Ulp1 enzyme double-antibody sandwich elisa kit and application

Abstract

The application belongs to the technical field of Ulp1 enzyme quantitative detection, and particularly relates to a Ulp1 enzyme double-antibody sandwich ELISA kit and application. The kit comprises an enzyme-labeled plate coated with anti-Ulp1 enzyme mouse monoclonal antibody, anti-Ulp1 enzyme rabbit polyclonal antibody, HRP-labeled goat anti-rabbit antibody, diluent, cleaning liquid, blocking liquid, coloring liquid and termination liquid. The application uses the complex of Ulp1 enzyme and SUMO tag protein as an immunogen, immunizes mice and New Zealand rabbits at the same time, and prepares Ulp1 enzyme antibodies of different species and good affinity. Then, by further screening the optimal reaction conditions, the application develops a double-antibody sandwich ELISA detection kit with high specificity, strong sensitivity and stability, which can more effectively solve the deficiencies of the prior art.

Description

Technical Field

[0001] This invention belongs to the field of Ulp1 enzyme quantitative detection technology, specifically relating to an Ulp1 enzyme double antibody sandwich ELISA kit and its application. Background Technology

[0002] Escherichia coli is one of the important prokaryotic expression hosts in biological research and industrial applications. Since the advent of the first biosimilar, human insulin, in 1982, E. coli has become a core tool for the production of recombinant protein drugs. Its advantages include: Rapid growth: doubling time is only 20-30 minutes, suitable for industrial scale-up; Clear genetic background: mature gene manipulation techniques and a wide variety of expression vectors; Low cost: simple culture medium and mature fermentation process; High expression levels: Recombinant proteins account for more than 20% of total cellular protein.

[0003] The main limitation of Escherichia coli as an expression host is the lack of eukaryotic post-translational modification functions (such as glycosylation) and an imperfect folding helper system, which may lead to mispairing of protein disulfide bonds or conformational abnormalities. Under high expression conditions, recombinant proteins often accumulate in the form of inactive aggregates (inclusion bodies).

[0004] Strategies for reducing inclusion body formation in E. coli include: 1. Lower the culture temperature: Lowering the culture temperature from the usual 37℃ to 16-25℃ can slow down the protein synthesis rate, prolong the correct folding time, and reduce aggregation caused by hydrophobic interactions.

[0005] 2. Control the induction intensity and timing: Use low concentrations of inducers (such as IPTG concentration) or mild induction strategies (such as automated induction medium) to avoid overexpression of exogenous proteins that could lead to aggregation.

[0006] 3. Co-expression of molecular chaperones and folding enzymes: Introducing chaperone systems such as GroEL / GroES, DnaK / DnaJ / GrpE, or folding enzymes such as thioredoxin (Trx) to assist in the correct folding of recombinant proteins.

[0007] 4. Add folding aids: Add osmotic pressure protectants such as sorbitol and sucrose, or an appropriate amount of L-arginine to the culture medium to improve the folding microenvironment.

[0008] 5. Optimize signal peptide design: Direct the protein into the periplasmic space through secretory expression (e.g., using the PelB signal peptide) and utilize the oxidative environment to promote proper disulfide bond pairing.

[0009] 6. Select suitable strains: Use protease-deficient strains (such as BL21(DE3)pLysS) to reduce protein degradation, or use engineered strains that express chaperone proteins (such as the Origami series).

[0010] 7. Optimize the composition of the culture medium: provide a nutrient-rich culture medium (such as adding glucose and trace elements), and strictly control the dissolved oxygen content (≥30%) and pH (6.8-7.4) to maintain cell metabolic homeostasis.

[0011] 8. Fusion soluble tags: Use tags such as GST, MBP or SUMO to enhance the solubility of the target protein and reduce the tendency to aggregate.

[0012] Table 1 Properties of common dissolving labels SUMO is a ubiquitin-like protein derived from Saccharomyces cerevisiae. Its ubiquitin-like domain can stabilize the folded intermediate state, promote the correct pairing of disulfide bonds, and reduce the exposure of hydrophobic regions, thereby improving the conformational integrity of complex proteins and significantly reducing the tendency of recombinant proteins to aggregate. It is particularly suitable for the expression of poorly soluble eukaryotic proteins in prokaryotic systems.

[0013] Table 2 Advantages of the SUMO Labeling System SUMO tags can improve solubility and stability during recombinant protein expression, but they can mask the target protein’s native active site or affect its higher-order structure. Therefore, they need to be precisely removed to obtain a target protein with intact structure and undisturbed function.

[0014] A specific protease (Ulp1 protease) can be used to hydrolyze the peptide bond after the second glycine in the C-terminal Gly-Gly-x sequence. The cleavage efficiency is higher than that of tool proteases such as Factor Xa, Thrombin, TEV, and 3C. Furthermore, after cleavage by Ulp1 protease, there are no redundant tag amino acids remaining at the N-terminus of the target protein, which is an advantage that other solubilizing tags do not possess.

