Monoclonal antibody of folic acid and folic acid receptor protein compound and application thereof

Abstract

The invention provides a monoclonal antibody of a folic acid and folic acid receptor protein compound and application of the monoclonal antibody. The sequences of heavy chain variable regions CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and the sequences of light chain variable regions CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. The antibody can be used for double sandwich method detection of folic acid, and has good detection sensitivity and detection linear range.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody of a folic acid-folic acid receptor protein complex and its application, belonging to the field of antibody technology. Background Technology

[0002] Folic acid, also known as vitamin B9, is a water-soluble vitamin composed of three parts: pteridine, para-aminobenzoic acid, and L-glutamic acid. It is also called pteroylglutamic acid. It was named folic acid because it was first discovered in spinach leaves.

[0003] Folic acid can be transported into cells through multiple pathways, among which the folate receptor 1 protein (FOLR1) has high affinity and can bind folate in the nanomolar range. Folate receptor 1 protein (FOLR1) is also known as folate receptor α or folate binding protein (FBP).

[0004] Folic acid plays a vital role in nervous system development, DNA synthesis, erythrocyte production, and amino acid metabolism. Folic acid deficiency can lead to megaloblastic anemia, cardiovascular disease, and neural tube defects in fetuses. Therefore, routine monitoring and testing of folic acid levels is of great importance.

[0005] Current folic acid test kits typically use a competitive method, where a folic acid conjugate (such as folic acid-BSA) competes with folic acid in the sample for binding sites on the folic acid receptor / antibody, thus reflecting the folic acid concentration in the sample. This method usually suffers from low sensitivity and a narrow detection range, necessitating the search for new alternative detection methods. Summary of the Invention This invention provides a monoclonal antibody of folic acid and folic acid receptor protein complex and its application, which can effectively solve the above problems.

[0006] A monoclonal antibody of a folic acid-folic acid receptor protein complex, wherein the sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. In some embodiments, the monoclonal antibody of the folic acid-folic acid receptor protein complex has a heavy chain variable region sequence as shown in SEQ ID NO:5 and a light chain variable region sequence as shown in SEQ ID NO:9.

[0007] In some embodiments, the monoclonal antibody of the folic acid and folic acid receptor protein complex has a heavy chain sequence as shown in SEQ ID NO:3 and a light chain sequence as shown in SEQ ID NO:4.

[0008] A folic acid assay kit comprising the monoclonal antibody described above.

[0009] A folic acid assay kit comprising the monoclonal antibody described above.

[0010] A non-disease diagnostic and therapeutic detection method for folic acid, comprising using the aforementioned monoclonal antibody to perform immunoassay for folic acid.

[0011] In some embodiments, the immune detection method is a sandwich method.

[0012] The application of the monoclonal antibody described above in the preparation of folic acid detection reagents.

[0013] The beneficial effects of this invention are: This invention provides a monoclonal antibody Ab1 that can recognize newly formed epitopes after FOLR1 binds to folic acid, enabling double-sandwich detection of folic acid.

[0014] The monoclonal antibody of the folic acid-folate receptor protein complex of the present invention exhibits high specificity, binding tightly only to the complex formed by folic acid and the folate receptor protein, without interacting with the folate receptor protein alone. This characteristic makes the monoclonal antibody particularly suitable for the double-sandwich assay of folic acid, enabling precise identification and quantification of the folic acid-folate receptor protein complex in complex biological samples, thereby ensuring the accuracy and reliability of the test results. Furthermore, the antibody demonstrates excellent sensitivity during the detection process, capable of detecting extremely low concentrations of the folic acid-folate receptor protein complex, while also exhibiting an extremely broad linear detection range, covering a wide range from low to high concentrations, providing a powerful tool for clinical diagnosis and research. Attached Figure Description

[0015] 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.

[0016] Figure 1 The image shows the purification results of FOLR1 in Example 1.

[0017] Figure 2 This is a linear graph of Ab1 activity detected by ELISA in Example 2.

[0018] Figure 3 This is a linear graph of the clinical detection using immunofluorescence in Example 3. Detailed Implementation

[0019] 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.

