An antibody targeting tetrahydrocannabinol (THC) and its application in the detection of THC.

Abstract

This invention relates to the field of antibody technology, and in particular to an antibody targeting tetrahydrocannabinol (THC) and its application in the detection of THC. This invention utilizes site-directed mutagenesis and phage screening techniques to mutate and screen THC-specific antibodies, obtaining an antibody (denoted as D16H) with higher affinity for THC. The D16H antibody provided by this invention exhibits stronger binding affinity to THC, with a half-maximal binding concentration (EC50) of [missing value]. 50 The concentration was 11 ng / ml, compared to the original antibody A12 (EC). 50 The half-binding concentration (51 ng / ml) was significantly improved, which can be used to prepare a kit for detecting tetrahydrocannabinol.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to an antibody targeting tetrahydrocannabinol (THC) and its application in the detection of THC. Background Technology

[0002] Tetrahydrocannabinol (THC) is a resin naturally found in the cannabis plant (Cannabis sativa), a member of the mulberry family. It is secreted by the female flowers of the cannabis plant and can be chemically synthesized. Long-term use may lead to mental decline and severe loss of work ability. If a person uses THC for an extended period, a urine test will generally show a positive result for THC. This substance can be detected in bodily fluids and hair after ingesting cannabis products.

[0003] Tetrahydrocannabinol (THC) has a molecular weight of 314.462. Rapid tests typically use colloidal gold assays with specific antibodies to detect the presence of this compound in bodily fluids. As a small organic molecule, THC has a weak binding affinity to most specific antibodies. Currently available commercially available kits contain antibodies with relatively weak binding affinity to THC, with a detection threshold of approximately 50 ng / ml. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an antibody targeting tetrahydrocannabinol (THC) and its application in the detection of THC. The antibody provided by this invention exhibits strong binding affinity to THC, with a half-maximal binding concentration (EC50) of [missing value]. 50 The concentration was 11 ng / ml, compared to the original antibody A12 (EC). 50 The half-binding concentration (51 ng / ml) was significantly improved, which can be used to prepare a kit for detecting tetrahydrocannabinol.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an antibody targeting tetrahydrocannabinol (THC), comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a polypeptide having more than 90% similarity to the HCDR region, and the light chain variable region comprises a polypeptide having more than 90% similarity to the LCDR region. The HCDR region comprises HCDR1, HCDR2, and HCDR3, and the LCDR region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.1; The amino acid sequence of HCDR2 is shown in SEQ ID NO.2; The amino acid sequence of HCDR3 is shown in SEQ ID NO.3; The amino acid sequence of LCDR1 is shown in SEQ ID NO.4; The amino acid sequence of LCDR2 is shown in SEQ ID NO.5; The amino acid sequence of LCDR3 is shown in SEQ ID NO.6.

[0006] Preferably, the amino acid sequence of the heavy chain variable region includes an amino acid sequence having more than 90% similarity to SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes an amino acid sequence having more than 90% similarity to SEQ ID NO.9.

[0007] Preferably, the coding sequence of the heavy chain variable region comprises a nucleotide sequence having more than 90% similarity to SEQ ID NO.8.

[0008] Preferably, the coding sequence of the light chain variable region comprises a nucleotide sequence having more than 90% similarity to SEQ ID NO.10.

[0009] Preferably, the antibody subtype is IgG1; the light chain is Kappa type.

[0010] This invention provides the application of the antibody described in the above technical solution in 1) or 2): 1) Testing for tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes; 2) Prepare reagents or kits for detecting tetrahydrocannabinol.

[0011] This invention provides a kit for detecting tetrahydrocannabinol (THC), comprising the antibody described in the above technical solution.

[0012] Preferably, the kit also includes reagents used in colloidal gold assays or ELISA assays.

[0013] This invention provides a method for detecting tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes, comprising: Tetrahydrocannabinol (THC) in the sample was detected using a colloidal gold method or an ELISA assay; the antibody used was the antibody described in the above technical solution.

[0014] Preferably, the sample to be tested includes one or more of the following: body fluids, hair, blood, and urine.