[0015] The Ulp1 enzyme needs to be completely removed after the SUMO tag is cleaved, mainly for the following four reasons: 1. Avoid non-specific protein degradation: As a cysteine ​​protease, Ulp1 retains its catalytic activity even after SUMO tag cleavage. If it remains in the sample, it may recognize and cleave other SUMO-modified proteins (such as endogenous SUMOylated proteins), leading to non-target protein degradation or functional abnormalities. Although commercially available Ulp1 enzymes claim to be "free from non-specific protease contamination," residual enzymes still pose a risk of accidental cleavage.

[0016] 2. Maintaining the purity and stability of the target protein: The Ulp1 enzyme has a molecular weight of approximately 45 kDa and contains a His tag (a common design feature of commercial enzymes). Residual enzyme molecules can contaminate the final product, especially when the target protein is intended for structural analysis, drug development, or functional experiments. Impurity enzymes may interfere with experimental results or reduce protein homogeneity. For example, residual His tags can affect the surface charge properties of the protein, and enzyme molecules may form dimers or multimers, increasing sample complexity.

[0017] 3. Preventing interference from downstream applications: In enzyme kinetics, cell experiments, or animal models, residual Ulp1 enzyme may continuously cleave SUMOylated proteins in the experimental system, altering signaling pathway activity; in addition, it may induce an immune response when applied in vivo.

[0018] 4. Compliance with pharmaceutical protein manufacturing standards: In the production of recombinant protein drugs, pharmacopoeias of various countries have strict limits on residual impurities (such as the requirement that residual host cell protein levels be below ppm). Even trace amounts of proteases can affect product safety and batch-to-batch consistency, and therefore must be eliminated.

[0019] Commercially available Ulp1 enzymes typically have a His tag (6× or 8× His tag) at their N-terminus. After enzymatic digestion, the mixture contains: target protein (without His tag), Ulp1 enzyme (His tag), and free SUMO tag (His tag). Secondary purification using a nickel column allows the target protein to flow through, while the Ulp1 enzyme and free SUMO tag are adsorbed and removed, reducing the risk of enzyme residue. Therefore, effectively detecting Ulp1 enzyme derived from prokaryotic recombinant expression of *Saccharomyces cerevisiae* has become a pressing technical problem in this field. Summary of the Invention

[0020] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a rapid, simple, and high-throughput double-antibody sandwich ELISA kit for detecting Ulp-1 enzyme residues in samples. This kit can effectively detect Ulp1 enzyme derived from prokaryotic recombinant expression in Saccharomyces cerevisiae. This invention utilizes a recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein as an immunogen to simultaneously immunize mice and New Zealand rabbits, preparing Ulp1 enzyme antibodies of different species with good affinity. Then, through further screening for optimal reaction conditions, a double-antibody sandwich ELISA detection kit with high specificity, high sensitivity, and stability was developed, effectively addressing the shortcomings of existing technologies.

[0021] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A sandwich ELISA kit containing two anti-Ulp1 enzyme antibodies includes: an enzyme-labeled plate coated with mouse monoclonal antibody against Ulp1 enzyme, rabbit polyclonal antibody against Ulp1 enzyme, HRP-labeled goat anti-rabbit antibody, diluent, washing buffer, blocking buffer, chromogenic buffer, and termination buffer.

[0022] Preferably, the Ulp1 enzyme double-antibody sandwich ELISA kit provided by the present invention includes: an ELISA plate coated with anti-Ulp1 enzyme mouse monoclonal antibody, anti-Ulp1 enzyme rabbit polyclonal antibody, HRP-labeled goat anti-rabbit antibody, diluent (PBST containing 0.1-0.5% BSA), washing buffer (PBST), blocking buffer (PBST containing 2-5% skim milk powder), chromogenic solution and stop solution.

[0023] HRP-labeled goat anti-rabbit antibodies are commercially available, for example, (model / catalog number D110058-0001, specification 1ml).

