Antibodies that specifically bind mycophenolic acid and uses thereof

Abstract

The application discloses an antibody specifically combined with mycophenolic acid and application thereof, and belongs to the technical field of genetic engineering. The antibody BA-IgG prepared by the method has better activity than BA-scFv, the IC 50 is 0.259 ng / mL, the linear range is 0.5-20 ng / mL, the detection limit is 0.410 ng / mL, and the method can be applied to the immune analysis of mycophenolic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to an antibody specifically binding to bongkrekic acid and its application, and belongs to the technical field of genetic engineering. BACKGROUND

[0002] Bongkrekic acid (BA) is a highly toxic biological toxin produced by B. typhonium. Its structure is very stable, and ordinary cooking methods are difficult to destroy. There is a lack of effective treatment for bongkrekic acid poisoning in clinical practice, and sudden food poisoning incidents often occur. After human ingestion, it can cause extensive organ damage and even death. It is urgent to develop a simple, rapid and on-site detection method for bongkrekic acid quantitative detection.

[0003] Recognition is the basis of detection. The most common recognition element is antibody. Antibodies have high affinity and specificity. In the prior art, the following schemes are used to detect bongkrekic acid by antigen-antibody:

[0004] Patent CN113801220B discloses a bongkrekic acid complex antigen, a bongkrekic acid antibody and its preparation method and an enzyme-linked immunosorbent assay kit. The bongkrekic acid antibody prepared by optimizing the structure of the antigen, immunizing animals and hybridoma cells has a sensitivity of 10 μg / L.

[0005] Patent CN114045266A discloses an anti-bongkrekic acid monoclonal antibody and its application. The bongkrekic acid antibody is prepared by immunizing animals and hybridoma cells. In this scheme, the detection limit of the obtained antibody for detecting bongkrekic acid by enzyme-linked immunosorbent assay is 5.79 ng / mL.

[0006] In summary, in the existing schemes, bongkrekic acid antibodies are prepared by hybridoma cells prepared after animal immunization. On the one hand, this method is complicated and costly. On the other hand, the detection limit of the antibodies provided by the prior art in the detection of bongkrekic acid is still high, which limits the application scenarios in actual detection. Therefore, it is urgent to provide a bongkrekic acid antibody with simple preparation, low cost and low detection limit. SUMMARY

[0007] [TECHNICAL PROBLEM]

[0008] The technical problem to be solved by the present application is to provide a bongkrekic acid antibody with simple preparation, low cost and low detection limit.

[0009] [TECHNICAL SCHEME]

[0010] To solve the above technical problems, the present application provides the following technical scheme:

[0011] In a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that specifically binds to mycophenolic acid, the antibody or the antigen-binding fragment thereof comprising a light chain variable region VL and a heavy chain variable region VH, the VL comprising LCDR1-LCDR3 having the amino acid sequences of SEQ ID NO: 6, GTS, SEQ ID NO: 7, respectively, and the VH comprising HCDR1-HCDR3 having the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, respectively.

[0012] In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 2 and the VH comprises the amino acid sequence of SEQ ID NO: 1.

[0013] In one embodiment, the antibody comprises a heavy chain antibody HC and a light chain antibody LC, the HC having the amino acid sequence of SEQ ID NO. 11 and the LC having the amino acid sequence of SEQ ID NO. 13.

[0014] In one embodiment, the antigen-binding fragment is selected from a Fab, a Fab', a F(ab')2, a Fv or a scFv.

[0015] In a second aspect, the present application provides a biological material comprising at least one of the following (a) to (c):

[0016] (a): a polynucleotide encoding the antibody or the antigen-binding fragment thereof of the first aspect;

[0017] (b): a nucleic acid construct comprising the polynucleotide of (a);

[0018] (c): a vector comprising the polynucleotide of (a) or the nucleic acid construct of (b).

[0019] In a third aspect, the present application provides a cell comprising the biological material of the second aspect.

