Anti-sp cas9 protein monoclonal antibody and detection kit and application

Abstract

The application belongs to the technical field of biological detection, and discloses an anti-SpCas9 protein monoclonal antibody, a detection kit and application, wherein the heavy chain variable region amino acid sequence of the anti-SpCas9 protein monoclonal antibody is shown as SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2; and a double-antibody sandwich ELISA detection kit is constructed by taking the monoclonal antibody as a capture antibody and an anti-SpCas9 protein polyclonal antibody as a detection antibody. The ELISA detection kit has the advantages of high sensitivity, strong specificity, good repeatability, high-throughput and rapid detection, can accurately quantify the expression and residual amount of SpCas9 protein in a gene editing biological sample, and provides strong support for the application and supervision of CRISPR / Cas9 gene editing technology.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and particularly relates to anti-SpCas9 protein monoclonal antibodies, detection kits and applications. Background Technology

[0002] The CRISPR / Cas9 system, initially discovered in bacteria, is currently the most widely used gene-editing technology. Guided by guide RNA (gRNA), the Cas9 protein specifically recognizes and cuts DNA. This system enables rapid, efficient, and precise gene editing and regulation in the genomes of various organisms and tissues, leading to its widespread application in basic research and applied sciences. Among them, Cas9 (Streptococcus pyogenes Cas9, SpCas9), derived from Streptococcus pyogenes, is one of the earliest Cas9 nucleases to achieve genome editing applications and is currently the most frequently used, with extremely wide applications in basic life science research, crop genetics and breeding, and biomedicine.

[0003] In basic research, the expression level of SpCas9 protein directly determines gene editing efficiency, and accurate detection of its expression level is a core step in optimizing gene editing conditions and improving experimental reproducibility. In industrial applications, the expression and residue of SpCas9 protein can be used to screen for varieties without transgenic components, effectively avoiding the accumulation of exogenous proteins and their long-term effects on host cells, and providing a scientific basis for risk assessment and regulation of gene editing.

[0004] Currently, there are few studies on the quantitative detection of SpCas9 protein. Existing detection methods usually rely on techniques such as immunoprecipitation and Western blot. Although these methods have certain specificity, they are cumbersome to operate, time-consuming, and cannot achieve high-throughput quantitative detection. They are difficult to meet the needs of rapid screening of large batches of samples, which greatly limits the large-scale application of CRISPR / Cas9 technology and the further development of related research.

[0005] Enzyme-linked immunosorbent assay (ELISA) is a commonly used protein detection technique. Its core is based on the preparation of highly specific antibodies, combined with signal changes generated by enzymatic reactions to achieve quantitative analysis of target molecule concentrations. This method boasts advantages such as high sensitivity, strong specificity, ease of operation, and high-throughput detection. However, current research on the preparation of paired antibodies for SpCas9 protein and corresponding quantitative ELISA kits is limited, failing to meet the practical application requirements for accurate, rapid, and high-throughput quantitative detection of SpCas9 protein. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes an anti-SpCas9 protein monoclonal antibody and its detection kit and application. Recombinant SpCas9 protein was prepared, and monoclonal and polyclonal antibodies specifically recognizing SpCas9 were obtained. These antibodies were used as detection and capture antibodies, respectively, to prepare a sandwich ELISA kit for the quantitative detection of SpCas9 protein. This kit offers advantages such as ease of operation, rapid detection, high sensitivity, high specificity, and good stability, meeting the needs for quantitative detection of SpCas9 protein and providing strong support for the widespread application of CRISPR / Cas9 gene editing technology.

[0007] To achieve the above objectives, the present invention provides a monoclonal antibody against SpCas9 protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0008] Preferably, the heavy chain subclass of the monoclonal antibody is IgG2a, and the light chain is the κ chain.

[0009] A SpCas9 protein detection kit is also provided, which includes a microplate and detection reagents, and the microplate contains an anti-SpCas9 protein monoclonal antibody.