[0015] Beneficial effects: This invention utilizes site-directed mutagenesis and phage screening techniques to mutate and screen THC-specific antibodies, resulting in an antibody (denoted as D16H) with higher affinity for THC. The D16H antibody provided by this invention exhibits stronger binding affinity to THC, with a half-maximal binding concentration (EC50) of [missing value]. 50 The concentration was 11 ng / ml, compared to the original antibody A12 (EC). 50The half-binding concentration (51 ng / ml) was significantly improved, which can be used to prepare a kit for detecting tetrahydrocannabinol. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results show the comparison of the affinity of each antibody for the antigen; Figure 2 This is a restriction enzyme digestion map of the VH multiple cloning site of the D16H antibody; Figure 3 This is the restriction enzyme digestion map of the VL multiple cloning site of the D16H antibody. Detailed Implementation

[0018] This invention provides an antibody targeting tetrahydrocannabinol (THC), wherein the heavy chain variable region comprises a polypeptide having more than 90% similarity to the HCDR region, and the light chain variable region comprises a polypeptide having more than 90% similarity to the LCDR region; the HCDR region comprises HCDR1, HCDR2, and HCDR3, and the LCDR region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.1; The amino acid sequence of HCDR2 is shown in SEQ ID NO.2; The amino acid sequence of HCDR3 is shown in SEQ ID NO.3; The amino acid sequence of LCDR1 is shown in SEQ ID NO.4; The amino acid sequence of LCDR2 is shown in SEQ ID NO.5; The amino acid sequence of LCDR3 is shown in SEQ ID NO.6.

[0019] In one embodiment, the amino acid sequence of the heavy chain variable region includes an amino acid sequence having more than 90% similarity to SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes an amino acid sequence having more than 90% similarity to SEQ ID NO.9.

[0020] In one embodiment, the coding sequence of the heavy chain variable region includes a nucleotide sequence having more than 90% similarity to SEQ ID NO.8.

[0021] In one embodiment, the coding sequence of the light chain variable region includes a nucleotide sequence having more than 90% similarity to SEQ ID NO.10.

[0022] In one embodiment, the antibody subtype is IgG1; the light chain is Kappa type.

[0023] In one embodiment, the heavy chain constant region of the antibody is shown in SEQ ID NO.11, and the light chain constant region of the antibody is shown in SEQ ID NO.12.

[0024] In one implementation, the present invention utilizes the Kabat antibody encoding system to identify the variable region of an antibody.

[0025] This invention utilizes site-directed mutagenesis and phage screening techniques to mutate and screen THC-specific antibodies, resulting in an antibody (denoted as D16H) with higher affinity for THC. The D16H antibody provided by this invention exhibits stronger binding affinity to THC, with a half-maximal binding concentration (EC50) of [missing value]. 50 The concentration was 11 ng / ml, compared to the original antibody A12 (EC). 50 The half-binding concentration (51 ng / ml) was significantly improved, which can be used to prepare a kit for detecting tetrahydrocannabinol.

[0026] Based on the above advantages, the present invention provides the application of the antibody described in the above technical solution in 1) or 2): 1) Testing for tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes; 2) Prepare reagents or kits for detecting tetrahydrocannabinol.

[0027] Based on the above advantages, the present invention provides a kit for detecting tetrahydrocannabinol (THC), comprising the antibody described in the above technical solution. As one embodiment, the kit further includes reagents used in colloidal gold assays or ELISA detection methods.

[0028] Based on the above advantages, the present invention provides a method for detecting tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes, comprising: Tetrahydrocannabinol (THC) in the sample was detected using a colloidal gold method or an ELISA assay; the antibody used was the antibody described in the above technical solution.

[0029] As one implementation method, the sample to be tested includes one or more of the following: body fluids, hair, blood, and urine.