[0020] In one embodiment, the cell is a host of a microorganism or a mammalian cell.

[0021] In a fourth aspect, the present application provides a kit comprising the antibody or the antigen-binding fragment thereof of the first aspect.

[0022] In a fifth aspect, the present application provides use of the antibody or the antigen-binding fragment thereof of the first aspect, the biological material of the second aspect or the cell of the third aspect in the preparation of a reagent for detecting mycophenolic acid in a sample.

[0023] In the sixth aspect, the application provides a method for preparing the antibody or antigen binding fragment thereof of the first aspect, comprising: culturing the cell of the third aspect to obtain the antibody or antigen binding fragment thereof of the first aspect.

[0024] In one embodiment, the method comprises the following steps: inoculating the cell into a culture medium, culturing to OD 600 = 0.6-0.8, adding IPTG, inducing culture, and collecting the bacteria; the cell takes E. coli as a host and takes plasmid pET-22b as an expression vector.

[0025] In one embodiment, the method comprises the following steps: inoculating the cell into a culture medium, culturing at 30-40 DEG C and under 5-10% CO2 for 6-7 days, collecting the culture supernatant; the cell takes Expi293F cell as a host and takes plasmid pcDNA3.1 as an expression vector.

[0026] In the seventh aspect, the application provides a method for detecting whether mycophenolic acid or its content exists in a sample, comprising the step of contacting the antibody or antigen binding fragment thereof of the first aspect with the sample.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] 1. The mycophenolic acid antibody provided by the application has a detection limit significantly lower than that of the prior art when used for detection, and can realize high-sensitivity detection of mycophenolic acid. The IC 50 of the mycophenolic acid antibody provided by the application is 0.259 ng / mL when detecting mycophenolic acid by a competitive ELISA detection method, the linear range is 0.5-20 ng / mL, and the detection limit is 0.410 ng / mL.

[0029] 2. The application realizes expression and purification of scFv by constructing pET-22b-BA plasmid, transfecting E. coli BL21, and inducing scFv expression by IPTG, and finds that the activity of recombinant full-length IgG antibody obtained by the mammalian cell Expi293F expression system is higher than that of scFv.

[0030] 3. The mycophenolic acid antibody provided by the application does not need animal immunization when prepared, and the preparation method is simple.

[0031] 4. The mycophenolic acid recombinant antibody expressed in the application can be genetically modified according to experimental purposes, and is widely applied to rapid preparation of antibodies of other targets. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Verification of artificial antigen of mycophenolic acid, wherein A: UV-visible spectrum of coated antigen BA-BSA, B: SDS-PAGE gel electrophoresis diagram of coated antigen BA-BSA, C: UV-visible spectrum of immunized antigen BA-KLH.

[0033] Figure 2 Agarose gel electrophoresis diagram of extracted total RNA.

[0034] Figure 3 Agarose gel electrophoresis diagram of antibody variable region gene.

[0035] Figure 4 Agarose gel electrophoresis diagram of scFv before and after enzyme digestion, wherein lane 1: before enzyme digestion, lane 2: after enzyme digestion.

[0036] Figure 5 Agarose gel electrophoresis diagram of pCANTAB-5E before and after enzyme digestion, wherein lane 1: before enzyme digestion, lane 2: after enzyme digestion.

[0037] Figure 6 Colony PCR identification of pCANTAB5E-scFv recombinant plasmid, lanes 1-12 represent different colonies.

[0038] Figure 7 Plate for determination of antibody library capacity titer.

[0039] Figure 8 Verification result of phage-ELISA.

[0040] Figure 9 SDS-PAGE electrophoresis diagram of BA-scFv antibody bacterial cell precipitate and supernatant, lane 1 represents fermentation supernatant, lanes 2-5 represent bacterial cell precipitate.

[0041] Figure 10 SDS-PAGE electrophoresis diagram of BA-IgG antibody bacterial cell precipitate and supernatant, lane 1 is cell precipitate, lane 2 is cell expression supernatant.