[0010] Preferably, in the microplate, the coating concentration of the anti-SpCas9 protein monoclonal antibody is 5 μg / mL, and the coating buffer is 0.05 M carbonate buffer at pH 9.6.

[0011] Preferably, the detection reagent comprises an anti-SpCas9 protein polyclonal antibody, an enzyme-labeled working solution, SpCas9 protein standards, a sample processing solution, a concentrated washing solution, a chromogenic agent, and a stop solution.

[0012] Preferably, the enzyme-labeled working solution is HRP-labeled goat anti-rabbit IgG; the concentrated washing solution is 20×PBST; the chromogenic agent is TMB chromogenic solution; and the stop solution is 2M sulfuric acid solution.

[0013] Preferably, the following components are used: 6 mL of anti-SpCas9 polyclonal antibody; 6 mL of enzyme-labeled working solution; 96 ng of SpCas9 protein standard; 50 mL of sample processing solution; 20 mL of concentrated washing solution; 6 mL of chromogenic reagent; 6 mL of stop solution; and the kit should be stored at 2-8℃.

[0014] It also provides an application of an SpCas9 protein detection kit for detecting SpCas9 protein.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention uses SpCas9 recombinant protein as an immunogen, which has good immunogenicity. The obtained monoclonal and polyclonal antibodies have advantages such as high titer and good specificity. The ELISA kit established using monoclonal antibody as capture antibody and polyclonal antibody as detection antibody has advantages such as high specificity and high sensitivity, and can perform large-scale sample detection, which can meet the needs of quantitative detection of SpCas9 protein.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 The image shows an SDS-PAGE electrophoresis result of the SpCas9 recombinant protein expression and purification process. In the figure, A represents the detection of recombinant protein expression form, B represents the detection of recombinant protein purification gradient elution, and C represents the purified SpCas9 recombinant protein. The black arrows indicate the SpCas9 recombinant protein bands.

[0018] Figure 2 The figure shows the recognition characteristics of FL5861-10 monoclonal antibody and rabbit polyclonal antibody on SpCas9 recombinant protein and rice materials. In the figure, M is the molecular weight of the protein, 1 and 2 are 10 ng and 2 ng of SpCas9 recombinant protein loaded, respectively, 3 and 4 are SpCas9 gene-edited rice samples, and 5 is wild-type rice sample. The black arrows indicate the location of the SpCas9 protein.

[0019] Figure 3 A standard curve for ELISA detection of SpCas9 recombinant protein.

[0020] Figure 4 The results of cross-reactivity of four Cas proteins, SaCas9, FnCas12a, BrCas12b, and LwaCas13a, were obtained by ELISA. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] Example 1 Expression and purification of SpCas9 recombinant protein.

[0026] S1. Construction of recombinant expression vector: Based on the SpCas9 gene sequence published by NCBI (GenBank accession number: 69900935), codons were optimized according to the E. coli expression system. The target gene was synthesized by Shanghai Qingke Biotechnology Co., Ltd., and then ligated into the pET30a expression vector to construct the pET30a-SpCas9 recombinant plasmid.

[0027] S2. Protein Induction Expression: The recombinant plasmid was transformed into *E. coli* Rosetta (DE3) competent cells and plated on LB solid medium containing 50 μg / mL kanamycin. The cells were incubated overnight at 37°C with inverted incubation. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The cells were incubated overnight at 37°C with shaking at 250 rpm to obtain the seed culture. The seed culture was then transferred at a 1:100 ratio to 1 L of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C with shaking at 250 rpm until OD (Organic Growth Rate) was reached. 600 =0.5-0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5mM, and induce expression at 18℃ for 15h.

[0028] S3. Cell lysis and supernatant collection: Centrifuge at 8000×g, 4℃ for 15 min, collect the cells, resuspend the cells in 60 mL of Ni-NTA binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH=8.0), and add benzyl sulfonyl fluoride (PMSF) to a final concentration of 1 mM. Lyse the cells using a Constant Systems high-pressure cell disruptor (30 kPsi), and ultracentrifuge at 40000×g, 4℃ for 30 min, then collect the supernatant.

