDFWYQQKPDGTVKLLIYDYVSRKVPSRFSASGSGTDYSLTISNLEQEDFATYFCWPLQEIRFGGGTKLEIKRTD AAAPVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:10) 9. Antibody expression and purification Balb / c mice were injected intraperitoneally with 0.5 ml of liquid paraffin. Ten days later, the selected hybridoma cell lines were inoculated with 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 was extracted in a single procedure.
[0040] Ascites supernatant was purified by protein A affinity chromatography, following the Cytiva handbook: Affinity Chromatography, Vol. 1: Antibodies. Anti-cortisol complex monoclonal antibody 10D1 was prepared from the purified supernatant. The purified SDS-PAGE image is shown below. Figure 1 The purified antibody has clear and single heavy and light chain bands, indicating high purity.
[0041] 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.
[0042] Example 2: ELISA detection of cortisol complex antibody activity 1. Coat each well with 100 ng of cortisol Ab1 antibody (purchased from HaiTai Biotechnology, catalog number 2C2cc, mouse anti-IgG2a) and 100 ng of cortisol Ab1 + 10 ng of cortisol small molecule complex, and coat the well with coating solution (10 mM phosphate buffer, pH 7.4, pH 9.4). Cover the wells and incubate overnight at 2–8°C.
[0043] 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.
[0044] 3. At room temperature, add 200 µL of blocking buffer (10% skim milk) to each well and block for 1 hour.
[0045] 4. Suction, invert the perforated plate and gently pat it on absorbent paper to remove residual liquid.
[0046] 5. Add 100 μL to the blocking buffer to dilute 10D1 solutions of different concentrations. Incubate at room temperature for 1 hour.
[0047] 6. Aspirate the liquid from each well and wash 6 times with 200 µL of washing buffer to each well.
[0048] 7. Add 50-100 μL of HRP-labeled goat anti-mouse antibody (catalog number D110087, Shanghai Sangon Biotech) diluted 10,000 times to each well, incubate at 37°C for 30 min, and wash 6 times.
[0049] 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.
[0050] 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 .
[0051] Table 1. ELISA results of 10D1 with cortisol antibody Ab1 and cortisol antibody Ab1 + cortisol complex
[0052] From Table 1 and Figure 2 Looking at the results: Cortisol 10D1 antibody and the complex antibody showed almost no binding. As the concentration of the complex increased, OD450 did not increase significantly. However, the complex antibody of cortisol Ab1 antibody and cortisol showed good linear correlation with the complex antibody, indicating that the complex antibody specifically binds to the complex antibody of cortisol Ab1 antibody and cortisol and has good activity. It can be used for the development of the cortisol reagent sandwich method.
[0053] Example 3: 1. Cortisol complex antibody 10D1 used in immunofluorescence reagents Cortisol complex antibody 10D1 was streaked onto a nitrocellulose membrane at a dosage of 0.2 μg / cm as the T line; cortisol hapten Cor-BSA (purchased from Modis, catalog number LA790) was streaked onto the nitrocellulose membrane at a dosage of 0.3 μg / cm as the C line. The membrane was dried at 55℃ for 4 days. Simultaneously, cortisol Ab1 antibody was conjugated to fluorescent microspheres and sprayed onto a sample pad at a dosage of 0.1 μg cortisol Ab1 antibody / cm binding pad. The microspheres were then vacuum-dried for 3 hours to assemble the test strips. Subsequently, different concentrations of clinical samples (serum samples) were added to the strips at room temperature and humidity (25℃, 50%-60% humidity), and the fluorescence values were read. The results are shown in Table 2 below. Figure 3 .
[0054] Table 2. Clinical detection results of cortisol using the immunofluorescence sandwich method
[0055] From Table 2 and Figure 3 As shown: the sandwich immunoassay reagent composed of cortisol complex antibody and cortisol Ab1 showed that the fluorescence value increased with increasing sample concentration, indicating high detection sensitivity and good overall linearity in clinical samples ranging from 1 to 600 ng / mL.
[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.
Claims
1. A cortisol complex antibody, characterized in that, The amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1-3; The amino acid sequences of its light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4-6.
2. The cortisol complex antibody according to claim 1, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:7; the amino acid sequence of its light chain variable region is shown in SEQ ID NO:
8.
3. The cortisol complex antibody according to claim 1, characterized in that, The amino acid sequence of its heavy chain is shown in SEQ ID NO:9; the amino acid sequence of its light chain is shown in SEQ ID NO:
10.
4. A reagent for detecting cortisol, characterized in that, Includes the cortisol complex antibody as described in any one of claims 1 to 3.
5. A kit for detecting cortisol, characterized in that, Includes the cortisol complex antibody as described in any one of claims 1 to 3.
6. A method for detecting cortisol in a non-disease diagnostic and therapeutic context, characterized in that, The cortisol complex antibody according to any one of claims 1 to 3 is used to perform sandwich immunoassay for cortisol.
7. The use of the cortisol complex antibody according to any one of claims 1 to 3 in the preparation of cortisol detection reagents.
8. A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the cortisol complex antibody according to any one of claims 1 to 3.
9. An expression vector comprising the nucleic acid molecule of claim 8.
10. A host cell comprising the expression vector of claim 9.