[0042] Figure 11 Verification of activity of BA-scFv and BA-IgG antibodies.

[0043] Figure 12 Ic-ELISA absorbance-concentration curve of BA-IgG antibody.

[0044] Figure 13 Standard curve of Ic-ELISA of BA-IgG antibody. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further illustrated below in conjunction with the accompanying drawings and through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations to the present application.

[0046] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the related terms are provided as follows.

[0047] In the present application, the amino acids at the corresponding positions are represented by the recognized IUPAC single-letter abbreviations, in which each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0048] In the present application, the term "about" or "approximately" should be understood to include all numerical values falling within the permissible range of measurement error.

[0049] In the present application, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleotide sequence", and "polynucleotide" can be used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or triple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, consisting of, or consisting essentially of, purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0050] In the present application, the term "nucleic acid construct" refers to a nucleotide sequence comprising a complete expression cassette capable of expressing a gene of interest or a protein of interest. In addition to the coding sequence encoding the gene of interest or the protein of interest, the nucleic acid construct can also comprise genetic elements such as promoters, enhancers, terminators, and / or polyadenylation signals for regulating gene expression.

[0051] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host or a nucleic acid molecule capable of inserting a target gene fragment into the host genome, which transfers the target gene fragment carried to the host cell and / or between host cells. The vector can include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having various functions described above. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

[0052] In the present application, the "light chain variable region" (VL) or "heavy chain variable region" (VH) is composed of "framework" regions separated by three "complementarity determining regions" or "CDRs". The framework region is used to align the CDRs that specifically bind to the epitopes of the antigen. The CDRs include the amino acid residues in the antibody that are mainly responsible for antigen binding. Both the VL domain and the VH domain comprise the following framework regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0053] Examples:

[0054] The following example section contains the following components and sources:

[0055] LB plate medium (Amp+): yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, ampicillin 100 μg / L;

[0056] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L;

[0057] Expi293F cells and BL21(DE3) competent cells were purchased from Thermo Fisher Scientific, USA.

[0058] The following example uses the following reagents (cationic transfection reagent, transfection enhancer I, enhancer II from Thermo Fisher Scientific, USA; TMB color developing solution A, TMB color developing solution B from Yikesheng Biotechnology (Shanghai) Co., Ltd.

[0059] Example 1: Preparation of murine single-chain antibody (scFv)

[0060] I. Experimental Methods

[0061] 1. Synthesis of immunizing antigen

[0062] Dissolve 10 mg mycophenolic acid, 10 mg EDC and 10 mg NHS in N,N-dimethylformamide (C3H7NO, DMF) and activate at room temperature for 4 h as A liquid; dissolve 50 mg BSA in 5 mL PBS buffer solution, and stir to dissolve to prepare B liquid; under magnetic stirring, add A liquid dropwise to B liquid, and magnetically stir at 4°C overnight. Dialyze the reaction liquid with PBS at 4°C for 3 days to obtain coated antigen BA-BSA, which is stored in a -20°C refrigerator for standby use. The synthesis method of immunizing antigen BA-KLH is the same as that of coated antigen, except that BSA is replaced by KLH (hemocyanin). The synthesized antigen is verified by ultraviolet spectrum, SDS-PAGE and ELISA.

[0063] 2. Mouse immunization

[0064] Mouse immunization is performed with immunizing antigen, and mouse serum titer is detected.

[0065] Mouse immunization: take 6-8 week old Balb / c mice, mix the prepared BA-KLH with a concentration of 1 mg / mL with an equal amount of Freund's adjuvant, completely emulsify, and then inject 100 μL into each mouse by multiple intraperitoneal injection to gradually stimulate the spleen B lymphocytes of the mice to produce BA antibody genes. The first immunization uses Freund's complete adjuvant (FCA), and the subsequent booster immunization uses Freund's incomplete adjuvant (FICA), with immunization every 2 weeks, and a total of 4 times of booster immunization.