[0029] S4. Protein Purification and Identification: The supernatant was purified using a Ni-NTA protein purification column, followed by gradient elution with elution buffers containing 50mM, 100mM, 200mM, 250mM, and 500mM imidazole. The target protein eluent was collected. A desalting column with a molecular weight cutoff of 10kDa was used for buffer replacement. The replacement buffer consisted of 50mM NaH₂PO₄, 100mM NaCl, 1mM DTT, and 10% glycerol, pH=7.5. The purified SpCas9 recombinant protein was collected.

[0030] S5. Protein purity and concentration detection: Protein purity was analyzed by SDS-PAGE electrophoresis on a 10% separating gel. After Coomassie brilliant blue staining, protein purity was determined by grayscale analysis. Protein concentration was determined by a BCA protein quantification kit. After aliquoting, the protein was stored at -80℃ for later use.

[0031] The results are as follows Figure 1 As shown, the SpCas9 recombinant protein was mainly expressed in soluble form in the supernatant. The theoretical molecular weight after including the His tag was approximately 162 kDa, consistent with the expected results. After purification by Ni-NTA affinity chromatography, high-purity SpCas9 recombinant protein was obtained by elution with 250 mM imidazole. After desalting and dialysis, the purified protein concentration reached 2.5 mg / mL, with a purity >95%, which can meet the requirements of subsequent animal immunization experiments.

[0032] Example 2 I. Preparation of SpCas9 protein monoclonal antibody.

[0033] 1) Animal immunization.

[0034] The SpCas9 recombinant protein prepared in Example 1 was used as an immunogen to immunize 6-week-old female BALB / c mice three times via subcutaneous injection. The immunization dose per mouse was 25 μg each time. The initial immunization was emulsified with an equal volume of Freund's complete adjuvant, while the second and third immunizations were emulsified with an equal volume of Freund's incomplete adjuvant. Ten days after the third immunization, blood was collected from the tail vein of the mice, and serum antibody titers were determined by indirect ELISA. Mice with higher antibody titers were selected for subsequent cell fusion. Three days before fusion, a booster immunization was performed with the same antigen dose. The specific immunization schedule is shown in Table 1.

[0035] Table 1. Immunization procedures for preparing monoclonal antibodies

[0036] The specific steps for detecting antibody titers using the indirect ELISA method are as follows.

[0037] S1. Antigen coating: Dilute SpCas9 recombinant protein to 1 μg / mL with 0.05M carbonate buffer (pH 9.6), add 100 μL / well to a 96-well microplate, and coat overnight at 4°C. S2, Blocking: Discard the coating solution, add 150µl of blocking solution (2% BSA), and incubate at 37°C for 1h; S3. Primary antibody incubation: The serum to be tested is serially diluted 3-fold starting from 1:1000. 100µl of the solution is added to the reaction well and incubated at 37°C for 1 h. S4. Wash 3 times with TBST, add 100µl / well HRP-goat anti-mouse secondary antibody (1:3000 dilution), and incubate at 37℃ for 1h; S5. Colorimetric reaction: Wash 3 times with TBST, add 100µl / well of TMB substrate colorimetric solution, and develop color at room temperature in the dark for 5-10 min. S6. Termination and Detection: Add 50 µl / well of stop solution and measure OD using a microplate reader. 450 .

[0038] II. Preparation and screening of hybridoma cell lines.

[0039] S1, Cell Fusion: Mice with the highest immune titer were selected (serum was collected as a positive control group). After disinfection of the body surface with 75% alcohol, the spleen was aseptically removed, the connective tissue was removed and the spleen cells were ground to prepare a suspension. The suspension was transferred to a 50mL centrifuge tube, and basal culture medium was added to 30mL. After centrifugation at 400×g for 10min, the supernatant was discarded, and the cells were washed twice and resuspended in fresh basal culture medium.