[0024] Preferably, to achieve the above objectives, the method of the present invention includes the following steps: Recombinant expression of Ulp1 enzyme and SUMO-tagged protein: Ulp1 enzyme (amino acid sequence: lvpelnekdddqvqkalasrentqlmnrdnieitvrdfktlaprrwlndtiieffmkyiekstpntvafnsffytnlsergyqgvrrwmkrkktqidkldkiftpinlnqshwalgiidlkkktigyvdslsngpnamsfailtdlqkyvmeeskhtigedfdlihldcpqqpngydcgiyvcmntlygsadapldfdykdairmrrfi) was synthesized via gene synthesis. The expression genes for the Ulp1 enzyme and the SUMO tag protein (amino acid sequence: mghhhhhmsdsevnqeakpevkpevkpethinlkvsdgsseiffkikkttplrrlmeafakrqgkemdslrflydgiriqadqtpedldmedndiieahreqigg) were constructed into the pET28a vector. The constructed pET28a vectors containing the expression genes for both the Ulp1 enzyme and the SUMO tag protein were then transformed into BL21(DE3) expression strains. The competent E. coli cells (BL21(DE3)) were removed from a -80°C freezer and slowly thawed on ice. 10 μL of plasmid DNA (approximately 100 ng) was added to 100 μL of competent cells and gently mixed, avoiding vigorous shaking that could damage the cells. Incubate on ice for 30 minutes, then heat shock in a 42°C water bath for 60-90 seconds to promote DNA entry into cells. Immediately return to ice to cool for 2 minutes, add 900 μL of antibiotic-free LB liquid medium, and incubate at 37°C with shaking for 45-60 minutes to allow bacteria to recover growth and express resistance genes. Spread an appropriate amount of bacterial culture onto LB agar plates containing the corresponding antibiotic (kanamycin, Kansei), and incubate upside down at 37°C for 12-16 hours to screen for positive transformants. Select independent single colonies from the plates and inoculate them into 5 mL of LB liquid medium containing antibiotics, and incubate overnight at 37°C (approximately 12-16 hours). Transfer the overnight bacterial culture to fresh LB medium at a ratio of 1:50 to 1:100 (V / V), and incubate at 37°C with shaking until the OD600 reaches 0.6-0.8. Add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1-1 mM, and induce at 37°C for 3-6 hours. After induction, collect the bacterial cells by centrifugation (e.g., at 4°C, 5000-8000 rpm, for 10 minutes), and discard the supernatant.

[0025] The collected bacterial sludge was resuspended and dissolved in equilibration buffer (25 mM Tris, 500 mM NaCl, 20% glycerol, pH 7.4) at a ratio of bacterial sludge to equilibration buffer of 1:10 (m / V). Lysis was performed using sonication: 200 W power, pulsed operation (5 seconds on, 5 seconds off), for 10-20 minutes, maintaining a low temperature. After centrifugation at 11000 rpm for 10-20 minutes, the supernatant was collected for purification. A Ni SmartBeads 6FF column was prepared and equilibrated with equilibration buffer (25 mM Tris, 500 mM NaCl, 20% glycerol, pH 7.4). The prepared sample was then loaded onto the Ni SmartBeads 6FF gravity column. After loading, the column was washed with washing buffer (25 mM Tris, 500 mM NaCl, 20% glycerol, 20 mM imidazole, pH 7.4). Elution with elution buffer (25 mM Tris, 500 mM NaCl, 20% glycerol, 200 mM imidazole, pH 7.4) yielded recombinant proteins of Ulp1 enzyme and SUMO-tagged protein, respectively.

[0026] The recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein was prepared by mixing 200 mg of recombinant SUMO-tagged protein with 1 mg of recombinant Ulp1 enzyme and allowing it to react at 2-8°C for 16 hours. Since the N-terminus of the SUMO-tagged protein carries a 6×His tag, affinity purification can be used. 10 ml of Ni NTA Beads 6FF was packed into a gravity chromatography column, and equilibrated with 100 ml of equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) at a flow rate of 3 ml / min. After the reaction, the sample was loaded into the Ni NTA Beads 6FF gravity column at a flow rate of 2 ml / min. After loading, 50 ml of washing buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) was used to wash away impurities. Elution was performed by adding elution buffer (50 mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, pH 8.0) to obtain a recombinant protein complex of Ulp1 enzyme and SUMO tag protein (electrophoresis confirmed the simultaneous presence of Ulp1 enzyme and SUMO tag protein in the elution).

[0027] The “recombinant protein complex of Ulp1 enzyme and SUMO tag protein” referred to in this application refers to a binding system / complex formed after the two are mixed and incubated in vitro, which can be eluted together by subsequent affinity purification and can be detected simultaneously by electrophoresis.

[0028] The recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein is mixed with a loading buffer containing SDS and a reducing agent (such as β-mercaptoethanol), and heated at 95-100°C for 5-10 minutes to denature the protein, break the disulfide bonds, and fully bind to SDS to form a negatively charged linear complex. A commercially available precast protein electrophoresis gel (SmartPAGE PrecastProtein Gel Plus 4-20% 12 Wells) is prepared. The processed sample is added to the wells of the gel using a micropipette, while pre-stained protein markers are added to adjacent wells as molecular weight references.