[0066] Detection of mouse serum titer: take 100-200 μL of tail tip blood from each immunized mouse after each immunization for 1 week, centrifuge at 12000 r / min for 15 min to take the upper serum, dilute by gradient, and then detect the titer by ELISA. Take the OD450 nm value of the mouse serum to be tested as P, and the OD450 nm value of the serum of a healthy mouse as N. When P / N≥2.1, the maximum dilution factor of the serum is the antibody titer 450 nm

[0067] 3. Construction of recombinant vector

[0068] ​The spleen cells of the immunized mice were taken for total RNA extraction, the integrity of the extracted total RNA was verified by agarose gel electrophoresis, and the first strand cDNA was obtained by reverse transcription. The obtained cDNA was used as a template, and the antibody VH and VL fragments were amplified by using the mouse antibody universal primer. The VH and VL were assembled into scFv by using nest PCR amplification, and Not I and Sfi I enzyme cutting sites were introduced at both ends of the scFv to construct a recombinant plasmid. The pCANTAB-5E vector was double-cut by using Not I and Sfi I endonucleases, and the scFv was connected to the pCANTAB-5E vector by means of T4 ligase to form a pCANTAB5E-scFv recombinant plasmid.

[0069] 4. Antibody screening

[0070] The recombinant plasmid pCANTAB5E-scFv was electroporated into the E. coli TG1 competent cells, and after infection and amplification by the helper phage M13K07, the BA phage antibody library was obtained, and affinity panning was performed. The wells with a sample absorbance value / negative control group absorbance value (P / N) > 2.1 times were selected as positive single colony phages, and the positive clones were obtained, and the plasmid was extracted, the bacteria were preserved, and were sent to Shanghai Sangon Biotech Company for sequencing.

[0071] II. Experimental results

[0072] The identification of the coated antigen BA-BSA and the immunizing antigen BA-KLH is shown in Figure 1 . The successful synthesis of the BA coated antigen was verified by ultraviolet-visible spectroscopy Figure 1 A) and SDS-PAGE gel electrophoresis Figure 1 B). The ultraviolet absorption spectrum of the BA-BSA product is between BA and BSA, and a new protein band appears in the SDS-PAGE gel electrophoresis at about 70 kDa, which has a molecular weight slightly larger than BSA. These results show that the coupling reaction between BA and BSA is successful. It can be known from Figure 1 C that the ultraviolet absorption spectrum of the BA-KLH product is between BA and KLH, which is relatively close to the KLH ultraviolet absorption curve, which may be caused by the excess of KLH molecules and the low concentration of BA-KLH. It is verified by ELISA that BA-BSA and BA-KLH both have good specific binding with mycophenolic acid antibody.

[0073] The results of the detection of the serum titer of the mice are shown in Table 1. It is determined that the highest serum titer of the immunized mice is 1:1000.

[0074] Table 1 ELISA detection of the titer of the tail blood of the immunized mice

[0075]

[0076] Figure 2 shows the agarose gel electrophoresis results of total RNA from the spleen of immunized mice. Clear 28S and 18S bands are visible, indicating good integrity of the total RNA, which can serve as a template for reverse transcription into cDNA. Electrophoresis results of antibody VH and VL fragment amplification are shown below. Figure 3 The VL band, approximately 300 bp, and the VH band, approximately 350 bp, are consistent with the expected single-chain antibody gene fragment sizes, indicating successful amplification of the light and heavy chain fragments, which can be used to assemble scFv. The scFv was identified by agarose gel electrophoresis, and the approximately 750 bp band was recovered and purified using a gel extraction kit. Verification results are shown in [link to verification results]. Figure 4 (Lane 1). As can be seen from the figure, the amplified scFv band is clear and its size matches the expected result, indicating that the scFv fragment was successfully assembled and can be used for the construction of recombinant plasmids. The agarose gel electrophoresis results after double enzyme digestion of the scFv fragment are as follows. Figure 4 As shown in lane 2, the size of the target band did not change significantly before and after enzyme digestion because scFv itself is linearized, and double digestion only removed a few bases. The lighter band color is due to the decrease in scFv concentration after gel extraction and enzyme digestion.