[0040] Spleen cells and SP2 / 0 myeloma cells (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were mixed at a cell ratio of 5:1. 50% PEG-1500 solution was added to induce fusion for 90s. After centrifugation at 400×g for 5min, the supernatant was discarded. The cells were washed with HAT selective medium and then pipetted into a single-cell suspension. The suspension was seeded into 96-well cell culture plates at 200μL / well and cultured at 37℃ in a 5% CO2 incubator for 7 days.

[0041] S2. Positive clone screening and subcloning: After monoclonal cell aggregates have formed in the wells, OD are screened using the above-mentioned indirect ELISA method. 450 Strongly positive wells with a value >1.0 were used to expand and culture the initially screened positive cells. The positive cells were diluted to 0.8 cells / well using a limiting dilution method. After confirming monoclonalness under a microscope, three rounds of subcloning were performed, ultimately obtaining 12 hybridoma cell lines with 100% positive cell lines, which were then expanded and used for further development.

[0042] III. Preparation and purification of monoclonal antibodies.

[0043] S1. Ascites preparation: Healthy female BALB / c mice aged 6-8 weeks were selected and sensitized by intraperitoneal injection of 0.5 mL of sterile liquid paraffin. One week later, 1-2 × 10⁻⁶ g of paraffin was injected into the peritoneum. 6 A number of positive hybridoma cells were collected. The abdominal condition of mice was observed daily. After the abdominal circumference increased significantly, the mice were sacrificed, and ascites fluid was collected aseptically. The fluid was centrifuged at 4000×g at room temperature for 15 min, and the supernatant was collected.

[0044] S2. Antibody Purification: The antibody was crudely purified using ammonium sulfate fractionation precipitation. Saturated ammonium sulfate solution was added dropwise to the ascites supernatant with stirring at 4°C until a final concentration of 50% was reached. The mixture was then centrifuged at 12000×g at 4°C for 30 min, and the precipitate was collected. The precipitate was reconstituted with 0.01M PBS (pH 7.4), and the salting-out process was repeated (final ammonium sulfate concentration 33%). The mixture was then dialyzed at 4°C for 24 h to remove residual ammonium sulfate. The antibody was further purified using a Protein G affinity chromatography column. The column was activated sequentially with 5 mL of ultrapure water and 5 mL of equilibration buffer (0.4 M PB, pH 7.0). After loading the sample, the flow rate was controlled to ≤1 mL / min to bind the antibody. Non-specific proteins were washed away with 10 mL of equilibration buffer. The antibody fraction was collected with 5 mL of elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately neutralized to neutral (pH 7.0) with 1 M Tris-HCl (pH 8.0). After sterilization by filtration through a 0.22 μm filter, the sample was aliquoted and stored at -80 °C.

[0045] IV. Characterization of Monoclonal Antibodies.

[0046] 1) Subtype identification: The subtypes of 12 monoclonal antibodies were determined using a mouse antibody subtype identification kit, as shown in Table 2.

[0047] Table 2 Antibody Type Identification

[0048] 2) Titer determination: The titers of the 12 purified monoclonal antibodies were determined using the indirect ELISA method described above, with the highest titer reaching 1:729000, as shown in Table 3.

[0049] Table 3 Antibody titer determination

[0050] Example 3 Preparation of polyclonal antibodies against SpCas9 protein.

[0051] Two-month-old female New Zealand white rabbits were selected and subjected to acclimatization for one week before immunization experiments were conducted. 500 µg of the SpCas9 recombinant protein prepared in Example 1 was dissolved in PBS (pH 7.2), and then fully emulsified with an equal volume of Freund's complete adjuvant (for the first immunization) or Freund's incomplete adjuvant (for the booster immunization). The mixture was then injected subcutaneously at multiple points (neck and back, 15°, 1.5 cm depth).