[0020] The materials used in the embodiments of this invention are as follows: 293F cells were purchased from Thermo Fisher Scientific; SMM 293-TII medium was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; SMS 293-SUPI feed medium was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; 6-8 week old SPF-grade Balb / c mice were purchased from Fuzhou Wu's Animal Experiment Center; RPMI 1640 basal culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; HAT culture medium was purchased from Sigma; DMEM serum-free culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.; goat anti-mouse antibody was purchased from Sigma.

[0021] Example 1: Preparation of Ab1 monoclonal antibody 1. Preparation of recombinant FOLR1 The amino acid sequence encoding FOLR1 (SEQ ID NO:1) was obtained from NCBI: CAG46816.1. A His tag was added to the N-terminus, and the nucleotide sequence was optimized according to human gene codon preferences (SEQ ID NO:2). The sequence was then synthesized into the pTT5 vector by Suzhou Genewise.

[0022] SEQ ID NO:1 MAQRMTTQLLLLLVWVAVVGEAQTHHHHHHRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS SEQ ID NO:2 ATGGCACAAAGAATGACAACACAGCTGCTGCTCCTGCTTGTATGGGTGGCGGTGGTCGGTGAAGCGCAGACACACCATCACCATCATCACAGAATAGCCTGGGCTCGAACCGAGTTGTTGAATGTGTGCATGAACGCCAAGCACCACAAAGAAAAGCCGGGGCCAGAAGACAAGCTGCACGAGCAGTGCAGACCATG GAGGAAGAATGCCTGTTGCTCAACCAACACAAGCCAGGAGGCCCATAAGGATGTTAGTTATCTGTACCGATTCAATTGGAATCATTGTGGGGAAATGGCACCTGCCTGCAAGCGCCATTTCATTCAAGACACCTGTCTGTACGAGTGCAGCCCCAATTTGGGCCCCTGGATACAACAGGTGGATCAGTCTTGGAGAA AAGAGAGAGTGTTGAATGTGCCACTCTGTAAGGAAGATTGCGAACAGTGGTGGGAGGATTGTCGGACCTCCTACACTTGTAAATCAAATTGGCACAAGGGCTGGAATTGGACAAGTGGCTTTAACAAGTGTGCAGTCGGTGCAGCATGCCAGCCTTTTCACTTCTATTTCCCCACTCCGACCGTTCTGTGCAACGAG ATCTGGACCCACAGCTACAAGGTGTCCAATTATTCACGGGGGTCTGGAAGGTGTATCCAAATGTGGTTTGACCCTGCCCAGGGTAATCCGAATGAGGAGGTCGCTCGATTTTATGCAGCAGCTATGAGTGGAGCCGGCCCCTGGGCCGCTTGGCCATTTCTTTTGTCTCTCGCACTGATGCTGCTGTGGCTGCTTTCT The constructed pTT5-FOLR1 plasmid was transfected into 293F cells using the PEI transfection method. After successful transfection, the cells were cultured in a shaker at 37°C, 8% CO2, and 120 rpm for 24 h. Then, 3.5% (v / v) feed medium was added, and the cells were returned to the shaker for another 96 h to obtain 293F cell culture medium expressing FOLR1.

[0023] Centrifuge at 12,000 rpm for 30 min at 4°C, collect the supernatant, and filter the cell supernatant through a 0.22 μm filter membrane. Equilibrate the NiFF column with Buffer A (20 mM PB, pH 7.4), and load the cell supernatant onto the NiFF column for purification. After loading, wash with Buffer A to equilibrate, elute with Buffer B (20 mM PB, 10 mM imidazole, pH 7.4) to remove contaminating proteins, and elute the target protein with Buffer C (20 mM PB, 200 mM imidazole, pH 7.4). Ultrafilter the purified FOLR1, concentrate, and store in PBS pH 7.4. Purification results are shown in [Figure number missing]. Figure 1 .

[0024] 2. Mouse immunization Folic acid and FOLR1 were mixed at a molar ratio of 2:1 and incubated at 37°C for 1 hour to form a folic acid-FOLR1 complex. This complex was then emulsified with an equal volume of Freund's complete adjuvant. 6-8 week old SPF-grade Balb / c mice were injected subcutaneously at multiple sites with 200 μg per mouse. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites with 100 μg per mouse. Two booster immunizations were administered. Three days before fusion, a pulse immunization was administered intraperitoneally.