[0029] Connect the power supply and set the voltage to 160-180V. Under the influence of the electric field, the protein-SDS complex migrates towards the positive electrode. Smaller proteins migrate faster, while larger proteins migrate slower. After electrophoresis, remove the gel, stain it with Coomassie Brilliant Blue, and then destain it with destaining solution until the background is clear. The protein bands can then be observed. The accuracy of the recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein is determined by comparing the migration distance of the recombinant protein complex band of Ulp1 enzyme and SUMO-tagged protein with that of the standard protein marker. Figure 3 As can be seen in the electrophoresis of the recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein, there is a clear band of Ulp1 enzyme recombinant protein (green arrow) and a band of SUMO-tagged protein recombinant protein (red arrow). The molar ratio of Ulp1 enzyme recombinant protein to SUMO-tagged protein recombinant protein in the Ulp1 enzyme-SUMO-tagged protein recombinant protein complex is 1:1.

[0030] The 3D structure of the recombinant protein complex can be referenced. Figure 4 The Ulp1 enzyme is in green, and the SUMO-tagged protein is in yellow.

[0031] (1) Mice were immunized with a recombinant protein complex of Ulp1 enzyme and SUMO tag protein. Then, multiple hybridoma cell lines were obtained through hybridoma. The desired clones were selected by affinity screening, and the antibody sequence was obtained by sequencing technology. The sequence number is 7H2 (see amino acid and gene sequence of mouse antibody). The sequence was used to construct a eukaryotic expression plasmid through gene synthesis and recombination technology, and recombinant expression was performed in Chinese hamster ovary cells. (2) Select healthy adult New Zealand white rabbits and immunize them with a recombinant protein complex of Ulp1 enzyme and SUMO tag protein. After the first immunization and multiple booster immunizations, determine the antibody titer in the serum and collect blood for purification of polyclonal antibodies. The obtained antibody has an SDS-PAGE purity of over 90%.

[0032] (3) After obtaining the two antibodies, prepare the double-antibody sandwich ELISA kit. The steps are as follows: 1. Dilute the anti-Ulp1 mouse monoclonal antibody to 6ug / ml with coating buffer (CBS), add 100ul to each well of the microplate for coating, and coat at 4℃ for 16-20h. 2. After cleaning each well three times with 100ul-300ul of cleaning solution, add sealing solution and seal at 37℃ for 1-2 hours, then pat dry.

[0033] 3. Dilute the Ulp1 enzyme to a standard concentration of 810 ug / ml using diluent. Using this concentration as a baseline, continue to dilute it in multiple gradients of 3-fold. Add the diluted Ulp1 enzyme to the coated microplate and incubate at 37°C for 1-2 hours.

[0034] 4. Clean each well three times with 100ul-300ul of cleaning solution, then pat dry.

[0035] 5. Add anti-Ulp1 enzyme rabbit polyclonal antibody dilution buffer to a final concentration of 6ug / ml, and incubate at 37℃ for 1 hour.

[0036] 6. Clean each well three times with 100ul-300ul of cleaning solution, then pat dry.

[0037] 7. Add HRP-labeled goat anti-rabbit antibody dilution buffer at a dilution ratio of 1:5000, and incubate at 37°C for 1-2 hours to bind.

[0038] 8. Clean each well three times with 100ul-300ul of cleaning solution, then pat dry.

[0039] 9. Add color developing solution and develop at 25°C in the dark for 5 minutes.

[0040] 10. Add stop solution to terminate the process, and take a reading at OD450nm.

[0041] 11. Based on the readings of the microplate reader, match them with the logarithmic values ​​of the Ulp1 enzyme standard concentration to obtain a standard curve.

[0042] Because the Ulp1 enzyme specifically recognizes the tertiary structure of the SUMO tag for enzymatic digestion, and forms a complex structure after digestion, this invention uses the Ulp1 enzyme and SUMO tag recombinant protein complex as an antigen for immunization. This ensures the structure of the Ulp1 enzyme during immunization, resulting in antibodies with better specificity and providing favorable conditions for kit development.

[0043] Preferably, the application of the enzyme-labeled plate coated with the anti-Ulp1 enzyme mouse monoclonal antibody, the anti-Ulp1 enzyme rabbit polyclonal antibody, and the HRP-labeled goat anti-rabbit antibody in detecting the prokaryotic recombinant expression of Ulp1 enzyme derived from Saccharomyces cerevisiae.

[0044] The beneficial effects of this invention are as follows: (1) The recombinant protein complex of Ulp1 enzyme and SUMO tag protein serves as an immunogen, ensuring that targeted antibodies can be generated in mice and rabbits. This ensures that the prepared genetically engineered mouse monoclonal antibody and the rabbit polyclonal antibody obtained by rabbit serum purification have specific recognition capabilities for Ulp1 enzyme, laying a good foundation for the subsequent development of a double-antibody sandwich ELISA kit with high specificity and high sensitivity.

[0045] (2) Antibodies obtained by recombinant expression or immunization are purified by antigen-specific affinity to ensure the specificity of the antibodies and to prevent them from being affected by host impurity proteins or other immunoglobulins.