[0052] The immunization schedule was as follows: a second immunization was administered 2 weeks after the first immunization, and a third immunization was administered 4 weeks after the second immunization. Blood was collected from the marginal ear vein on the 10th day after each immunization, and the antibody titer was detected by indirect ELISA (coating antigen concentration 2 µg / ml). When the titer was ≥729000, cardiac puncture was performed to collect blood. The purification steps were the same as in Example 2, and the results are shown in Table 4. The titer of the purified polyclonal antibody was 1:243000.

[0053] Table 4. Determination of the titer of purified polyclonal antibodies

[0054] Example 4 Construction and performance validation of the SpCas9 protein sandwich ELISA kit.

[0055] I. Construction of the SpCas9 protein sandwich ELISA kit.

[0056] 1) Screening with paired antibodies.

[0057] A double-antibody sandwich ELISA method was used, with 12 monoclonal antibodies prepared in Example 2 as capture antibodies and rabbit polyclonal antibodies prepared in Example 3 as detection antibodies, for pairing and screening. The specific steps are as follows: S1. Coating: Dilute each monoclonal antibody to 5 μg / mL with 0.05 M carbonate buffer (pH 9.6), add 100 μL / well to the microplate, and coat at 37°C for 3 h.

[0058] S2. Sample addition: Wash the plate 3-5 times with PBST, pat dry, and add 1000 ng / mL, 100 ng / mL, and 10 ng / mL of SpCas9 standard respectively. Add PBS as a blank control. Add 100 µl per well and react at 25℃ for 45 min.

[0059] S3. Add detection antibody: Wash the plate 3-5 times with PBST, pat dry, add rabbit polyclonal antibody (diluted 5000 times), 100 μL per well, and react at 25℃ for 30 min.

[0060] S4. Add enzyme-labeled antibody: Wash the plate 3-5 times with PBST, pat dry, add HRP-goat anti-rabbit IgG (diluted 5000 times), 100μL per well, and react at 25℃ for 30min.

[0061] S5. Color development and reading: Wash the plate 3-5 times with PBST, pat dry, add 100μL of color development reagent, and develop color at 25℃ in the dark for 15min; add 100μL of stop solution, and read the OD value at 450nm wavelength using an ELISA reader.

[0062] The test results are shown in Table 5. The monoclonal antibody with the number FL5861-10 showed the highest detection signal, while the blank control also showed a low signal. This monoclonal antibody was selected for the development of the ELISA kit.

[0063] Table 5. Pairing screening of monoclonal antibodies and polyclonal antibodies

[0064] Note: # indicates that the signal value exceeds the instrument's detection range.

[0065] The hybridoma cell line numbered FL5861-10 was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0066] The amino acid sequence of the heavy chain variable region is as follows: EVKLVESGPELVKPGASVKMSCKASGYTFSRYVMHWVKQKPGQGLEWIGYINPYNDYTKYNERFKGKATLTSDKSSSTAYMELRRLTSEDSSVYYCASATSYGSSRDWGQGTLVTVSA (SEQ ID No. 1); The amino acid sequence of the light chain variable region is as follows: DIQMTQSPAIMSASPGEKVTMTCSASSSVNYMHWYQQKSGTSPKRWIHDTSKLASGVPARFSGRGSGTSYSLTISSMEAEDAATYYCQQWSSDPPTFGGGTKLELK (SEQ ID No. 2).

[0067] 2) Antibody specificity detection.

[0068] SpCas9 gene-edited and wild-type rice leaf proteins were extracted separately and subjected to SDS-PAGE electrophoresis, with a positive control of recombinant SpCas9 protein. After electrophoresis, the proteins were transferred to an NC membrane. Primary antibodies were FL5861-10 monoclonal antibody (1:1000) and rabbit polyclonal antibody (1:1000), respectively; secondary antibodies were HPR-labeled goat anti-mouse secondary antibody (1:10000) and HPR-labeled goat anti-rabbit secondary antibody (1:10000), respectively. ECL luminescent substrate was added, and protein bands were detected using a chemiluminescence imaging system. Experimental results are shown below. Figure 2As shown, the FL5861-10 monoclonal antibody and the rabbit polyclonal antibody can specifically recognize the SpCas9 recombinant protein and the Cas9 protein expressed in rice.