[0025] 3. Preparation of feeder cells BALB / c mouse peritoneal macrophages were used as feeder cells. One day before cell fusion, BALB / c mice were euthanized by cervical dislocation and then immersed in 75% alcohol. Under aseptic conditions in a laminar flow hood, the abdominal skin was incised to expose the peritoneum. Then, 5 mL of RPMI 1640 basal culture medium was injected into the peritoneal cavity using a syringe. After repeated rinsing, the rinsing fluid was collected. The rinsing fluid was centrifuged at 1000 rpm for 5 minutes, and the pellet was collected. The pellet was resuspended in RPMI 1640 complete culture medium containing HAT, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 The sample was collected at a concentration of 150 μL / mL and then added to a 96-well plate. The plate was then incubated overnight at 37°C with 5% CO2.

[0026] 4. Preparation of immune spleen cells Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a petri dish, and rinsed once with RPMI 1640 basal culture medium. Then, the spleen was ground and filtered through a nylon mesh in a small beaker to prepare a cell suspension. The cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and finally, cell counting was performed.

[0027] 5. Cell fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12000) respectively, and place them in a 37°C water bath for preheating.

[0028] (2) Take mouse myeloma cells Sp2 / 0 (2 - 5 × 10⁻⁵) respectively. 7 (10) of the above-mentioned immune spleen cells 8 Add the suspension to a 50mL centrifuge tube and mix well. Then add DMEM serum-free culture medium to a final volume of 40mL. Centrifuge for 10 minutes, discard the supernatant, and mix again.

[0029] (3) Place the centrifuge tube in water preheated to 37°C, take 0.7 mL of preheated 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of serum-free culture medium preheated to 37°C.

[0030] (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. Mix well with a pipette and add 2 drops to each of the four wells of a 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.

[0031] 6. Selection, culture, and clone screening of hybridoma cells On days 1, 3, 5, and 7 after cell fusion, the cells were cultured in the aforementioned HAT medium for medium replacement to screen for true hybrid cells. Subsequently, three rounds of subcloning were performed to screen for single clones that could specifically recognize the folic acid and FOLR1 complex, resulting in a total of 10 clones.

[0032] 7. Expression and purification of ascites fluid Balb / c mice were injected intraperitoneally with 0.5 mL of liquid paraffin. Ten days later, the selected hybridoma cell lines (1×10⁻⁶) were... 6 (Number of mice) were inoculated intraperitoneally into Balb / c mice. After approximately 10 days, the mice's abdomens began to swell. At this point, the mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and ascites fluid was extracted once. The ascites fluid was purified using a Protein A affinity column to obtain the monoclonal antibody. Example 2: Determining the detection linear range and sensitivity using ELISA Dissolve FOLR1 at a concentration of 1 μg / ml in 20 mM PB pH 7.4 coating buffer, add 100 μL (i.e., 100 ng / well) to the corresponding well, and incubate overnight at 4°C. Aspirate the liquid from the wells and wash three times with 300 μL of washing buffer.

[0033] Add 300 μL of blocking buffer (5% skim milk powder, prepared with PBST) to each well and incubate at 37°C for 1 hour. Aspirate the liquid from the wells and wash three times with 300 μL of washing buffer.

[0034] Add 100 μL of serially diluted folic acid sample (2-fold) to each well and incubate at 37°C for 1 h. Aspirate the liquid from the wells, wash three times with 300 μL of wash buffer, and then add 100 μL of 1 μg / ml detection antibody to each well.

[0035] Remove the liquid from the wells, wash three times with 300 μL of washing buffer, then add 100 μL of HRP-labeled rabbit anti-mouse antibody (1:5000, diluted with PBST) to each well and incubate at 37°C for 30 min.

[0036] Remove the liquid from the wells and wash five times with 300 μL of washing buffer. Add 100 μL of chromogenic solution to each well and incubate at 37°C for 10 min. Then add 50 μL of 2 mol / L H2SO4 stop solution. Within 20 min after adding the stop solution, read the OD450 value using a microplate reader.