[0046] (3) The kit prepared by the present invention has the advantages of high sensitivity (the lowest concentration in the sample can be detected at 0.02 ng / ml), wide detection range, good repeatability, high specificity (low blank background of ELISA), and good stability. It can reduce detection costs and make up for the defects of Ulp1 enzyme in the production process of drug protein. Attached Figure Description

[0047] Figure 1 This is an SDS-PAGE electrophoresis image of rabbit polyclonal antibody antigen affinity purification provided in a specific embodiment of the present invention; Figure 2 This is a standard curve of the Ulp1 enzyme provided in a specific embodiment of the present invention; Figure 3 This is an electrophoresis diagram of the recombinant protein complex of the Ulp1 enzyme and the SUMO-tagged protein; Figure 4 This is a 3D structural diagram of the recombinant protein complex of the Ulp1 enzyme and the SUMO-tagged protein. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0049] Example 1: Preparation of mouse monoclonal antibodies This experiment employed the BALB / c mouse immunohybridoma technique, completing monoclonal antibody preparation over approximately 5 months. Recombinant expression was then achieved after antibody sequencing. Key stages included: antigen emulsification, animal immunization, cell fusion, hybridoma screening, antibody sequencing, and validation of recombinant expression.

[0050] I. Mouse Immunization Phase Day 1-7 Antigen Preparation: The recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein (150 μg) was mixed with an equal volume of complete Freund's adjuvant and emulsified by injecting 20 times through a three-way valve under ice bath conditions to form a water-in-oil structure. Emulsification Verification: 5 μl of the emulsion was dropped into clear water, and a complete emulsion droplet that maintained non-diffusion for 30 seconds was considered qualified.

[0051] Day 8 First Immunization: Intraperitoneal injection of 50 μl of emulsified antigen (containing 50 μg of protein), and the mice were separately raised after injection to observe allergic reactions.

[0052] Day 22 & Day 36 Booster Immunization: Incomplete Freund's adjuvant was used instead, and the dose was reduced to 30 μg per injection. The injection site was rotated to the subcutaneous area of the back.

[0053] Day 50 Titer Detection: Blood was collected from the orbital cavity to isolate serum, and the titer was detected by indirect ELISA. When the titer was ≥1:6400, the mice entered the sprint immunization stage.

[0054] Day 53 Sprint Immunization: Intravenous injection of 100 μg of adjuvant-free antigen solution.

[0055] II. Hybridoma Preparation Stage Day 56-58 Cell Fusion: The mice were sacrificed by cervical dislocation, and the spleen was aseptically taken to prepare a single cell suspension (5×10 7 cells), which was mixed with SP2 / 0 myeloma cells at a ratio of 5:1. 50% PEG-1500 was used to promote fusion for 90 seconds, and screening was carried out with HAT medium. The cells were cultured in a 37°C / 5% CO2 incubator. Day 59-87 Hybridoma Screening: On the 7th day of culturing in a 37°C / 5% CO2 incubator, ELISA was used for primary screening of positive wells (OD450nm > 1.0). On the 14th day, limited dilution cloning was carried out (an average of 0.8 cells per well). After three subclonings, a stable secreting cell line was obtained.

[0056] III. Antibody Sequencing Stage Day 88-101 Gene Cloning: TRIzol method was used to extract hybridoma RNA, and cDNA was obtained by reverse transcription; specific primers for IgG were designed to amplify the variable regions of the heavy and light chains; after TA cloning, it was sent for Sanger sequencing, and the sequence information was analyzed by software.

[0057] IV. Recombinant Expression Stage Day 102-122 Vector Construction: The heavy and light chain genes were separately cloned into the pcDNA3.4 vector; 293F cells were transfected (by electroporation), and the supernatant was collected 72 hours after transfection.

[0058] Days 123-129 Purification and Identification: Purification was performed using an rProtein A column, and the purity was verified to be >95% by SDS-PAGE. Binding activity was also verified by indirect ELISA.

[0059] Example 2: Preparation of rabbit polyclonal antibodies A recombinant protein complex of Ulp1 enzyme and SUMO-tagged protein was used as an immunogen to immunize New Zealand white rabbits via subcutaneous injection at multiple sites on the back. The initial immunization consisted of 400 μg, with booster immunizations of 200 μg every 14 days. The initial immunization used an equal volume of Freund's complete adjuvant and antigen, while the booster immunization used an equal volume of Freund's incomplete adjuvant and antigen. One week after the fourth immunization, blood was collected from the marginal ear vein to measure serum antibody titers. When the serum titer met the requirements (ELISA detection of antibody activity in serum, with an OD450 absorbance value of ≥0.5 at a 1:64000 dilution), blood was collected from the middle ear artery, the animals were euthanized, and the serum was separated by centrifugation. Anti-Ulp1 enzyme rabbit polyclonal antibodies were obtained through antigen-specific purification. (See electrophoresis image below.) Figure 1 .