[0069] 3) Preparation of sandwich ELISA kit.

[0070] Microplates pre-coated with capture antibodies: 48 wells / plate, coated with FL5861-10 monoclonal antibody at a concentration of 5 μg / mL, coated with 0.05 M carbonate buffer (pH 9.6), coated at 4°C for 12 h, washed 3 times with PBST (0.05% Tween-20), then blocked with blocking buffer (1% BSA + 5% sucrose, 200 μL / well), blocked at 37°C for 3 h, discarded the blocking buffer, dried at 37°C for 30 min, and then packaged in aluminum foil bags (with desiccant inside, humidity <20%) and vacuum sealed. Rabbit polyclonal antibody working solution (dilution ratio 1:5000, containing 1% BSA), 6mL / vial; Enzyme-labeled working solution: 6 mL / bottle, which is a 1:5000 dilution of HRP-labeled goat anti-rabbit IgG working solution; SpCas9 protein standard (96 ng): The concentration is 96 ng / mL after reconstitution with 1 mL of sample processing solution; Sample processing solution: 50mL / bottle, the main component is PBST; 20× Concentrated Washing Solution: 20mL / bottle, containing 20×PBST, diluted 20 times with ultrapure water before use; Colorimetric reagent: 6 mL / bottle, in TMB form; Stop solution: 6 mL / bottle, is a 2M sulfuric acid solution; Board sticker: 1 sheet.

[0071] Take one portion of each of the above-mentioned test reagents and consumables, assemble them into one kit, and store at 2-8℃.

[0072] The reagent kit testing steps are as follows: S1. Take the required test reagents and microplates out of the refrigerated environment and allow them to equilibrate at room temperature (20-25℃) for 30 minutes.

[0073] S2. Add sample / standard: First, prepare the test sample. Take 0.2g of rice leaves, put them into a 1.5mL centrifuge tube and crush them. Then add 1mL of sample processing solution (equivalent to a 5-fold dilution), shake and mix for 5min; centrifuge at 4000rpm for 3min, and take 100µL of supernatant for detection. Next, prepare the standard by dissolving it in the sample extract at an initial concentration of 48 ng / mL, followed by a 2-fold serial dilution to 1.5 ng / mL. Add 100 µL of the test sample, standard, and sample extract (blank control) to each well, gently vortex to mix, and incubate at 25°C in the dark for 45 min. Shake off the liquid in the wells, add 250 mL of PBST per well, and wash thoroughly 4-5 times, with 10-second intervals between each wash. Pat dry with absorbent paper.

[0074] S3. Add enzyme-labeled working solution: 100µL / well, gently shake to mix, react at 25℃ in the dark for 30 min, and repeat the plate washing step.

[0075] S4. Color development: Add 100µL of color developer per well and react in a 25℃ dark environment for 15 min.

[0076] S5. Measurement: Add 100µL of stop solution per well, gently shake to mix, and set the microplate reader to 450nm to measure the OD value of each well. Plot a standard curve based on the standard readings, and calculate and convert the SpCas9 concentration by substituting the absorbance values ​​of the test samples into the standard curve.

[0077] II. Performance verification of the SpCas9 protein sandwich ELISA kit.

[0078] 1) Reagent kit sensitivity test.

[0079] Following the above kit detection steps, the SpCas9 protein standard was diluted to gradient concentrations of 48, 24, 12, 6, 3, and 1.5 ng / mL using sample processing buffer. Three replicates were set up for each concentration, and 20 blank control wells were also included. The OD of each well was measured. 450 Value. With the concentration of the standard as the x-axis, OD... 450 The value is the ordinate, and a four-parameter logistic regression equation is used to fit the curve, yielding the standard curve equation. The results are as follows: Figure 3 As shown, the standard curve fitting equation is: .

[0080] Among them, the correlation coefficient R 2 =0.9984, indicating excellent fit. (OD value was used as a blank control.) 450 The limit of detection (LOD) of the kit is calculated to be 0.56 ng / mL based on the mean (n=20) plus three times the standard deviation, demonstrating extremely high detection sensitivity.