[0030] The amino acid or nucleotide sequences involved in this invention are as follows: HCDR1: QYYIY, SEQ ID NO.1; HCDR2: GVQPDNGQTTFNDKFRT, SEQ ID NO.2; HCDR3: YEAF, SEQ ID NO.3; LCDR1:RASNEIGSSLQ, SEQ ID NO.4; LCDR2: GTSSLDT, SEQ ID NO.5; LCDR3: LQFASSPYT, SEQ ID NO.6; The amino acid sequence of the heavy chain variable region of D16H is shown in SEQ ID NO.7, as follows: ELVKPGTSVRLSCKASGFTFTQYYIYWVKQRPGQGLEWIGGVQPDNGQTTFNDKFRTKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCTIYEAFWGQGTSVTVSS; The nucleotide sequence of the heavy chain variable region of D16H is shown in SEQ ID NO.8, as follows: GAGCTGGTTAAACCCGGCACGAGCGTGCGCCTTTCCTGTAAAGCCTCAGGGTTCACCTTTACCCAGTACTACATCTATTGGGTGAAACAGCGCCCTGGTCAGGGACTTGAGTGGATTGGCGGGGTCCAGCCTGACAATGGACAAACGACCTTCAAC GACAAATTCAGGACGAAGGCCACCCTGACAGTTGACAAGTCTTCTTCCACTGCCTACATGCAACTGTCATCACTGACCAGTGAAGACTCCGCGGTGTATTATTGCACGATCTATGAGGCCTTTTGGGGTCAAGGTACCTCTGTCACCGTGAGTTCC.

[0031] The amino acid sequence of the light chain variable region of D16H is shown in SEQ ID NO.9, as follows: DIQMTQSPSSLSASLGERVSLTCRASNEIGSSLQWLQQEPDGTIKRLIYGTSSLDTGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQFASSPYTFGGGTKLEIR; The nucleotide sequence of the light chain variable region of D16H is shown in SEQ ID NO.10, as follows: GATATCCAGATGACACAGAGCCCTTCTAGCCTCTCAGCTTCTCTGGGTGAACGCGTGAGCCTTACCTGCCGGGCCTCCAATGAGATTGGGTCCAGTCTCCAGTGGCTGCAACAGGAGCCAGATGGCACCATTAAGCGACTGATCTATGGAACTAGCAGCCTGGACACAGGCGTGCCCAAGAGATTTTCAGGGTCTCGCAGCGGATCCGATTACTCTCTGACCATTTCATCCCTGGAATCCGAAGACTTCGTGGATTATTACTGCCTGCAGTTCGCATCTTCACCTTACACCTTTGGGGGGGGGACTAAGTTGGAGATACGG.

[0032] The heavy chain constant region of D16H antibody is shown in SEQ ID NO. 11, which is specifically as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0033] The light chain constant region of D16H antibody is shown in SEQ ID NO. 12, which is specifically as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes an antibody targeting tetrahydrocannabinol (THC) and its application in the detection of THC, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Parental antibody A12: BALB / c mice were immunized with THC antigen (Biodragon; catalog number: ZD6538). Following routine hybridoma cell fusion and limiting dilution subcloning, a hybridoma cell line stably secreting anti-THC specific antibodies was obtained. The secreted anti-THC specific antibody is designated A12. The amino acid sequence of the heavy chain variable region of A12 is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region of A12 is shown in SEQ ID NO.14, as detailed below: SEQ ID NO.13: ELVKPGTSVRLSCKASGFTFTDYYVYWVKQRPGQGLEWIGGIYWDNSQTTFNDKFRTKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCTIGLAFWGQGTSVTVSS; SEQ ID NO.14: DIQMTQSPSSLSASLGERVSLTCRASNEFGSSLQWLQQEPDGTIKRLIYGTSSLDTGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQFAGSPYTFGGGTKLEIR.

[0036] Using the heavy chain variable region (VH) and light chain variable region (VL) encoding genes of parental antibody A12 as templates, degenerate primer site-directed mutagenesis technology was employed to introduce random base mutations into key antigen-binding regions of the heavy chain CDR1 / CDR3 and light chain CDR2 / CDR3. A full-length single-chain antibody (scFv) fragment was then assembled via overlap extension PCR. The nucleotide sequence is shown in SEQ ID NO.15, as follows: .