[0037] The obtained monoclonal antibody and FOLR1 were used as the detection antibody and coating material, respectively. A double-sandwich ELISA method was used to detect diluted folic acid samples. Finally, an antibody with the best detection range and sensitivity was selected and named Ab1. The detection results are shown in Table 1 and [Table data would be inserted here]. Figure 2 Within the range of 0.16–40 ng / mL, the paired detection of folic acid by Ab1 and FOLR1 showed a good linear relationship. Table 1

[0038] The sequence of the monoclonal antibody Ab1, after sequencing, is as follows: Heavy chain: EVQIMQPSADLLKPGTGVKLSCKASGYTFTGKFLTWVKQRPGQGLEWIGDIEWAESHLQYGDYKSKGHLTIEKSSGTGYMDLTALTSNDSAVRYCTRGGQEHTIEYPAQGTVLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO:3) Light chain: ELQMKNGPSALSASLGAKVTITCRGTNEIEKQLSWYETKPSKGPLLIWFVGHISPGIPSRFGTASVREYSFSLSDVEPEDIGTYYCVTKEQVPKTFGSAHNLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:4) Heavy chain variable region: EVQIMQPSADLLKPGTGVKLSCKASGYTFTGKFLTWVKQRPGQGLEWIGDIEWAESHLQYGDYKSKGHLTIEKSSGTGYMDLTALTSNDSAVRYCTRGGQEHTIEYPAQGTVLTVSS (SEQ ID NO:5) Heavy chain variable region CDR1: GKFLT (SEQ ID NO:6) Heavy chain variable region CDR2: DIEWAESHLQYGDYKS (SEQ ID NO:7) Heavy chain variable region CDR3: GGQEHTIEY (SEQ ID NO:8) Light chain variable region: ELQMKNGPSALSASLGAKVTITCRGTNEIEKQLSWYETKPSKGPLLIWFVGHISPGIPSRFGTASVREYSFSLSDVEPEDIGTYYCVTKEQVPKTFGSAHNLEIK (SEQ ID NO:9) Light chain variable region CDR1: RGTNEIEKQLS (SEQ ID NO:10) Light chain variable region CDR2: FVGHISP (SEQ ID NO:11) Light chain variable region CDR3: VTKEQVPKT (SEQ ID NO:12) Example 3: Ab1 and FOLR1 applied to immunofluorescence reagents Ab1 and folic acid antigen were applied to nitrocellulose membranes at concentrations of 0.04 μg / cm and 0.08 μg / cm, respectively, and dried at 55°C for 3 days. Simultaneously, FOLR1 was conjugated and labeled with fluorescent microspheres, and sprayed onto the sample pad at a concentration of 0.02 μg antibody / cm binding pad. The pads were then vacuum-dried for 3 hours to assemble the reagent strips.

[0039] Subsequently, clinical samples of different concentrations were added to an environment with normal temperature and humidity (25℃, 50%-60% humidity), and the fluorescence values ​​were read. The results are shown in Table 2 below. Figure 3 As shown, within the range of 0.7–50 ng / mL, the detection value increased with increasing sample concentration, exhibiting a good linear gradient. Table 2

[0040] 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 monoclonal antibody against a folic acid-folic acid receptor protein complex, characterized in that, The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

2. The monoclonal antibody against the folic acid-folic acid receptor protein complex according to claim 1, characterized in that, The heavy chain variable region sequence is shown in SEQ ID NO:5, and the light chain variable region sequence is shown in SEQ ID NO:

9.

3. The monoclonal antibody against the folic acid-folic acid receptor protein complex according to claim 1, characterized in that, Its heavy chain sequence is shown in SEQ ID NO:3, and its light chain sequence is shown in SEQ ID NO:

4.

4. A folic acid detection reagent, characterized in that, Includes the monoclonal antibody as described in any one of claims 1 to 3.

5. A folic acid detection kit, characterized in that, Includes the monoclonal antibody as described in any one of claims 1 to 3.

6. A method for detecting folic acid in non-disease diagnostic and therapeutic applications, characterized in that, The monoclonal antibody according to any one of claims 1 to 3 is used to perform an immunoassay for folic acid.

7. The detection method according to claim 6, characterized in that, The immunoassay method is the sandwich method.

8. The use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of folic acid detection reagent.

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