[0081] 2) Reproducibility test of the kit.

[0082] Six SpCas9 standards with concentrations of 48, 24, 12, 6, 3, and 1.5 ng / mL were selected, with three replicates for each concentration. Testing was performed within the same batch, and the measured concentrations were calculated based on the standard curve. The intra-batch coefficient of variation (CV) was then calculated. Inter-batch precision: The above six concentrations of SpCas9 standards were repeatedly tested in three different batches (on different days), and the coefficient of variation (CV) was calculated. The CV calculation formula is: CV = (SD / Mean) × 100%.

[0083] Where SD represents the standard deviation of the test results, and Mean represents the average value of the test results.

[0084] The results are shown in Table 6. The measured concentrations were in good agreement with the theoretical concentrations. The intra-batch CV was 0.98%-3.90%, all <5%; the inter-batch CV was 0.70%-7.34%, all <10%. This indicates that the kit has excellent intra-batch and inter-batch repeatability and the detection results are stable and reliable.

[0085] Table 6. Coefficients of variation for intra- and inter-batch repeatability tests of the reagent kit

[0086] 3) Matrix interference verification.

[0087] Wild-type rice extract was used as the matrix. Diluted solutions of stock solution, 1:2, 1:4, 1:8, 1:16, and 1:32 were established, and standards (12 ng / mL) were added to each solution. Three replicates were performed for sandwich ELISA detection. The results are shown in Table 7. The recoveries of the standards ranged from 96.0% to 110.5%, indicating that this method has good tolerance to the rice matrix, can directly detect the extract without dilution, and is easy to operate.

[0088] Table 7 Evaluation of the matrix effect of rice leaf extract

[0089] 4) Spike recovery verification.

[0090] Wild-type rice extract was used as the matrix, and six concentrations of standards (48, 24, 12, 6, 3, and 1.5 ng / mL) were added. Three replicates were set up for sandwich ELISA detection. The results are shown in Table 8, with recoveries ranging from 97.0% to 109.1%.

[0091] Table 8 Recovery rate of spiked SpCas9 protein in rice leaf extract

[0092] 5) Dilution linearity verification.

[0093] Two extracts of SpCas9 gene-edited rice leaves were used, and sandwich ELISA was performed at dilutions of stock solution, 1:2, 1:4, and 1:8, with three replicates. The results are shown in Table 9. At a 1:2 dilution, the recoveries were 102.9% and 109.0%, respectively; at a 1:4 dilution, the recoveries were 102.9% and 109.0%, respectively; at a 1:8 dilution, the recoveries were 97.5% and 111.8%, respectively; and at a 1:8 dilution, the recoveries were 96.8% and 114.1%, respectively, all within the range of 80-120%.

[0094] Table 9. Linearity evaluation of dilution of SpCas9 gene-edited rice leaf extract

[0095] 6) Specificity verification.

[0096] The specificity of the kit was investigated by detecting SaCas9, FnCas12a, BrCas12b, and LwaCas13a proteins (final concentration 48 ng / mL). Triple replicates were performed using sandwich ELISA. Results are shown below. Figure 4 As shown, the detection signals of the four Cas proteins were all below the detection limit, indicating that the kit did not cross-react with the four Cas proteins.

[0097] Example 5 The kit was used to detect the SpCas9 protein in the leaves of gene-edited rice.

[0098] Leaves from 16 T2 generation SpCas9 gene-edited rice plants and 2 wild-type rice plants were selected, and the SpCas9 protein content in the rice leaves was detected using the kit detection steps described in Example 4.

[0099] Sample pretreatment: Take 0.2g of each rice leaf sample, put them into a 1.5mL centrifuge tube and crush them thoroughly. Add 1mL of sample treatment solution, shake and mix for 5min, centrifuge at 8000rpm for 3min, and take the supernatant as the sample solution to be tested.