[0037] The scFv fragment was linearized and ligated into the pCANTAB5e phage vector using the In-fusion Seamless Cloning Kit. The ligation product was electroporated into E. coli TG1 competent cells, and the library volume was determined by serial dilution and plating. The final library volume was 3.2 × 10⁻⁶. 9 CFU scFv mutant phage library. After library amplification, M13KO7 helper phage was added for superinfection, and the library was cultured overnight to prepare the primary phage library stock solution.

[0038] The ELISA experimental procedure is as follows: 1) Purchased THC-BSA (Biodragon; item number: ZD6538); 2) Coat THC-BSA in a 96-well plate at 100 ng / well and incubate overnight at 4°C; 3) Wash the plate three times with PBST; block with 5% BSA-PBS; 4) Dilute the parental antibody A12 and the test antibody in 8 serial dilutions from 50 ng / ml, add 50 μl / well to a 96-well plate, and incubate at 37℃ for 1 h. 5) Wash the plate three times with PBST; 6) Add HRP-labeled goat anti-human IgG (Shanghai Sangon Biotech) and incubate at 37°C for 1 h; 7) Wash the plate three times with PBST; 8) Develop color using 50 μl of TMB colorimetric solution per well, and incubate at room temperature for 5 min; 9) Microplate reader reading at 450 nm.

[0039] Four-wheel solid phase pressure screening solution: Round 1: 96-well ELISA plates were coated with 1 μg / well of THC-BSA antigen, blocked, and then incubated with phage stock solution. The plates were washed 5 times with PBST to elute the bound phage and then infected with TG1 bacteria for amplification. Rounds 2-4: Gradually reduce the amount of coating antigen, from 500 ng to 200 ng to 100 ng / well, while simultaneously increasing the number of PBST washes (from 8 to 12 to 15) for pressure screening, gradually eliminating low-affinity phage clones. The screening enrichment rate is shown in Table 1.

[0040] Table 1. Enrichment results after four rounds of screening

[0041] After four rounds of screening, 96 monoclonal phage strains were randomly selected and soluble scFv was prepared for indirect ELISA affinity screening. Secondary screening was performed by determining the half-conjugation concentration (EC50) using quantitative ELISA. 50 Three antibodies, D16H, D21C, and G15E, were obtained through screening. The optimal mutant clone was ultimately determined to be D16H, with its EC50... 50 =11 ng / mL, compared to the original parent A12 (EC) 50 =51 ng / mL), antigen-binding affinity increased by approximately 4.6 times. ELISA results for different antibodies are shown in Table 2. Comparison of the affinity of each antibody for the antigen is shown in [Table 2]. Figure 1 .

[0042] Table 2. ELISA results of different antibodies

[0043] The heavy chain variable region of D21C (SEQ ID NO.16): QVQKPGTSVRLSCKASGFTFTQYYSYWVKQRPGQGLEWIGGVQPDNGQTTFNDKFRTKATLTVDKSSSTAYMQLYSLTSEDSAVYYCMIYEGFWGQGTSVTVSS; The light chain variable region of D21C (SEQ ID NO.17): DIQMTQSPSSLSAPLGERVSFTCRASNEIGSSLQWLQQEPDGTIKRLISGTSGLDTGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQFASGPYTFGGGTKLEIR; The heavy chain variable region of G15E (SEQ ID NO.18): ELVKPGTSVRLSCKASGFTFTQYYIYWVKQRPGQGLEWIGGVQKDNGQTTFNDKFRTKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCTIYEAFWGQGTSVTVSS; The light chain variable region of G15E (SEQ ID NO.19): DIQMTQSPSSLSASLGERVSLTCRASNEFGSSLQWLQQEPDGTIKRLIYGTSSLDTGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQFASGPYTFGGGTKLEIR.

[0044] Example 2 The heavy and light chain nucleic acid sequences of the phage-derived D16H single-chain antibody were determined by Sanger gene sequencing, and the gene was synthesized. The sequences are as follows: The nucleotide sequence of the heavy chain of the D16H single-chain antibody is shown in SEQ ID NO.20, as follows: The nucleotide sequence of the light chain of the D16H single-chain antibody is shown in SEQ ID NO.21, as follows: .

[0045] The nucleic acid sequences of the heavy and light chains were respectively processed... EcoR I and BamH The I site was constructed into two pTT5 vectors, resulting in two recombinant plasmids.