[0060] Example 3: Establishment and optimization of the double-antibody sandwich ELISA method 1. Basic steps of the testing method 1) Coating the microplate: Dilute the anti-Ulp1 mouse monoclonal antibody to 6ug / ml with coating buffer (CBS), add 100ul to each well of a 96-well microplate for coating, and coat at 4℃ for 16h. Then wash three times with 300ul PBST per well, washing for 1-2min each time. Pat the microplate dry on absorbent paper. 2) Blocking the microplate: Use 100 μL of blocking buffer per well (blocking buffer is PBST with 2-5% skim milk powder added. PBST composition: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM, Tween-20 0.1% (w / v)) and block at 37℃ for 1 h. After incubation, discard the blocking buffer and wash three times with 300 μL of PBST per well, washing for 1-2 min each time. Pat the microplate dry on absorbent paper. 3) Add Ulp1 enzyme standard curve: Dilute the Ulp1 enzyme stock solution to a standard concentration of 810ug / ml with diluent, and then perform multiple serial dilutions at a 3-fold gradient. Add the diluted Ulp1 enzyme to the coated microplate and incubate at 37°C for 1 hour. After incubation, remove the blocking solution and wash three times with 300ul PBST per well for 1-2 minutes each time. Pat the microplate dry on absorbent paper.

[0061] 4) Add detection antibody: Dilute the anti-Ulp1 enzyme rabbit polyclonal antibody with diluent, add 100ul of diluted antibody to each well to ensure a final concentration of 6ug / ml, incubate at 37℃ for 1h, remove blocking solution after incubation, and then wash three times with 300ul PBST per well for 1-2 minutes each time. Pat dry the ELISA plate on absorbent paper.

[0062] 5) Add enzyme-labeled antibody: Dilute the HRP-labeled goat anti-rabbit antibody with diluent at a ratio of 1:5000. Add 100 μL to each well of the microplate and incubate at 37°C for 1-2 hours. After incubation, remove the blocking solution and wash three times with 300 μL PBST per well for 1-2 minutes each time. Pat the microplate dry on absorbent paper.

[0063] 6) Add colorimetric solution: Add 100 μL of TMB colorimetric solution to each well of the microplate and incubate at 25°C in the dark for 5 min.

[0064] 7) Add stop solution: Add 100 μL of 0.5 M phosphate stop solution to each well of the microplate to stop the color development, and take the reading at OD 450 nm.

[0065] 8) Reading: Read and record the values ​​using a microplate reader at OD 450nm wavelength. Calculation and analysis results are shown below. Figure 2 .

[0066] 2. Condition Optimization 1) Selection of optimal temperature and time for antibody coating With 500 ng of mouse monoclonal antibody against Ulp1 enzyme per well, four conditions were selected: 37℃ for 1 h, 37℃ for 2 h, 37℃ for 3 h, and 4℃ for 16 h. The final concentration of rabbit polyclonal antibody against Ulp1 enzyme was measured to be 2.5 ug / ml. Taking into account the N value, the condition with the largest P / N value was determined as the optimal coating condition. The results are shown in Table 3. These results prove that the optimal coating condition is 4℃ for 16 h.

[0067] Table 3 Selection of Plate Temperature and Time .

[0068] 2) Selection of the optimal concentration of antibody for plate coating With the coating antibody concentrations set at 150 ng, 300 ng, and 600 ng per well for the anti-Ulp1 mouse monoclonal antibody, and the coating temperature and time selected as 37℃ for 1 h, considering the N value, the condition with the largest P / N value was determined as the optimal coating condition. The results are shown in Table 4. These results demonstrate that the optimal antibody concentration for coating is 600 ng / well.

[0069] Table 4 Selection of Antibody Concentration for Plate Packaging .

[0070] 3) Screening of sealing fluid types We screened various blocking solutions, including 2.5% (W / V) skim milk powder, 5% (W / V) skim milk powder, 7.5% (W / V) skim milk powder, 2% (W / V) gelatin, 5% (W / V) gelatin, 2% (W / V) BSA, 5% (W / V) BSA, 7.5% (W / V) BSA, 2% (W / V) NH4Cl, 5% (W / V) NH4Cl (all aqueous solutions), and fetal bovine serum. Blocking was performed at 37℃ for 1 hour. Considering the nitrogen (N) value, the solution with the largest P / N ratio was selected as the optimal blocking condition. The results are shown in Table 5. 2.5% (W / V) skim milk powder was chosen as the blocking solution.

[0071] Table 5 Screening of Sealing Fluid Types 4) Selection of closing time Using 5% (W / V) skim milk powder, the sealing time was determined to be 1h, 1.5h and 2h at 37℃, taking into account the N value and selecting the condition with a larger P / N value. The results are shown in Table 6. Considering the need to save detection time, the sealing time was selected as 1h.

[0072] Table 6 Screening of Closure Time .