[0100] Detection and Result Calculation: The detection was completed according to the kit detection steps. The content of SpCas9 protein in each sample was calculated based on the standard curve. The results are shown in Table 10. The detection results of wild-type rice leaf samples were all negative (below the detection limit), while SpCas9 protein was detected in the leaves of all 16 SpCas9 gene-edited rice plants.

[0101] Table 10 Results of SpCas9 protein detection in gene-edited rice leaves

[0102] Note: Data are expressed as mean ± standard deviation (n=3); LOD (limit of detection) = 0.56 ng / mL. 1-16 represent SpCas9 gene-edited rice, and 17-18 represent wild-type rice.

[0103] In summary, this invention utilizes a monoclonal antibody as the capture antibody and a rabbit-derived polyclonal antibody as the detection antibody, and through system optimization, a double-antibody sandwich ELISA detection kit has been constructed. This kit exhibits a detection limit as low as 0.56 ng / mL, demonstrating extremely high detection sensitivity; intra-assay coefficient of variation is less than 5%, and inter-assay coefficient of variation is less than 10%; the detection results are stable, reliable, and highly reproducible; simultaneously, it can achieve simultaneous detection of dozens to hundreds of samples in a single batch, with simple operation and a short detection cycle. It completely overcomes the shortcomings of traditional methods such as Western blot and immunoprecipitation, which are unable to perform high-throughput detection and are cumbersome and time-consuming, fully meeting the needs for rapid screening and accurate quantification of SpCas9 protein in large batches of samples.

[0104] The monoclonal antibody and detection kit provided by this invention have a wide range of applications and strong adaptability. They can provide a stable and reliable detection tool for quantitative analysis of SpCas9 protein expression levels, optimization of gene editing conditions, and research on the correlation between editing efficiency and protein expression levels in basic life science research. They can also provide precise technical means for screening SpCas9 protein residues in gene-edited crops and selecting non-GMO edited varieties in the field of crop genetics and breeding. Furthermore, they can provide scientific quantitative evidence for quality control of gene-edited products in the biopharmaceutical field, biosafety risk assessment of gene-editing technology applications, and industry regulation.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A monoclonal antibody against SpCas9 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The anti-SpCas9 protein monoclonal antibody according to claim 1, characterized in that, The heavy chain subclass of the monoclonal antibody is IgG2a, and the light chain is the κ chain.

3. A SpCas9 protein detection kit, characterized in that, The kit comprises a microplate and a detection reagent, wherein the microplate contains the anti-SpCas9 protein monoclonal antibody as described in claim 1.

4. The SpCas9 protein detection kit according to claim 3, characterized in that, In the microplate, the coating concentration of the anti-SpCas9 protein monoclonal antibody is 5 μg / mL, and the coating buffer is 0.05 M carbonate buffer at pH 9.

6.

5. The SpCas9 protein detection kit according to claim 3, characterized in that, The detection reagent includes an anti-SpCas9 protein polyclonal antibody, enzyme-labeled working solution, SpCas9 protein standard, sample processing solution, concentrated washing solution, chromogenic agent, and stop solution.

6. The SpCas9 protein detection kit according to claim 5, characterized in that, The enzyme-labeled working solution is HRP-labeled goat anti-rabbit IgG; the concentrated washing solution is 20×PBST; the chromogenic agent is TMB chromogenic solution; and the stop solution is 2M sulfuric acid solution.

7. The SpCas9 protein detection kit according to claim 5, characterized in that, The amount of the anti-SpCas9 protein polyclonal antibody is 6 mL; the amount of the enzyme-labeled working solution is 6 mL; the amount of the SpCas9 protein standard is 96 ng; the amount of the sample processing solution is 50 mL; the amount of the concentrated washing solution is 20 mL; the amount of the chromogenic reagent is 6 mL; the amount of the stop solution is 6 mL; and the kit is stored at a temperature of 2-8℃.

8. The application of the SpCas9 protein detection kit according to any one of claims 3-7 in the detection of SpCas9 protein.

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