[0046] After sequencing verification confirmed the recombinant plasmid was free of mutations and endotoxins, the plasmid was extracted. Following a plasmid:PEI transfection reagent ratio of 1:3 (w / w), the light and heavy chain plasmids were co-transfected into suspension-cultured HEK293F cells at a molar ratio of 1:1. The initial cell culture density was 2 × 10⁶ cells / year. 6Cells / mL were cultured in FreeStyle 293 serum-free medium at 37°C and 120 rpm with shaking. On day 3 after transfection, 5% glucose was added as a nutrient supplement, and the cells were cultured for 5 days. Afterward, the cells were centrifuged at 8000 rpm and 4°C for 30 min to remove the cell pellet, and the supernatant was collected. The supernatant was filtered through a 0.45 μm aqueous filter to remove impurities and then loaded onto a pre-equilibrated Protein A affinity chromatography column. The equilibration buffer was 20 mM PBS buffer (pH 7.4), and the loading flow rate was 1 mL / min. After loading, the column was washed with PBS to remove contaminating proteins, and the bound antibody was eluted with 0.1 M citrate buffer (pH 3.0). The eluted fraction was immediately neutralized to neutral with 1 M Tris-HCl (pH 8.0). The collected purified antibody solution was analyzed by UV spectrophotometry. 280 Protein quantification: Using blank PBS as a reference, absorbance was measured at 280 nm. Antibody protein concentration was calculated based on the extinction coefficient of IgG antibody of 1.35 mL / (mg·cm). After aliquoting, the protein was stored at -80℃ for later use.

[0047] In this embodiment, the VH and VL sequences and corresponding constant region sequences of the D16H antibody were resynthesized, and then... EcoR I and BamH I site was constructed into the pTT5 vector ( Figure 2 , Figure 3 It can achieve efficient secretory expression of IgG with a yield of up to 511 mg / L, which can meet the needs of subsequent downstream experiments such as in vitro binding and functional verification.

[0048] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibody targeting tetrahydrocannabinol (THC), comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region includes a polypeptide with more than 90% similarity to the HCDR region, and the light chain variable region includes a polypeptide with more than 90% similarity to the LCDR region; the HCDR region includes HCDR1, HCDR2 and HCDR3, and the LCDR region includes LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.1; The amino acid sequence of HCDR2 is shown in SEQ ID NO.2; The amino acid sequence of HCDR3 is shown in SEQ ID NO.3; The amino acid sequence of LCDR1 is shown in SEQ ID NO.4; The amino acid sequence of LCDR2 is shown in SEQ ID NO.5; The amino acid sequence of LCDR3 is shown in SEQ ID NO.

6.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region includes an amino acid sequence with more than 90% similarity to SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes an amino acid sequence with more than 90% similarity to SEQ ID NO.

9.

3. The antibody according to claim 1 or 2, characterized in that, The coding sequence of the heavy chain variable region includes a nucleotide sequence with more than 90% similarity to SEQ ID NO.

8.

4. The antibody according to claim 1 or 2, characterized in that, The coding sequence of the light chain variable region includes a nucleotide sequence that has more than 90% similarity to SEQ ID NO.

10.

5. The antibody according to claim 1, characterized in that, The antibody subtype is IgG1; the light chain is Kappa type.

6. The use of the antibody according to any one of claims 1-5 in 1) or 2): 1) Testing for tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes; 2) Prepare reagents or kits for detecting tetrahydrocannabinol.

7. A kit for detecting tetrahydrocannabinol (THC), characterized in that, Includes the antibody as described in any one of claims 1-5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes reagents used in colloidal gold or ELISA detection methods.

9. A method for detecting tetrahydrocannabinol (THC) for non-diagnostic and non-therapeutic purposes, characterized in that, include: Tetrahydrocannabinol (THC) in the test sample was detected using colloidal gold method or ELISA. The antibody used is the antibody described in any one of claims 1-5.

10. The method according to claim 9, characterized in that, The test sample includes one or more of the following: body fluids, hair, blood, and urine.

Images