[0077] The agarose gel electrophoresis results of the pCANTAB-5E vector plasmid and its NotⅠ and SfiⅠ double enzyme digestion are as follows: Figure 5 Lanes 1 and 2 are shown in the figure. It can be seen from the figure that the bands of the vector change significantly before and after enzyme digestion, becoming a slightly smaller molecular weight digested vector, indicating successful digestion and suitability for ligation and transformation.

[0078] The constructed recombinant plasmid pCANTAB5E-scFv was electroporated into TG1 competent cells, and the cells grew well on 2×YT-Amp plates, indicating successful transformation. Twelve transformant single colonies were randomly selected for colony PCR identification, and the results are as follows: Figure 6 As shown in the figure, all 11 colonies amplified the target fragment, with a positive insertion rate of 91.67%. The 11 monoclonal bacterial cultures containing the correct-sized target fragment were sent to Sangon Biotech in Shanghai for sequencing. Sequencing analysis revealed that all colonies successfully inserted different target gene sequences of approximately 750 bp between the Not I and Sif I restriction sites.

[0079] The titer analysis showed that the constructed BA single-chain antibody library had a volume of approximately 8 × 10⁻⁶. 9 pfu / 10μL (titer assay plate, e.g.) Figure 7 (As shown). After amplification by helper phage M13K07, the BA phage antibody library had a capacity of approximately 8 × 10⁻⁶. 12pfu / mL, which can be used for subsequent screening. The titer of the elution product and the amplified product was determined after each round of screening, and the number of enriched phages in each round was calculated. The specific titer determination results are shown in Table 2.

[0080] Table 2 Titer determination of BA-scFv particles in the screening process

[0081]

[0082] As can be seen from Table 2, with the increase of the number of panning, the phages were obviously enriched, each round was increased by about 10 times than the last round, indicating that the screening method was feasible. Thirty single colonies were randomly selected, expanded, infected with M13K07, and monoclonal phages were prepared for phage-ELISA identification. The clones with P / N value greater than 2.1 were selected as positive clones. The verification results are shown in Figure 8 As can be seen from the figure, the color development effect and P / N value of No. 3 clone are obviously better than those of other single colonies, so No. 3 clone (scFv3) is determined as a positive clone, and its plasmid extraction, bacteria preservation and sequencing are carried out by Shanghai Sangon Biotech Company. Analysis shows that the full length of scFv3 is 720 bp, in which the size of VH is 351 bp, the size of VL is 324 bp, and the length of Linker is 45 bp. Through NCBI BLAST comparison, the homology of VH gene sequence with mouse immunoglobulin heavy chain variable region gene (GenBank: AAA16584.1) is 93.28%, and the homology of VL gene sequence with mouse immunoglobulin light chain kappa variable region gene (GenBank: AAA92406.1) is 100%. According to the amino acid sequence of the variable region of the mouse-derived antibody published in the NCBI database, the ScFv3 is annotated using the IMGT coding rule (the heavy chain CDR region is bold and underlined, and the light chain CDR region is italic and underlined), and the specific annotation results are as follows:

[0083] Heavy chain variable region VH amino acid sequence (BA-ScFv3_VH):

[0084]

[0085] Among them, the heavy chain CDR region (HCDR) is as follows:

[0086] Heavy chain CDR1 region, SEQ ID NO: 3: GFNIKDTY;

[0087] Heavy chain CDR2 region, SEQ ID NO: 4: IDPANGNT;

[0088] Heavy chain CDR3 region, SEQ ID NO: 5: ASYYGSSYDY.