[0073] 5) Selection of antibody concentration for detection The selected antibody concentrations for detecting the Ulp1 enzyme were rabbit polyclonal antibodies at 1.5 ug / ml, 3.0 ug / ml, and 6.0 ug / ml. The antibody was incubated at 37°C for 1 hour. Taking into account the N value, the optimal concentration of the detection antibody was determined by selecting the condition with the largest P / N value. The results are shown in Table 7. These results prove that the optimal concentration of the detection antibody is 6.0 ug / ml.

[0074] Table 7 Screening for antibody concentration detection .

[0075] 6) Selection of enzyme-labeled antibody dilution ratio The enzyme-labeled antibody was diluted at ratios of 1:5000, 1:10000, and 1:20000, and incubated at 37°C for 1 hour. Taking into account the N value, the optimal enzyme-labeled antibody dilution ratio was determined by selecting the condition with the largest P / N value. The results are shown in Table 8, which proves that the dilution ratio is 1:5000.

[0076] Table 8 Screening of enzyme-labeled antibody concentration .

[0077] 7) Selection of color development time Using TMB colorimetric solution, color development was performed at 25℃ for 5 min, 10 min, 15 min, and 20 min respectively. The optimal color development time was determined by considering the smaller N value and the larger P / N value. The results are shown in Table 9. Based on the results, the color development time of 5 min was selected.

[0078] Table 9. Selection of color development time Through condition optimization, we obtained the following optimal preparation and usage conditions for the kit: the optimal coating time and temperature is 16 hours, the coating concentration at 4℃ is 600 ng / well, the blocking buffer is 2.5% (w / v) skim milk powder, and the blocking time is 1 hour. The optimal antibody concentration is 6.0 ug / ml. The enzyme-labeled antibody dilution ratio is 1:5000, and the color development time is 5 minutes. Only by considering these conditions can the advantages of the kit be fully realized.

[0079] 3. Experimental verification 1) Sensitivity verification The Ulp1 enzyme was serially diluted three times to 13.72 ng / ml, 4.57 ng / ml, 1.52 ng / ml, 0.51 ng / ml, 0.17 ng / ml, 0.06 ng / ml, and 0.02 ng / ml. The sensitivity of the kit was verified using the sandwich ELISA method of this invention (plate coating time and temperature: 16 h, 4 °C; plate coating concentration: 600 ng / well; blocking buffer: 2.5% (w / v) skim milk powder; blocking time: 1 h; detection antibody concentration: 6.0 ug / ml; enzyme-labeled antibody dilution ratio: 1:5000; color development time: 5 min). The detection data are shown in Table 10 below. At a concentration of 0.02 ng / ml, the P / N value reached above 2.1; therefore, the sensitivity of the kit is 0.02 ng / ml.

[0080] Table 10 Sensitivity Verification .

[0081] 2) Intra-batch stability verification Using ELISA plates coated from the same batch, the Ulp1 enzyme standard was diluted into multiple gradients, with three replicates for each concentration, to verify the stability of the same batch. The test results were used to calculate the coefficient of variation (CV) of the intra-batch experiments. The formula for calculating the CV is: CV (%) = SD / Mean × 100%. Where: SD is the standard deviation; Mean is the mean. The intra-batch repeatability verification results are shown in Table 11, with intra-batch differences less than 10%.

[0082] Table 11 In-batch stability verification 3) Inter-batch stability verification Ulp1 enzyme standard was diluted to multiple gradients using microplates coated with different batches, with each concentration tested in triplicate to verify batch-to-batch stability. The test results were used to calculate the coefficient of variation (CV) between batches. The formula for calculating the CV is: CV (%) = SD / Mean × 100%. Where: SD is the standard deviation; Mean is the mean. The batch-to-batch repeatability verification results are shown in Table 12, with batch-to-batch differences less than 10%.

[0083] Both intra-batch and inter-batch differences were less than 10%, indicating that the method has good repeatability.

[0084] Table 12 Inter-batch stability verification .

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

[0086] Mouse monoclonal antibody amino acid sequence Heavy Chain MGWSWILLFLLSVTAGVHSQVQLQQSGAELVRPGASVTLSCKASGYRFTDYEMHWVKQTPVHGLEWIGAIDPETGDTAYN QKFKGKATLTADKSSSTAYMDFRSLTSEDSAVYYCTPPGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLV KGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQD WLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYK NTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light Chain DIVMTQSPLTLSVTIGQPASISCKSSQSLLASDGETYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKI SRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Mouse monoclonal antibody light chain gene sequence, vector pcDNA3.4 5’ -GACATCGTGATGACCCAGAGCCCCCTGACACTGTCCGTGACAATCGGCCAGCCCGCTTCTATCAGCTGCAAGTCCAGCCAGAGCCTGCTGGCTTCCGACGGAGAAACATACCTGAATTGGCTGCTGCAGAGGCCTGGCCAGAGCCCTAAAAGACTGATCTACCTGGTGAGCAAGCTGGATTCCGGCGTGCCTGATAGATTCACCGGCTCCGGAAGCGGCACAGACTTTACACTGAAGATCTCCAGGGTGGAGGCCGAGGACCTGGGAGTGTATTACTGCTGGCAGGGCACCCACTTCCCCCAAACATTTGGCGGCGGCACCAAGCTGGAGATCAAGAGAGCTGATGCTGCACCTACCGTGTCCATTTTCCCTCCAAGCAGCGAACAGCTTACAAGCGGAGGAGCCAGCGTCGTCTGTTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGATCTGAGAGGCAGAACGGAGTGCTGAACTCTTGGACCGACCAGGACAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACTTGCGAGGCCACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGCAACGAGTGC-3’ Mouse monoclonal antibody heavy chain gene sequence, vector pcDNA3.4