[0089] Heavy chain variable region VH amino acid sequence (BA-ScFv3_VH):

[0090]

[0091] Wherein, the light chain CDR region (LCDR) is as follows:

[0092] Light chain CDR1 region, SEQ ID NO: 6: SSISSSN;

[0093] Light chain CDR2 region amino acid sequence: GTS;

[0094] Light chain CDR3 region, SEQ ID NO: 7: QQWSSYPLT.

[0095] Example 2: Construction and biological expression of recombinant IgG full-length antibody plasmid of mycophenolic acid single-chain antibody ScFv

[0096] I. Experimental method

[0097] The mycophenolic acid ScFv3 sequence obtained by screening in Example 1: the BA-ScFv3_VH coding gene nucleotide sequence is shown in SEQ ID NO: 8, and the BA-ScFv3_VL coding gene nucleotide sequence is shown in SEQ ID NO: 9.

[0098] In the recombinant IgG full-length antibody, the full-length antibody heavy chain HC amino acid sequence is shown in SEQ ID NO: 11, and the coding gene nucleotide sequence is shown in SEQ ID NO: 10; the full-length antibody light chain LC amino acid sequence is shown in SEQ ID NO: 13, and the coding gene nucleotide sequence is shown in SEQ ID NO: 12.

[0099] The sequence of ScFv3 of Mycophenolic acid obtained by screening of Example 1 was introduced into pET-22b expression vector between NcoI and XhoI two enzyme cutting sites in the form of seamless cloning, to construct plasmid pET-22b-BA (VL and VH were connected by linker, linker was GS connecting peptide: GGGGSGGGGSGGGGS), and recombinant single-chain antibody (BA-scFv) was expressed in E. coli BL21; in the same way, the target gene was cloned into pcDNA3.1 vector between EcoRI and BamHI (when used for gene expression, start codon was added at 5' end of the gene, and stop codon was added at 3' end of the gene, so that the gene could be successfully expressed) two enzyme cutting sites, to construct plasmids pcDNA3.1-HC and pcDNA3.1-LC, respectively, and full-length recombinant antibody (pcDNA3.1-HC: pcDNA3.1-LC = 1:1.5) was expressed in mammalian cells Expi293F.

[0100] The specific steps are as follows:

[0101] (1) Prokaryotic expression of single-chain antibody BA-scFv:

[0102] The recombinant bacteria containing pET-22b-BA plasmid were streaked and cultured at 37°C overnight. A single colony was inoculated in LB liquid medium and cultured overnight. The next day, the plasmid was extracted and quantified by Nanodrop. 5 μg of plasmid was added to BL21(DE3) competent cells, which were then incubated in ice water for 30 min, heated at 42°C for 90 s, and then immediately placed in ice water for 5 min. 900 μL of LB culture solution (preheated at 37°C) was added to the above competent cells, which were then incubated at 37°C, 220 r / min for 40 min, and then cultured overnight. A single colony was inoculated in LB / Amp liquid medium and cultured to OD 600 >1. The bacterial solution was added to 100 mL of LB / Amp medium and cultured to OD 600 = 0.6-0.8, and 0.1 mM IPTG was induced at 18°C, 220 r / min overnight. The next day, centrifugation was performed at 12000 r / min, 4°C for 5 min, and SDS-PAGE gel electrophoresis was performed on the bacterial precipitate and supernatant, respectively.

[0103] (2) Purification of single-chain antibody BA-scFv:

[0104] His-tag protein purification kit to purify the liquid containing the target protein: filter through 0.22 μm membrane, take 1 mL His-tag purification resin into the affinity chromatography empty tube column, add 0.5 times the column volume of non-denaturing lysis buffer for 2-3 times. Add the liquid to be purified into the affinity chromatography column, collect the flow-through, and cycle 2-3 times. Elute with gradient concentration of imidazole solution, collect the target protein. Take 20 μL of the purified sample and verify it by 12.5% SDS-PAGE gel electrophoresis. Dialyze in 0.01 M, pH 7.4 PBS at 4°C for 3 days, and change the dialysis solution 2-3 times a day.

[0105] (3) Expression and purification of recombinant full-length antibody BA-IgG:

[0106] Recovery and subculture of Expi293F cells were performed in advance to ensure cell viability. On the day of transfection, the total number of cells reached 7.5 x 10 7 cells, and the cell volume was adjusted with fresh Expi293 serum-free medium. Add 30 μg of plasmid (pcDNA3.1-HC: pcDNA3.1-LC = 1:1.5) to 1.5 mL of Opti-MEM I serum-free medium and mix well. Add 81 μL of cationic transfection reagent to 1.5 mL of Opti-MEM I serum-free medium and mix well, and let stand at room temperature for 5 min. Mix the above suspension, and after 20 min at room temperature, add 3 mL to the cells, and incubate at 37°C, 8% CO2, 125 r / min on a cell shaker. After 12 h of transfection, add 150 μL of transfection enhancer I and 1.5 mL of enhancer II, mix well, and continue to culture for 6-7 d, then collect the target protein. Purification of recombinant full-length antibody BA-IgG is the same as above (2).

[0107] (4) Evaluation of genetically engineered antibodies BA-scFv and BA-IgG:

[0108] Add coated antigen BA-BSA and BSA to the 96-well plate, respectively, and coat at 37°C for 2 h. Wash the plate three times with 0.05% PBST and pat dry. Add 350 μL of 3% skim milk powder to each well, and block at 37°C for 2 h, wash the plate three times and pat dry. Add 100 μL of antibody BA-scFv or BA-IgG to each well, and incubate at 37°C for 1 h, wash the plate three times and pat dry. Add 100 μL of anti-His-HRP secondary antibody and incubate at 37°C for 30 min, wash the plate three times and pat dry. Add 50 μL of TMB color developing liquid A and B, respectively, and react at 37°C for 15 min, then add the stop solution, and measure OD 450nmOptical density. 0.125, 0.25, 1, 2 μg / mL BA-BSA was coated in the wells of the enzyme-labeled plate, respectively, and after blocking according to the above operation, different concentrations of antibody BA-scFv or BA-IgG were added, and the subsequent steps were the same as above.

[0109] II. Experimental results

[0110] Prokaryotic expression of single-chain antibody BA-scFv: The SDS-PAGE gel electrophoresis identification result of the single-chain antibody BA-scFv expressed and purified by E. coli is shown in Figure 9 Lane 1 is the fermentation supernatant, and from lane 1 it can be seen that the expression product does not exist in the supernatant, lanes 2-5 are the target proteins eluted in turn, and the results show that the single-chain antibody BA-scFv expressed by E. coli exists in the bacterial precipitate. The theoretical molecular weight of BA-scFv is about 29 kDa, which is consistent with the single band in lanes 2-5.

[0111] Recombinant full-length antibody BA-IgG cell expression: The SDS-PAGE result is shown in Figure 10 Under reducing conditions, the disulfide bond connecting the heavy chain and the light chain is destroyed, so two separate bands will appear on the gel. Lane 1 is the cell precipitate, which does not contain antibody protein, and lane 2 is the cell expression supernatant, which shows obvious bands after purification. The band with a molecular weight of about 25 kDa is the light chain, and the band with a molecular weight of about 55 kDa is the heavy chain. Figure 10 The band color is obviously Figure 9 deeper, indicating that the expression yield of BA-IgG is higher than that of BA-scFv.

[0112] Evaluation of antibodies BA-scFv and BA-IgG: After dialysis of the purified antibody protein in PBS, the same concentration of BA-BSA and BSA was used to coat the 96-well plate, respectively, and the activity of the antibody was verified by non-competitive ELISA. The results are shown in Figure 11 When the same concentration of antigen and antibody is added, the optical density of BA-IgG at 450 nm is significantly higher than that of BA-scFv, indicating that the activity of BA-IgG is better. Therefore, BA-IgG is selected for the establishment of the subsequent enzyme-linked immunoassay method.