Claims

1. An Ulp1 enzyme double antibody sandwich ELISA kit, characterized in that, include: ELISA plates coated with mouse monoclonal antibody against Ulp1 enzyme, rabbit polyclonal antibody against Ulp1 enzyme, HRP-labeled goat anti-rabbit antibody, diluent, washing buffer, blocking buffer, chromogenic buffer, and stop solution.

2. The reagent kit according to claim 1, characterized in that, The diluent is PBST containing 0.1-0.5% BSA.

3. The reagent kit according to claim 1, characterized in that, The cleaning solution is PBST, the blocking solution is PBST containing 2-5% skim milk powder; the color developing solution is TMB color developing solution; and the stop solution is 0.5M-0.6M phosphate stop solution.

4. The reagent kit according to claim 1, characterized in that, The anti-Ulp1 enzyme mouse monoclonal antibody and the anti-Ulp1 enzyme rabbit polyclonal antibody are prepared by immunization with a complex protein of Ulp1 enzyme and SUMO tag protein. The sequence number of the anti-Ulp1 enzyme mouse monoclonal antibody is 7H2. The mass ratio of SUMO tag protein to Ulp1 enzyme is 200:

1.

5. The use of the kit according to any one of claims 1 to 4 in detecting Ulp1 enzyme derived from prokaryotic recombinant expression of Saccharomyces cerevisiae.

6. Application of anti-Ulp1 enzyme mouse monoclonal antibody, anti-Ulp1 enzyme rabbit polyclonal antibody and HRP-labeled goat anti-rabbit antibody in the detection of prokaryotic recombinant expression of Ulp1 enzyme from Saccharomyces cerevisiae.

7. The application of the kit according to claim 5 in detecting Ulp1 enzyme derived from prokaryotic recombinant expression of Saccharomyces cerevisiae, characterized in that, Includes the following steps: (1) Dilute the anti-Ulp1 enzyme mouse monoclonal antibody to 6-8ug / ml with coating buffer, add 100-150ul to each well of the microplate for coating, and coat at 4-37℃ for 16-20h. (2) After cleaning each well three times with 100ul-300ul of cleaning solution, add sealing solution and seal at 37℃ for 1-2 hours, then pat dry; (3) Dilute the Ulp1 enzyme to a standard concentration of 800-810 ug / ml with diluent. Using this concentration as a reference, continue to dilute it in multiple gradients according to a 3-fold gradient. Add the diluted Ulp1 enzyme to the coated microplate and incubate at 37°C for 1-2 hours. (4) Clean each well three times with 100ul-300ul of cleaning solution, then pat dry; (5) Add anti-Ulp1 enzyme rabbit polyclonal antibody dilution buffer to a final concentration of 1.5-6 ug / ml, and incubate at 37℃ for 1 h for binding; (6) Clean each well three times with 100ul-300ul of cleaning solution, then pat dry; (7) Add HRP-labeled goat anti-rabbit antibody dilution solution and incubate at 37°C for 1-2 hours to bind; (8) Clean each well three times with 100ul-300ul of cleaning solution, then pat dry; (9) Add color developing solution and develop color at 25°C in the dark; (10) Add the stop solution to terminate the process and take a reading at OD450nm; (11) Based on the reading of the microplate reader, the standard curve is obtained by matching the logarithmic value of the Ulp1 enzyme standard concentration.

8. The application according to claim 7, characterized in that, The blocking solution is selected from one or more of the following: 5% (W / V) skim milk powder, 7.5% (W / V) skim milk powder, 2% (W / V) gelatin, 5% (W / V) gelatin, 2% (W / V) BSA, 5% (W / V) BSA, 7.5% (W / V) BSA, 2% (W / V) NH4Cl, 5% (W / V) NH4Cl, and fetal bovine serum.

9. The application according to claim 7, characterized in that, The concentration of the coated antibody was 600 ng / well.

10. The application according to claim 7, characterized in that, The dilution ratio in step (7) is 1:5000-1:20000; the color development time in step (9) is 5-20 min.

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