[0113] The above results show that the choice of host and molecular weight have a significant impact on the activity and affinity of the antibody. For the mycophenolic acid antibody sequence provided by the present application, the expression of single-chain antibody in E. coli will result in a lack of activity, and the activity of the recombinant full-length antibody prepared in mammalian Expi293F cells is better.

[0114] Example 3: ELISA kit for mycophenolic acid

[0115] Establishment of competitive ELISA detection method:

[0116] (1) The optimal BA-BSA coating concentration and the optimal antibody concentration were determined by chessboard titration method. The mycophenolic acid antigen and the genetically engineered antibody were diluted to 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL and 2 μg / mL respectively, and the subsequent operations were the same as (4) in Example 2. OD 450nm The antigen-antibody concentration corresponding to the value close to 1 was the optimal concentration.

[0117] (2) After 100 μL of the optimal concentration of BA-BSA coating and blocking were taken, 50 μL of different concentrations of mycophenolic acid (0.0125-100 ng / mL) standard and 50 μL of twice the optimal concentration of genetically engineered antibody BA-IgG were added, and the subsequent operations were the same as (4) in Example 2. The standard curve was established with the BA standard concentration as the abscissa and the inhibition rate B / B0 as the ordinate. Among them, B was the absorbance value corresponding to different concentrations of mycophenolic acid standard; B0 was the absorbance value corresponding to the mycophenolic acid standard concentration of 0.

[0118] Experimental results:

[0119] According to the chessboard titration method, the optimal BA-BSA coating concentration was 2 μg / mL, and the optimal genetically engineered antibody concentration was 2 μg / mL. The competition standard curve of BA concentration in the range of 0.0125-100 ng / mL is shown in Figure 12 and Figure 13 The IC50 value of the standard curve was calculated to be 0.259 ng / mL. When the concentration of BA was 0.5-20 ng / mL, the logarithmic value of BA concentration and B / B0 were linearly related, and the linear equation was Y=-13.564LgX+30.142, R 2 =0.946, and the LOD was 0.410 ng / mL.

[0120] The applicant declares that the detailed process flow of the present application is illustrated by the above examples, but the present application is not limited to the above detailed process flow, that is, it does not mean that the present application must rely on the above detailed process flow to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to bongkrekic acid, said antibody or antigen fragment comprising a light chain variable region VL and a heavy chain variable region VH, characterized in that, The VL contains the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NO:6, GTS, and SEQ ID NO:7, respectively, and the VH contains the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The VL contains the amino acid sequence shown in SEQ ID NO:2 and the VH contains the amino acid sequence shown in SEQ ID NO:

1.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody comprises an antibody heavy chain HC and an antibody light chain LC, wherein the HC has the amino acid sequence shown in SEQ ID NO.11 and the LC has the amino acid sequence shown in SEQ ID NO.

13.

4. A biomaterial, characterized in that, The biomaterial includes at least one of the following (a) to (c): (a): A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; (b): Nucleic acid constructs containing the polynucleotides described in (a); (c): A vector containing the polynucleotide described in (a) or the nucleic acid construct described in (b).

5. A cell, characterized in that, Contains the biomaterial described in claim 4.

6. The cell according to claim 5, characterized in that, The cells are selected from microbial or mammalian cells.

7. A reagent kit, characterized in that, The kit contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the biological material as described in claim 4, or the cell as described in claim 5 or 6 in the preparation of a reagent for detecting bongkrekic acid in a sample.

9. A method for preparing the antibody or antigen-binding fragment of any one of claims 1 to 3, characterized in that, include: By culturing the cells described in claim 5 or 6, an antibody or its antigen-binding fragment as described in any one of claims 1 to 3 can be prepared.

10. A method for detecting the presence or content of bongkrekic acid in a sample, characterized in that, The method includes the step of contacting the antibody or its antigen-binding fragment according to any one of claims 1 to 3 with the sample.

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